Method for screening lipid-lowering active substances from natural extracts by improving enzyme curing efficiency

Abstract

The invention discloses a method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency, compared with the prior art, a polypropylene hollow fiber membrane is used as an adsorption carrier, glutaraldehyde is used as a cross-linking agent, pancreatic lipase is covalently cross-linked to the polypropylene hollow fiber membrane, and the lipid-lowering active substances are screened from the natural extracts. The enzyme loading amount, the repeated utilization rate and the storage stability of the polypropylene hollow fiber membrane are obviously improved; the enzyme immobilization efficiency and the enzyme activity are improved; furthermore, liquid chromatography-quadrupole time-of-flight mass spectrometry and a molecular network analysis technology are combined to screen 31 active ligand structures from the wild chrysanthemum flower extract with numerous and jumbled components, so that the method can be popularized and used for efficiently screening active ligands in natural products with complex components; the material basis of the lipid-lowering effect of the natural-source active ingredients can be deeply illuminated, and the active ligand has certain lipid-lowering potential and can be used as a pilot chemical of lipid-lowering drugs.

Description

Technical Field

[0001] The invention relates to the field of drug screening and evaluation methods, and in particular to a method for screening lipid-lowering active substances from natural extracts by improving enzyme solidification efficiency. Background Art

[0002] Currently, obesity has become a major health challenge affecting people in different regions of my country, and the age of people suffering from obesity is getting younger year by year. The World Health Organization (WHO) points out that adults can be considered obese when their BMI index reaches or exceeds 30. The common fat metabolism disorder in obese patients will lead to an increase in the prevalence of other chronic diseases, such as diabetes, cardiovascular disease, hypertension and cancer.

[0003] Exploring active ingredients with significant efficacy and few side effects from natural products will provide promising candidate compounds for new drug development and has become an alternative strategy for the development of drugs for the treatment of obesity. In recent years, screening technologies based on enzyme and ligand binding affinity have made significant progress in the screening of active ingredients in complex matrices, surpassing traditional screening methods such as affinity ultrafiltration screening-liquid chromatography-mass spectrometry in terms of screening efficiency, selectivity and sensitivity. However, this technology still has certain limitations, such as the enzyme is in a free state and cannot be reused during the affinity ultrafiltration process, and the ultrafiltration tube cannot be reused. These problems increase the cost of affinity ultrafiltration screening technology and limit its application.

[0004] Immobilized enzyme technology is a technology that uses physical or chemical methods to fix free enzymes on a carrier or confine them to a certain area for enzymatic catalytic reactions. Immobilized enzymes are highly stable, reusable, and easy to recycle. Currently, immobilized enzyme technology has been widely used in the fields of medicine, chemical industry, environment, food, etc., especially in the field of medicine. Immobilized enzyme technology is often used for targeted screening of active ingredients in natural products. Currently, a variety of disease-related enzymes have been used as immobilized target enzymes, such as α-glucosidase, acetylcholinesterase, pancreatic lipase, trypsin, etc., providing innovative ideas for the development of new drugs.

[0005] Polypropylene hollow fiber membrane is a porous membrane with rich surface micropores and a cortex with a special cross-section. It has been widely used in wastewater treatment and sample preparation due to its large specific surface area, excellent chemical stability and corrosion resistance, low cost, and good sample enrichment effect. Immobilizing biological enzymes on hollow fiber membranes can improve the stability and reusability of enzymes and quickly capture target components from complex natural products.

[0006] For example, the invention application with publication number CN102824756A discloses a method for screening triglyceride inhibitors from plant extracts, which is used to quickly screen compounds with triglyceride inhibitory activity in plant extracts, by using polypropylene hollow fibers as adsorption carriers, and using adsorption carriers with triglyceride enzymes combined with liquid chromatography-mass spectrometry to affinity screen potential natural active ligands from natural product extracts and perform structural identification. However, this method fixes the biological enzyme on the surface of the hollow fiber membrane by non-covalent adsorption, resulting in limited enzyme loading and the reuse rate and enzyme activity during the screening process need to be improved.

[0007] Molecular Network (MN) technology based on LC-MS is a tool that has emerged in recent years to quickly visualize and process mass spectrometry data. It uses the similarity of mass spectrometry fragments to efficiently classify complex mass spectrometry data and cluster similar compounds, thereby quickly predicting the structure of unknown compounds and their analogs through the mass spectrometry database. Currently, molecular network analysis technology has been widely used in the rapid identification of complex chemical components of traditional Chinese medicine.

[0008] For example, reference 1 (Rapid analysis of Aronia nigra fruit components based on UPLC-Orbitrap-MS / MS technology combined with GNPS molecular network, 2024, Journal of Mass Spectrometry) uses UPLC-MS / MS molecular network analysis technology to quickly identify 42 chemical components from Aronia nigra fruit, including 20 anthocyanins, 12 flavonols, 7 organic acids and 3 proanthocyanidins. This method can quickly and accurately classify and identify Aronia nigra fruit components, providing a basis for the research and development of its efficacy material basis.

[0009] Although there are some methods for screening lipid-lowering active substances in the field of drug screening, the problems of increasing enzyme loading, enzyme reuse rate, enzyme activity and stability need to be solved urgently. In addition, affinity screening of active substances from natural products or extracts to provide a basis for the basic research and development and utilization of their efficacy materials is the focus of research in the field of drug screening. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency, based on the immobilization of biological enzymes on glutaraldehyde cross-linked polypropylene hollow fiber membranes, and combining liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis technology to affinity screen active ligands from wild chrysanthemum extracts and perform structural identification, which is used for rapid screening of lipid-lowering active substances and elucidating the material basis of the lipid-lowering effect in natural extracts.

[0011] A method for improving enzyme solidification efficiency and screening lipid-lowering active substances from natural extracts, characterized in that it comprises the following steps:

[0012] (1) immersing the polypropylene hollow fiber membrane in an organic solvent for activation to obtain an activated polypropylene hollow fiber membrane;

[0013] (2) taking the activated polypropylene hollow fiber membrane and placing it in a glutaraldehyde aqueous solution, shaking and mixing it, and washing it with ultrapure water to obtain a glutaraldehyde-crosslinked polypropylene hollow fiber membrane;

[0014] (3) preparing a pancreatic lipase solution using a phosphate buffer, placing the glutaraldehyde cross-linked polypropylene hollow fiber membrane in the pancreatic lipase solution, and stirring and incubating to obtain a glutaraldehyde cross-linked polypropylene hollow fiber membrane of pancreatic lipase;

[0015] (4) The polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde was incubated in an aqueous solution of wild chrysanthemum extract, then rinsed with phosphate buffer, and then placed in an organic solution for dissociation to obtain a dissociation solution. The active ligands were screened from the dissociation solution and the active component analysis was performed using liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis technology.

[0016] The invention adopts a polypropylene hollow fiber membrane cross-linked with glutaraldehyde as an adsorption carrier, wherein the polypropylene hollow fiber membrane has the advantages of large specific surface area, excellent chemical stability and corrosion resistance, low cost, good sample enrichment effect and the like; glutaraldehyde, as a cross-linking agent, contains two aldehyde groups, can covalently bind to the enzyme and the amino groups on the immobilized carrier to form a stable cross-linking structure, thereby increasing the enzyme loading and activity while improving the reuse rate; based on the polypropylene hollow fiber cross-linked with glutaraldehyde for pancreatic lipase, liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis technology are combined to affinity screen active ligands from wild chrysanthemum extract to improve the screening efficiency.

[0017] Preferably, the inner diameter of the polypropylene hollow fiber membrane is 300 μm to 400 μm, the wall thickness is 40 μm to 60 μm, the pore size is 0.1 μm to 0.3 μm, and the porosity is 60% to 80%.

[0018] The polypropylene hollow fiber membrane of the above specifications has a large specific surface area, excellent chemical stability and corrosion resistance, good sample enrichment effect, is not easy to break or be damaged during operation, and can increase the adsorption amount of the enzyme.

[0019] Further preferably, the inner diameter of the polypropylene hollow fiber membrane is 350 μm, the wall thickness is 45 μm, the pore size is 0.2 μm, and the porosity is 70%.

[0020] Preferably, the organic solvent is methanol or ethanol.

[0021] A suitable organic solvent is selected to activate the polypropylene hollow fiber membrane, so that the activated polypropylene hollow fiber membrane is bonded with the cross-linking agent or lipase.

[0022] More preferably, the organic solvent is methanol.

[0023] Preferably, the mass percentage of the glutaraldehyde aqueous solution is 0.125% to 2%.

[0024] Based on the chemical cross-linking method, the cross-linking agent glutaraldehyde is introduced to form a strong covalent bond between the enzyme and the adsorption carrier. The use of glutaraldehyde aqueous solution in the above-mentioned mass concentration range as a cross-linking agent can form a stable cross-linking structure with pancreatic lipase, thereby improving the activity of the enzyme and the immobilization stability.

[0025] More preferably, the mass percentage of the glutaraldehyde aqueous solution is 1%.

[0026] Preferably, the ionic strength of the phosphate buffer is 50-1000 mM, and the pH of the phosphate buffer is 6.2-8.0.

[0027] Buffer pH is crucial to enzyme activity. The function and conformation of the enzyme are highly dependent on the ionic environment around it. Changes in pH will affect the activity and structure of the enzyme. Buffer solutions within the above range can improve the structural stability of the enzyme and reduce the impact of pH changes on enzyme activity.

[0028] More preferably, the phosphate buffer has an ionic strength of 100 mM and a pH of 7.4.

[0029] Preferably, the phosphate buffer solution in step (3) and step (4) is rinsed 1 to 3 times, and more preferably, the phosphate buffer solution is rinsed 3 times.

[0030] In order to ensure that the polypropylene hollow fiber membrane effectively cross-links pancreatic lipase under the action of glutaraldehyde, the enzyme loading capacity of the polypropylene hollow fiber membrane cross-linked with glutaraldehyde for pancreatic lipase is investigated before affinity screening and separation of active substances. The investigation method includes: ultraviolet spectrophotometer detection based on substrate catalytic color development, scanning electron microscope microscopic morphology analysis and Fourier transform infrared spectrometer determination; wherein the substrate is p-nitrophenyl palmitate;

[0031] The substrate-catalyzed color development-based ultraviolet spectrophotometer detection is performed by incubating the polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde with a solution of p-nitrophenyl palmitate at 37° C. for 5 minutes, using an ultraviolet spectrophotometer to detect the absorbance value of the solution at 405 nm, and calculating the pancreatic lipase loading amount based on a standard curve of the product p-nitrophenol;

[0032] The scanning electron microscope microscopic morphology analysis visually examines the cross-linking of pancreatic lipase from the scanning electron microscope image;

[0033] The Fourier transform infrared spectrometer determines different chemical bonds and functional groups based on the specific absorption of infrared radiation by different chemical bonds and functional groups by identifying characteristic absorption peaks. When molecules are irradiated with infrared radiation, they absorb radiation of specific wavelengths, thereby causing the chemical bonds inside the molecules to vibrate. The energy changes generated by these vibrations are recorded to form an infrared spectrum. At the same time, the elements are in different chemical environments, and their binding energies will have slight differences, which in turn cause changes in chemical shifts. Therefore, the chemical shift values ​​can be used to analyze the valence and existence form of the elements, thereby further determining the formation of chemical bonds and changes in functional groups during the reaction.

[0034] Preferably, the concentration of the pancreatic lipase solution is 0.5-3 mg / mL.

[0035] The pancreatic lipase concentration within the above range can completely bind the enzyme to the carrier and will not cause oversaturation of the enzyme binding to the adsorption carrier, resulting in enzyme waste.

[0036] More preferably, the concentration of the pancreatic lipase solution is 2 mg / mL.

[0037] Preferably, the incubation time in step (3) and step (4) is 0.1 to 3 hours, and the incubation temperature is 15 to 37°C.

[0038] Incubation time and incubation temperature within the above range can ensure that the enzyme is fully immobilized without reducing the activity of the enzyme, and can bind to the active ingredients in the natural extract to the greatest extent through affinity.

[0039] More preferably, the incubation time in step (3) is 1 h and the incubation temperature is 37°C.

[0040] Preferably, the organic solution is a methanol aqueous solution or an acetonitrile aqueous solution.

[0041] For the adsorption carrier after enzyme immobilization, methanol aqueous solution or acetonitrile aqueous solution can inactivate pancreatic lipase, and the active ligand bound to pancreatic lipase with high affinity is released and eluted for active component analysis.

[0042] Further preferably, the volume percentage of methanol in the methanol aqueous solution is 90%, and the volume percentage of acetonitrile in the acetonitrile aqueous solution is 10%.

[0043] Selecting the above-mentioned polar organic solution for elution to obtain the active ingredients can release more active ingredients for subsequent identification of the active ligand structure, thereby improving the possibility and accuracy of active ligand identification.

[0044] Preferably, the method for obtaining the wild chrysanthemum extract comprises: collecting and grinding air-dried wild chrysanthemum plant samples, extracting and ultrasonicating the ground wild chrysanthemum plant samples with an organic solution, filtering and freeze-drying the extract to obtain the wild chrysanthemum extract.

[0045] Preferably, the organic solution is an ethanol aqueous solution with a volume percentage of 75% ethanol, the extraction time is at least 12 hours, and the ultrasound is performed at least 3 times.

[0046] Long-term immersion and multiple ultrasound treatments can fully release the active ingredients in wild chrysanthemum plant samples, thereby improving the possibility of subsequent active substance identification and the accuracy of active ingredient analysis.

[0047] Further preferably, the extraction time is 12 hours, and the ultrasonic treatment is performed 3 times, each time for 30 minutes.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] The invention discloses a method for improving enzyme solidification efficiency and screening lipid-lowering active substances from natural extracts. A polypropylene hollow fiber membrane is used as an adsorption carrier, and glutaraldehyde is used as a cross-linking agent to covalently cross-link pancreatic lipase to the polypropylene hollow fiber membrane, thereby significantly improving the enzyme loading capacity, reuse rate and storage stability of the polypropylene hollow fiber membrane, and improving the enzyme immobilization efficiency and enzyme activity. Liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis technology are further combined to screen and analyze the structures of 31 active ligands from a wild chrysanthemum extract with complex components. The method can be widely used for efficient screening of active ligands in natural products with complex components, and is helpful to deeply clarify the material basis of the lipid-lowering effect of active ingredients from natural sources. The active ligand has a certain lipid-lowering potential and can be used as a potential lead compound for lipid-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A flow chart of a method for improving enzyme solidification efficiency and screening lipid-lowering active substances from natural extracts provided by the present invention;

[0051] Figure 2 The results of optimizing the conditions for preparing polypropylene hollow fiber membrane for glutaraldehyde cross-linked pancreatic lipase are shown in FIG. Figure 2 A in it is the effect of pancreatic lipase concentration on the amount of pancreatic lipase loaded; Figure 2 B in the figure is the effect of incubation time on the amount of pancreatic lipase loaded; Figure 2 C in the figure is the effect of pH value of phosphate buffer on the amount of pancreatic lipase loaded; Figure 2 D in the figure is the effect of glutaraldehyde concentration on the amount of pancreatic lipase loaded;

[0052] Figure 3The scanning electron micrograph of the polypropylene hollow fiber membrane with covalently cross-linked pancreatic lipase, wherein: Figure 3 A in the figure is a scanning electron microscope image of a blank polypropylene hollow fiber membrane; Figure 3 B is a scanning electron micrograph of a polypropylene hollow fiber membrane with non-covalent adsorption of pancreatic lipase; Figure 3 C in the figure is a scanning electron micrograph of polypropylene hollow fiber cross-linked with pancreatic lipase by glutaraldehyde;

[0053] Figure 4 Fourier transform infrared spectra of blank polypropylene hollow fiber membrane, polypropylene hollow fiber membrane with non-covalent adsorption of pancreatic lipase, and polypropylene hollow fiber membrane with glutaraldehyde cross-linked pancreatic lipase;

[0054] Figure 5 Reusability of polypropylene hollow fiber membranes for glutaraldehyde cross-linking of pancreatic lipase;

[0055] Figure 6 Storage stability of polypropylene hollow fiber membrane cross-linked with pancreatic lipase by glutaraldehyde;

[0056] Figure 7 This is the molecular network diagram of all compounds of wild chrysanthemum extract;

[0057] Figure 8 This is the molecular network diagram of potential pancreatic lipase ligands among all compounds of wild chrysanthemum extract;

[0058] Fig. 9 The extracted ion chromatogram (EIC) results of 35 clustered ligands (black line) in wild chrysanthemum extract and incubated with activated (red line) and inactivated (blue line) pancreatic lipase;

[0059] Fig.10 The representative pancreatic lipase ligands of wild chrysanthemum extract have inhibitory activity on pancreatic lipase. DETAILED DESCRIPTION

[0060] The specific implementation of the present invention is further described in detail below in conjunction with the drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be pointed out that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The pancreatic lipase involved in the present invention was purchased from Yuanye Biotechnology Co., Ltd.; polypropylene hollow fiber membrane was purchased from Hangzhou Xinhui Membrane Technology Co., Ltd.; BCA protein concentration determination kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; p-nitrophenyl palmitate (p-NPP, 98%) and product p-nitrophenol (p-NP, 98%) were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; glutaraldehyde aqueous solution (25%) was purchased from Sinopharm Chemical Reagent Co., Ltd.; wild chrysanthemum active ingredient standard products caffeic acid ethyl ester, chlorogenic acid ethyl ester, neochlorogenic acid methyl ester and positive control proanthocyanidin B2 (98%) were purchased from Wuhan Zhongbiao Technology Co., Ltd.

[0061] Example 1

[0062] Preparation of glutaraldehyde cross-linked polypropylene hollow fiber membrane:

[0063] (1) 10 polypropylene hollow fiber membranes, each with a length of 2 cm, were soaked in methanol for activation for 1 hour;

[0064] (2) taking out the polypropylene hollow fiber membrane and washing it with deionized water for 10 minutes to rinse the methanol away, thereby obtaining an activated polypropylene hollow fiber membrane;

[0065] (3) Take 10 activated polypropylene hollow fiber membranes and place them in a 2 mL EP tube, add 1 mL of 1% glutaraldehyde aqueous solution by mass E ratio, shake and mix at 37°C for 0.5 h, then add 1 mL of ultrapure water to rinse once, and obtain glutaraldehyde cross-linked polypropylene hollow fiber membrane.

[0066] Preparation of polypropylene hollow fiber membrane for pancreatic lipase cross-linked with glutaraldehyde:

[0067] A pancreatic lipase solution was prepared with a 0.1 mol / L phosphate buffer solution (PBS buffer) with a pH of 7.4, and 1 mL of the pancreatic lipase solution with a concentration of 2.0 mg / mL was added to immerse the polypropylene hollow fiber membrane cross-linked with glutaraldehyde, and the membrane was incubated with stirring for 60 minutes at a temperature of 37° C. to obtain a polypropylene hollow fiber membrane cross-linked with glutaraldehyde;

[0068] 200 μL of the pancreatic lipase solution before and after immobilization was taken, and the protein concentration was measured using a BCA protein detection kit to calculate the amount of pancreatic lipase cross-linked on each polypropylene hollow fiber membrane.

[0069] Preparation of wild chrysanthemum extract aqueous solution:

[0070] (1) collecting air-dried wild chrysanthemum plant samples, grinding them, extracting them with ethanol aqueous solution for 12 hours and ultrasonicating them three times, each time for 30 minutes, filtering and freeze-drying the extract to obtain a wild chrysanthemum extract, wherein the volume percentage of the ethanol aqueous solution in this step is 75%;

[0071] (2) 30 mg of the prepared wild chrysanthemum extract was taken and dissolved in a phosphate buffer solution to a concentration of 10 mg / mL wild chrysanthemum extract aqueous solution.

[0072] Activity analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis:

[0073] (1) 1 mL of wild chrysanthemum extract aqueous solution was placed in an EP tube, 10 polypropylene hollow fiber membranes cross-linked with pancreatic lipase by glutaraldehyde were added, and the mixture was incubated at 37°C and 130 rpm / min for 1 h;

[0074] (2) After the incubation, the polypropylene hollow fiber membrane cross-linked with glutaraldehyde pancreatic lipase was rinsed with 1 mL of phosphate buffer, and the process was repeated three times to remove components not bound to the polypropylene hollow fiber membrane cross-linked with glutaraldehyde pancreatic lipase;

[0075] (3) transferring the sample rinsed in step (2) into a methanol-water solution and soaking for 10 min to inactivate the enzyme and release the captured components, and collecting the dissociation solution for liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) and molecular network analysis;

[0076] The liquid chromatography parameters are as follows: Chromatographic separation was performed on an Agilent 1290UPLC system (Agilent, USA) equipped with a Waters Sunniest C 18HT column (100×2.1 mm, 2 μm). The column temperature was 25°C, the injection volume was 5 μL, and the flow rate was 0.2 mL / min. The mobile phases were ultrapure water (A) and acetonitrile (B) containing 0.1% formic acid, and the liquid phase elution gradients were 0-40 min, 5%-95% (mobile phase B), 40.1-45 min, 95% (mobile phase B), and 45.1-50 min, 5% (mobile phase B).

[0077] The mass spectrometry parameters are as follows: Mass spectrometry analysis was performed using an Agilent 6530Q-TOF-MS / MS system (Agilent, USA). In negative ion mode, mass spectrometry information was collected using electrospray ionization mode. Inert nitrogen was used as drying, nebulizing, and collision gas; drying gas flow rate was 8 L / min; ESI spray voltage was 3.5 kV; nebulizer pressure was 35 psi; capillary temperature was 350 °C; collision energy was 10-40 eV. Primary and secondary mass spectrometry information was collected in the mass range of m / z 50-1000; mass spectrometry data analysis was completed by Mass Hunter workstation B.08.01.00.

[0078] Molecular network technology is an effective tool for quickly integrating and identifying similar compounds. This technology uses the similarity of mass spectrometry fragments to process large quantities of mass spectrometry data. The basic principle is that similar compounds produce similar fragment ions under the same mass spectrometry conditions. Molecular network analysis technology clusters similar compounds by evaluating the similarity of these fragment ions and establishes a visual molecular network diagram. In addition, the efficiency of compound structure identification can be improved based on the molecular network diagram.

[0079] To reduce false positive results caused by nonspecific adsorption, the same screening steps were performed using boiled inactivated pancreatic lipase, and the binding affinity between different compounds and pancreatic lipase was evaluated by comparing the binding degree values, which were calculated by the characteristic peak areas of the compounds obtained after screening using hollow fiber membranes cross-linked with active enzymes and cross-linked with inactivated enzymes on a liquid chromatograph.

[0080] Example 2

[0081] The steps of Example 2 are the same as those of Example 1, except that during the preparation of the glutaraldehyde cross-linked polypropylene hollow fiber membrane, the mass percentage of the glutaraldehyde aqueous solution is 0.125%.

[0082] Example 3

[0083] The steps of Example 3 are the same as those of Example 1, except that during the preparation of the glutaraldehyde cross-linked polypropylene hollow fiber membrane, the mass percentage of the glutaraldehyde aqueous solution is 0.25%.

[0084] Example 4

[0085] The steps of Example 4 are the same as those of Example 1, except that during the preparation of the glutaraldehyde cross-linked polypropylene hollow fiber membrane, the mass percentage of the glutaraldehyde aqueous solution is 0.50%.

[0086] Example 5

[0087] The steps of Example 5 are the same as those of Example 1, except that during the preparation of the glutaraldehyde cross-linked polypropylene hollow fiber membrane, the mass percentage of the glutaraldehyde aqueous solution is 2.0%.

[0088] Example 6

[0089] The steps of Example 6 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane of glutaraldehyde-crosslinked pancreatic lipase, the pH value of the phosphate buffer for preparing the pancreatic lipase solution is 6.2.

[0090] Example 7

[0091] The steps of Example 7 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde, the pH value of the phosphate buffer for preparing the pancreatic lipase solution is 6.8.

[0092] Example 8

[0093] The steps of Example 8 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde, the pH value of the phosphate buffer for preparing the pancreatic lipase solution is 8.0.

[0094] Example 9

[0095] The steps of Example 9 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linked with glutaraldehyde for pancreatic lipase, the concentration of the pancreatic lipase solution is 0.5 mg / mL.

[0096] Example 10

[0097] The steps of Example 10 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linked with glutaraldehyde for pancreatic lipase, the concentration of the pancreatic lipase solution is 1.0 mg / mL.

[0098] Embodiment 11

[0099] The steps of Example 11 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linked with glutaraldehyde for pancreatic lipase, the pancreatic lipase solution is 1.5 mg / mL.

[0100] Example 12

[0101] The steps of Example 12 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linked with pancreatic lipase by glutaraldehyde, the concentration of the pancreatic lipase solution is 3.0 mg / mL.

[0102] Example 13

[0103] The steps of Example 14 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linking pancreatic lipase with glutaraldehyde, the membrane was incubated with stirring for 30 minutes at a temperature of 37°C.

[0104] Embodiment 14

[0105] The steps of Example 15 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linking pancreatic lipase with glutaraldehyde, the membrane was incubated with stirring for 90 minutes at a temperature of 37°C.

[0106] Embodiment 15

[0107] The steps of Example 16 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane of glutaraldehyde-crosslinked pancreatic lipase, the mixture was incubated with stirring for 120 minutes at a temperature of 37°C.

[0108] Example 16

[0109] The steps of Example 17 are the same as those of Example 1, except that during the preparation of the polypropylene hollow fiber membrane cross-linking pancreatic lipase with glutaraldehyde, the membrane was incubated with stirring for 150 minutes at a temperature of 37°C.

[0110] Comparative Example 1

[0111] Preparation of glutaraldehyde cross-linked polypropylene hollow fiber membrane:

[0112] (1) Take 10 2 cm long polypropylene hollow fiber membranes and soak them in methanol for 1 hour for activation;

[0113] (2) taking out the polypropylene hollow fiber membrane and washing it with deionized water for 10 minutes to rinse the methanol away, thereby obtaining an activated polypropylene hollow fiber membrane;

[0114] (3) Take 10 activated polypropylene hollow fiber membranes and place them in a 2 mL EP tube, add 1 mL of a 1% by mass glutaraldehyde aqueous solution, shake and mix at 37°C for 0.5 h, then add 1 mL of ultrapure water to rinse once, and obtain a glutaraldehyde-crosslinked polypropylene hollow fiber membrane.

[0115] Comparative Example 2

[0116] Preparation of polypropylene hollow fiber membrane for non-covalent adsorption of pancreatic lipase:

[0117] (1) Take a 2 cm long polypropylene hollow fiber membrane and soak it in methanol for 1 hour for activation;

[0118] (2) Take out the polypropylene hollow fiber and wash it with deionized water for 10 minutes to rinse out the methanol;

[0119] (3) Take 10 activated polypropylene hollow fibers and place them in a 2 mL EP tube. Prepare pancreatic lipase solution with 0.1 mol / L PBS buffer with a pH value of 7.4, add 1 mL of 2.0 mg / mL pancreatic lipase solution to immerse the polypropylene hollow fiber membrane, stir and incubate for 60 minutes at a temperature of 37°C.

[0120] Test Example 1

[0121] Based on the above embodiments and comparative examples, the conditions in the enzyme immobilization process were optimized using an ultraviolet spectrophotometer:

[0122] The polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde was incubated with p-nitrophenyl palmitate solution at 37°C for 5 minutes; the absorbance value of the solution at 405nm was detected by ultraviolet spectrophotometer, and the pancreatic lipase activity was calculated by the standard curve of the product p-nitrophenol, which is the enzyme loading amount of pancreatic lipase.

[0123] like Figure 2 As shown in A, when the pancreatic lipase concentration was 0.5-3.0 mg / mL, the amount of pancreatic lipase on the hollow fiber gradually increased with the increase of pancreatic lipase concentration. However, when the pancreatic lipase concentration was 2.0 mg / mL, the growth rate of the enzyme loading slowed down, so the optimal pancreatic lipase concentration was determined to be 2.0 mg / mL.

[0124] like Figure 2 As shown in B, within 0.5-2.5 h, the amount of immobilized pancreatic lipase gradually increased with the increase of incubation time. After more than 1 h, the growth rate of the enzyme loading slowed down, so the incubation time was determined to be 1 h.

[0125] like Figure 2 As shown in Figure C, adjusting the pH value of the phosphate buffer from 6.2 to 8.0 had no significant effect on the loading amount of the enzyme, indicating that the immobilized pancreatic lipase had good pH stability;

[0126] like Figure 2 As shown in D, when the glutaraldehyde concentration was 1%, the enzyme immobilization amount reached a maximum value (0.5±0.03μg), which increased by 1.5 times compared with the enzyme immobilization amount when glutaraldehyde was not added (0.2±0.03μg), indicating that the introduction of glutaraldehyde as a cross-linking agent formed a stronger covalent bond between pancreatic lipase and polypropylene hollow fiber membrane, resulting in a stronger bond between pancreatic lipase and polypropylene hollow fiber membrane, further increasing the enzyme loading amount.

[0127] Test Example 2

[0128] Based on the above examples and comparative examples, the microscopic morphology of the polypropylene hollow fiber membrane before and after glutaraldehyde cross-linking of pancreatic lipase was characterized by scanning electron microscopy.

[0129] from Figure 3 As can be seen from A in the figure, the surface of the blank polypropylene hollow fiber membrane is clean, with some pits of different sizes. Figure 3 As can be seen from C, after pancreatic lipase was immobilized on the polypropylene hollow fiber membrane using glutaraldehyde as a crosslinker, some floccules of different sizes were attached to the surface, some of which filled in the small pits and some were directly adsorbed on the surface. The results show that pancreatic lipase was effectively immobilized on the polypropylene hollow fiber membrane through the glutaraldehyde crosslinker. Figure 3 In B, using glutaraldehyde as a cross-linking agent can effectively increase the enzyme loading capacity.

[0130] Test Example 3

[0131] Based on the above examples and comparative examples, Fourier transform infrared spectrometer was used to perform infrared spectroscopy characterization on blank polypropylene hollow fiber membrane, polypropylene hollow fiber membrane with non-covalent adsorption of pancreatic lipase, and polypropylene hollow fiber membrane with glutaraldehyde cross-linked pancreatic lipase. The results are as follows: Figure 4 shown.

[0132] Depend on Figure 4 It can be seen that the Fourier transform infrared spectrum of the blank polypropylene hollow fiber membrane has a peak at wave number 2800 cm -1 ~3000cm -1 The characteristic absorption peak of polypropylene appears at (black line);

[0133] In the Fourier transform infrared spectrum of the polypropylene hollow fiber membrane with non-covalent adsorption of pancreatic lipase, in addition to the characteristic absorption peak of polypropylene, there is also a peak at 1650cm -1 and 3400cm -1 There are two obvious characteristic absorption peaks, among which 1650cm -1 The absorption peak at 3400 cm -1 The absorption peak at is attributed to the effect of -OH stretching vibration;

[0134] After adding glutaraldehyde as a cross-linking agent, the polypropylene hollow fiber membrane of glutaraldehyde cross-linked pancreatic lipase was -1 The absorption peak at is significantly enhanced, which is related to the stretching vibration of C=O in the aldehyde group;

[0135] Based on the above results, it was shown that pancreatic lipase was successfully immobilized on the polypropylene hollow fiber membrane, and glutaraldehyde as a cross-linking agent could significantly enhance the covalent binding between pancreatic lipase and the carrier.

[0136] Test Example 4

[0137] The reusability and storage stability of immobilized enzymes are key properties that affect their practical applications. In order to investigate the reusability of covalently cross-linked pancreatic lipase hollow fiber membranes, p-NPP hydrolysis reactions catalyzed by immobilized enzymes were carried out several times.

[0138] Based on the above examples and comparative examples, the reusability and storage stability of the polypropylene hollow fiber membrane of glutaraldehyde cross-linked pancreatic lipase were investigated.

[0139] Reusability: The polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde was incubated with a solution of p-nitrophenyl palmitate at 37°C for 5 minutes; the absorbance value of the solution at 405nm was detected by an ultraviolet spectrophotometer, and the pancreatic lipase activity was calculated by the standard curve of the product p-nitrophenol; the polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde was taken out and rinsed three times with phosphate buffer, and the above incubation process was repeated 6 times; the enzyme activity obtained by the first incubation was defined as 100%, and the percentage ratio of the enzyme activity calculated after each repeated incubation to the enzyme activity calculated by the first incubation was used to evaluate the reusability of the immobilized enzyme.

[0140] Storage stability: The polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde is incubated with a solution of p-nitrophenyl palmitate at 37°C for 5 minutes; the absorbance value of the solution at 405nm is detected by an ultraviolet spectrophotometer, and the pancreatic lipase activity is calculated by the standard curve of the product p-nitrophenol; the above hollow fiber membrane is taken out from the solution and rinsed three times with phosphate buffer, and then continued to be stored in phosphate buffer for 2 hours, 6 hours, and 10 hours, respectively, and the hollow fiber membrane is taken out and the above incubation process is repeated; the enzyme activity obtained by the first incubation is defined as 100%, and the percentage ratio of the enzyme activity calculated after incubation at each time point to the enzyme activity calculated by the first incubation is used to evaluate the storage stability of the immobilized enzyme.

[0141] Reproducibility results such as Figure 5 As shown, the polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde still maintained 51.7±0.6% of the initial activity after 6 consecutive cycles, indicating that the hollow fiber membrane of covalently cross-linked pancreatic lipase has good reusability;

[0142] Storage stability results are as follows Figure 6 As shown, after the polypropylene hollow fiber membrane of glutaraldehyde cross-linked pancreatic lipase was stored in phosphate buffer for 10 h, its residual relative activity still remained at 56.7 ± 0.9%;

[0143] The above results show that the polypropylene hollow fiber membrane of glutaraldehyde cross-linked pancreatic lipase prepared by this method has good reusability and storage stability. In addition, the glutaraldehyde cross-linking agent further strengthens the covalent bonding between pancreatic lipase and the carrier, thereby improving the stability of enzyme immobilization, helping to reduce the application cost of the enzyme, and having good application value.

[0144] Test Example 5

[0145] There are a variety of compounds in wild chrysanthemum. In order to further identify the potential lipid-lowering compounds in wild chrysanthemum, UPLC-Q-TOF-MS / MS combined with molecular network technology was used to analyze the screening results. Figure 7 As shown in the figure, each node represents a quasi-molecular ion peak. A total of 425 nodes are formed in the molecular network diagram of wild chrysanthemum, of which 89 nodes have a binding strength (BD) value greater than 0, which can be used as potential pancreatic lipase ligands. Among them, 35 nodes form 10 clusters due to the similarity between mass spectral fragments ( Figure 8 ).

[0146] The retention time, quasi-molecular ion peak, secondary mass spectrometry fragments and binding strength of the compounds represented by these 35 cluster nodes are shown in Table 1 and Fig. 9 The BD values ​​of these potential ligands ranged from 0.7% to 72.2%.

[0147] Table 1 Retention time, quasi-molecular ion peak, secondary mass spectrometry fragments and binding strength of the compounds represented by the 35 cluster nodes

[0148]

[0149]

[0150]

[0151]

[0152] By comparing the quasi-molecular ion peaks and secondary fragments of these compounds with the mass spectrometry data in the corresponding standards, references and databases, the structures of 31 active ligands were analyzed and identified from the 35 clustered compounds of wild chrysanthemum. These active ligands are mainly divided into three categories, namely flavonoids, phenolic acids and their ester derivatives, and phospholipid compounds.

[0153] The compounds with higher binding strength are mainly flavonoids and phenolic acids and their ester derivatives. The three representative ligands are: chlorogenic acid ethyl ester, luteolin 7-O-glucuronide and caffeic acid ethyl ester.

[0154] Test Example 6

[0155] The half inhibitory concentration (IC 50 Value) determination:

[0156] The inhibitory activity of three representative ligands (chlorogenic acid ethyl ester, luteolin 7-O-glucuronide and caffeic acid ethyl ester) on pancreatic lipase was further detected through in vitro experiments, including: preparing different concentrations of the test sample solution and pancreatic lipase solution with phosphate buffer, taking 100 μL of each sample, adding 60 μL of 0.25 mg / mL pancreatic lipase solution and 40 μL of phosphate buffer, placing in a 96-well plate, shaking and mixing at 37°C for 10 minutes; adding 20 μL of p-nitrophenyl palmitate, shaking and mixing at 37°C for 20 minutes, using an ultraviolet spectrophotometer to measure the absorbance at 405 nm, and calculating the pancreatic lipase inhibitory activity, wherein the known pancreatic lipase inhibitor proanthocyanidin B2 was used as a positive control.

[0157] like Fig.10 The test results showed that the three flavonoid and phenolic acid ester derivatives screened and verified by glutaraldehyde cross-linking pancreatic lipase polypropylene hollow fiber membrane were potential inhibitors of pancreatic lipase, namely chlorogenic acid ethyl ester, luteolin-7-O-glucuronide and caffeic acid ethyl ester, all of which had pancreatic lipase inhibitory activity, and their half inhibition concentration (IC 50 values) were 195.8 μM, 217.3 μM and 293.3 μM, respectively.

Claims

1. A method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency, characterized in that: The following steps are involved: (1) immersing the polypropylene hollow fiber membrane in an organic solvent for activation to obtain an activated polypropylene hollow fiber membrane; (2) taking the activated polypropylene hollow fiber membrane and placing it in a glutaraldehyde aqueous solution, shaking and mixing it, and washing it with ultrapure water to obtain a glutaraldehyde-crosslinked polypropylene hollow fiber membrane; (3) preparing a pancreatic lipase solution using a phosphate buffer, placing the glutaraldehyde cross-linked polypropylene hollow fiber membrane in the pancreatic lipase solution, and stirring and incubating to obtain a glutaraldehyde cross-linked polypropylene hollow fiber membrane of pancreatic lipase; (4) The polypropylene hollow fiber membrane of pancreatic lipase cross-linked with glutaraldehyde was incubated in an aqueous solution of wild chrysanthemum extract, then rinsed with phosphate buffer, and then placed in an organic solution for dissociation to obtain a dissociation solution. The active ligands were screened from the dissociation solution and the active component analysis was performed using liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular network analysis technology.

2. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The inner diameter of the polypropylene hollow fiber membrane is 300 μm to 400 μm, the wall thickness is 40 μm to 60 μm, the pore size is 0.1 μm to 0.3 μm, and the porosity is 60% to 80%.

3. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The mass percentage of the glutaraldehyde aqueous solution is 0.125% to 2%.

4. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The ion strength of the phosphate buffer is 50-1000 mM, and the pH of the phosphate buffer is 6.2-8.

0.

5. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The concentration of the pancreatic lipase solution is 0.5-3 mg / mL.

6. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The incubation time in step (3) and step (4) is 0.1 to 3 hours, and the incubation temperature is 15 to 37°C.

7. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The organic solution is a methanol aqueous solution or an acetonitrile aqueous solution.

8. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 7, characterized in that: The volume percentage of methanol in the methanol aqueous solution is 90%, and the volume percentage of acetonitrile in the acetonitrile aqueous solution is 10%.

9. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 1, characterized in that: The method for obtaining the wild chrysanthemum extract comprises: collecting and grinding air-dried wild chrysanthemum plant samples, extracting and ultrasonicating the ground wild chrysanthemum plant samples with an organic solution, filtering and freeze-drying the extract, and obtaining the wild chrysanthemum extract.

10. The method for screening lipid-lowering active substances from natural extracts by improving enzyme immobilization efficiency according to claim 8, characterized in that: The extraction time is at least 12 hours, and the ultrasound is performed at least 3 times.

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