Method for high-throughput screening of alpha-glucosidase inhibitor in boxthorn leaves through multi-shell MOF immobilized enzyme

Abstract

The invention provides a method for high-throughput screening of an alpha-glucosidase inhibitor from boxthorn leaves based on a multi-shell MOF immobilized enzyme, and aims to overcome the bottlenecks of low throughput, tedious operation, high cost and the like of a traditional screening method. The core process of the method comprises the following steps: preparing a boxthorn leaf sample solution and pre-evaluating the in-vitro alpha-glucosidase inhibitory activity; a multi-shell MOF immobilized enzyme technology is coupled with a UPLC-QTOF-MS / MS technology, so that targeted screening, efficient separation and accurate identification of active components are realized; verifying the alpha-glucosidase inhibitory activity of the target compound; and predicting the binding site of the small molecule compound and the enzyme through a molecular docking technology. The method has the core advantages of high specificity, high sensitivity, high flux, repeated utilization of enzyme receptors and the like by depending on the inherent characteristics of an immobilized enzyme technology, can synchronously screen and identify various inhibitors, effectively integrates the resource advantages of a boxthorn leaf natural product and the high efficiency of a modern high-flux screening technology, and has a wide application prospect. And a key technical support is provided for diversified development of diabetes treatment medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analytical chemistry, specifically to a method for high-throughput screening of α-glucosidase inhibitors from natural products using multi-shell MOF immobilized enzymes, particularly a method for high-throughput screening of α-glucosidase inhibitors from wolfberry leaves. Background Technology

[0002] Diabetes mellitus (DM), a rapidly growing metabolic disease worldwide, has become a major public health problem posing a serious threat to human health. Its pathogenesis stems from insulin secretion defects or impaired biological action. Long-term hyperglycemia can lead to serious complications such as retinopathy, kidney failure, and cardiovascular disease, significantly increasing the risk of disability and death in patients. Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia, is the most common type of diabetes, accounting for over 90% of cases globally.

[0003] α-Glucosidase (α-Glu) in the brush border of the small intestinal mucosa hydrolyzes oligosaccharides and disaccharides into glucose, which, after absorption by the small intestine, directly leads to an increase in blood glucose levels. Therefore, α-Glu is widely recognized as a major target enzyme in the prevention and treatment of type 2 diabetes mellitus (T2DM). Inhibiting its activity can delay the body's absorption of glucose, making it an effective way to prevent postprandial hyperglycemia and treat T2DM. Currently, α-Glu inhibitors such as acarbose, miglitol, and voglibose are commonly used clinically and are effective drugs for treating T2DM. However, long-term use can cause adverse reactions such as abdominal pain, diarrhea, and cramps, severely limiting their application. Therefore, in recent years, the search for safe, effective, and low-toxicity α-Glu inhibitors from natural products has become a research hotspot in the pharmaceutical field.

[0004] Natural products, due to their structural diversity and rich bioactivity, have always been an important treasure trove for new drug development. Many plant, animal, and microbial extracts contain components that inhibit α-Glu activity, potentially offering novel treatment strategies for metabolic diseases such as diabetes. However, the chemical composition of natural products is extremely complex, containing not only a variety of compounds with different structures but also potentially exhibiting synergistic or antagonistic interactions, posing a significant challenge to efficient screening. Traditional screening methods, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA), while providing some analytical capabilities, suffer from low throughput, cumbersome operation, and high cost, making it difficult to rapidly and accurately screen α-Glu inhibitors from a large number of natural products in a short time, greatly limiting the efficiency of discovering active ingredients in natural products.

[0005] Lycium barbarum leaves are from the Lycium genus of the Solanaceae family, specifically the Ningxia Lycium Lycium barbarum L.) or wolfberry ( L. chinese The tender stems and leaves of *Lycium barbarum* (goji berry) have a long history of use in my country for both food and medicine. Ancient medical texts such as *Mingyi Bielu* record its effects of "tonifying deficiency and replenishing essence, clearing heat and improving eyesight," and its primary use in treating symptoms such as fever due to deficiency and thirst. Modern research shows that *Lycium barbarum* leaves contain various active ingredients, including polysaccharides, flavonoids, phenolic acids, and alkaloids, exhibiting antioxidant and hypoglycemic effects. Aqueous extracts of *Lycium barbarum* leaves can effectively reduce blood glucose levels in diabetic model animals and improve liver and kidney damage through insulin receptor signal transduction and the arachidonic acid metabolic pathway. Our research group found that *Lycium barbarum* leaf extract has strong inhibitory activity against α-Glu, but the specific active ingredient responsible for this inhibitory effect remains unclear. Therefore, screening for natural α-Glu from *Lycium barbarum* leaves is of significant importance.

[0006] In recent years, various enzyme inhibitor screening technologies have emerged based on molecular targeting strategies, such as biochromatography, ultrafiltration mass spectrometry, microfluidic chip technology, and immobilized target enzyme screening technology. Among these, immobilized target enzyme screening technology has seen rapid development due to its advantages such as good enzyme stability, strong pH and temperature tolerance, reusability, and ease of separation from the reaction mixture. Compared to other immobilized enzyme materials, metal-organic frames (MOFs) exhibit unique advantages: their large specific surface area and high porosity allow for high enzyme loading; their open pore structure allows for free transport and delivery of substrates and products; and the diversity of metal ions and organic ligands allows for targeted design and optimization of carrier materials based on enzyme properties and application purposes, thereby improving enzyme immobilization efficiency. Therefore, immobilizing α-Glu in MOFs can significantly improve the enzyme's stability, temperature and pH tolerance, and reusability, providing a new approach for the efficient screening of natural α-Glu inhibitors and demonstrating great application potential in the field of drug development. Summary of the Invention

[0007] This invention provides a high-throughput screening method for α-glucosidase inhibitors from wolfberry leaves based on multi-shell MOF immobilized enzymes, aiming to solve the problems of cumbersome operation, low efficiency, and high cost of traditional screening methods. The method first uses a ligand fishing strategy to screen for active ingredients that specifically bind to α-glucosidase from the sample; then, it uses LC-QTOF-MS / MS technology for separation and structural identification to obtain α-glucosidase inhibitors; subsequently, it verifies the inhibitory activity of the screened active ingredients against α-glucosidase, and uses Autodock software for molecular docking to simulate and predict the binding sites and binding energies of small molecule active compounds with α-glucosidase. Attached Figure Description

[0008] Figure 1Inhibitory activity of Lycium barbarum leaf extract against α-glucosidase.

[0009] Figure 2 Comparison of total ion chromatograms of wolfberry leaf extract (A) and the active ingredient (B) after incubation and dissociation of wolfberry leaf extract with multi-shell MOF immobilized α-glucosidase.

[0010] Figure 3 The active ingredient N obtained through screening 1 -N 10 Mass spectrum of dicaffeoyl spermidine.

[0011] Figure 4 N 1 -N 10 Dicaffeoylsperidin inhibits α-glucosidase activity.

[0012] Figure 5 Molecular docking prediction N 1 -N 10 The binding site of dicaffeoylspermidine to α-glucosidase.

[0013] Figure 6 Fluorescence Spectroscopy StudyN 1 -N 10 Mechanism of action of dicaffeoylspermidine and α-glucosidase.

[0014] Specific technical solutions 1. Preparation of multi-shell MOF immobilized α-glucosidase: Metal salts (e.g., zinc nitrate / zinc acetate) and imidazole ligands (e.g., 2-methylimidazole) were dissolved separately in ultrapure water and heated or sonicated for 20–50 min to ensure complete dissolution. At 20–35°C, the α-glucosidase solution was slowly added to the metal salt solution, and after thorough mixing, the imidazole ligand solution was added. The mixture was magnetically stirred for a period of time, and then a suitable amount of stabilizer was added for 10–60 min to stabilize the reaction. After the reaction was complete, the immobilized enzyme was collected by centrifugation and washed 1–5 times with PBS buffer to obtain single-shell MOF immobilized α-glucosidase. A suitable amount of the single-shell immobilized enzyme was then used as the core framework, and the above steps were repeated to obtain MOF immobilized α-glucosidase with a multi-shell structure.

[0015] 2. Screening of Active Components by Multi-Shell MOF Immobilized Enzymes: Samples at specific concentrations (10–1000 μg / mL) were incubated with multi-shell MOF immobilized enzyme materials at 0–50 °C for 10–60 min. The active components bound to α-Glu to form a complex. The multi-shell MOF immobilized enzyme materials were separated from the incubation system by centrifugation at 5000–15000 rpm / min for 10–30 min. The enzymes were washed 1–5 times with 10–200 mM PBS buffer (pH 5.0–8.0), followed by dissociation of the complex with 30–95% organic solvent (methanol / ethanol / acetonitrile-water) to release the active components. The dissociation solution was dried, and the residue was collected and reconstituted with 10–100% organic solvent (methanol / ethanol / acetonitrile-water). The reconstituted enzymes were then analyzed and identified by UPLC-QTOF-MS / MS to obtain UPLC-QTOF-MS / MS data.

[0016] 3. Identification of active ingredients: (1) Chromatographic conditions: Column: Agilent ZORBAX SB-C18 (2.1 mm × 100 mm, 1.8 μm); Mobile phase: A: 0.1% formic acid / acetic acid-water, B: 0.1% formic acid / acetic acid-methanol / acetonitrile. Elution program settings: 0–80 min, 2–98% B. Flow rate: 0.1–1 mL / min; Column temperature: 10–30℃; Sample tray temperature: 10–30℃; Injection volume: 1–10 μL.

[0017] (2) Mass spectrometry conditions: Agilent 6546 LC / Q-TOF, scan range: m / z 50~1200, Desolventization temperature: 400℃, ion source temperature: 120℃, sheath gas (N2) flow rate: 50 L / h, drying gas flow rate: 600 L / h, capillary voltage: 2.5kV, collision energy (CE): 10 ~ 60 eV.

[0018] 4. Detection of in vitro α-glucosidase inhibitory activity: using p-nitrophenyl-α-D-glucopyranoside (… p Using NPG as a substrate, the inhibitory activity of the screened active ingredient against α-glucosidase was verified. The formula for calculating the α-glucosidase inhibition rate is: In the formula, A1 represents the absorbance without the test sample; A2 represents the absorbance without the enzyme and the test sample; A3 represents the absorbance with the test sample and the enzyme; and A4 represents the absorbance without the enzyme.

[0019] 5. To further verify the enzyme inhibitory activity of the screened compounds, molecular docking was performed using Autodock software to simulate and predict the binding sites and binding energies of the active small molecule compounds with α-glucosidase.

[0020] 6. Advantages of the present invention: (1) The screening method for α-glucosidase inhibitors has the characteristics of high specificity, high sensitivity, high throughput and the target receptor can be used repeatedly. It is suitable for screening and identifying multiple α-glucosidase inhibitors from natural products at the same time. It can give full play to the advantages of multi-shell MOF immobilized enzymes and combine modern high-throughput analysis and identification technology (LC-QTOF-MS / MS) to improve the discovery efficiency of natural enzyme inhibitors, providing an important path for the diversified development of diabetes treatment drugs. (2) Compared with chemically synthesized drugs, α-glucosidase inhibitors screened from natural products have the advantages of better biocompatibility, lower toxicity and side effects, and lower cost. They can reduce the incidence of side effects, improve the risk of complications, and bring greater benefits to diabetic patients. (3) Discovering natural α-glucosidase inhibitors from wolfberry leaves not only provides natural and safe potential candidate drugs for the prevention and treatment of diabetes, but also provides a scientific basis for the in-depth development and high-quality utilization of wolfberry leaf resources.

[0021] The scope of protection of this invention is not limited to the screening of α-glucosidase inhibitors from natural products based on multi-shell MOF immobilized enzymes. Any technical solutions derived by those skilled in the art through logical analysis, deduction, and experimentation based on the ideas of this invention fall within the scope of protection of the claims.

Claims

1. A method for high-throughput screening of α-glucosidase inhibitors from Lycium chinense leaves using multi-shell MOF-immobilized enzymes, characterized by, The method comprises the following steps: (1) Incubation: Incubate the sample with the multi-shell MOF immobilized α-glucosidase, and bind the active ingredients to α-Glu to form an enzyme-inhibitor complex.

2. (2) Dissociation: Rapidly separate the multi-shell MOF immobilized enzyme from the incubation system by high-speed centrifugation, wash thoroughly with a PBS buffer solution, and then dissociate the complex with an organic solvent (methanol / ethanol-water) to release the active ingredients.

3. (3) LC-QTOF-MS / MS analysis: Blow dry the dissociation solution, collect the residue and re-dissolve it with an organic solvent, and then analyze it by LC-QTOF-MS / MS to obtain LC-QTOF-MS / MS data.

4. (4) Data statistical analysis: Analyze the LC-QTOF-MS / MS data, screen and identify α-Glu inhibitors, verify the inhibitory activity of the screened compounds by an in vitro α-Glu activity inhibition detection method, and perform molecular docking using Autodock software to simulate and predict the binding site and binding energy of the active small molecule and α-Glu.

5. The method of claim 1, wherein, In step (1), the sample used is a natural product extract or a chemical component in a natural product, and the enzyme used is a multi-shell MOF immobilized enzyme.

6. The method of claim 2, wherein, The sample used is an extract of wolfberry leaves and different parts (seeds, leaves and root bark) of wolfberry.

7. The method of claim 1, wherein, In step (3), in the LC-QTOF-MS / MS analysis step, the chromatographic conditions include: Chromatographic column: Agilent ZORBAX SB-C18 (2.1 mm x 100 mm, 1.8 μm), Mobile phase: A: water-0.1% formic acid / acetic acid, B: methanol / acetonitrile-0.1% formic acid / acetic acid, Elution program includes: 0-80 min, 2-98% B, Flow rate: 0.1-1 mL / min, Column temperature: 10-30℃, Sample tray temperature: 10-30℃; Injection volume: 1-10 μL.

8. The method of claim 1, wherein, In step (4), molecular docking is also performed using Autodock software to simulate and predict the binding site of the active small molecule and α-glucosidase.

9. The method of claim 1, wherein, Components such as spermidine, flavonoids, and alkaloids, such as nitrogen, found in different parts of wolfberry (fruit, leaves, and root bark). 1 -N 10 Dicaffeoyl spermidine, N 1 -Caffeoyl,N 3 -Dicaffeoylsperidin, N 1 -N 8 Active compounds such as dicaffeoyl spermidine, coumaroyl caffeoyl spermidine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid C, rutin, kaempferol-3-O-rutin, and rhamnoside-3-O-rutin have the potential to be developed and prepared as α-glucosidase inhibitors.

Images