Method for rapidly screening and identifying alpha-glucosidase inhibitor in stevia rebaudiana

Abstract

The invention discloses a method for rapidly screening and identifying an alpha-glucosidase inhibitor from stevia rebaudiana by combining a magnetic MOF immobilized enzyme technology with an ultra-high performance liquid chromatography-time-of-flight mass spectrometry technology. The method is based on an immobilized enzyme technology, screening of the alpha-glucosidase inhibitor in stevia rebaudiana is carried out by means of the affinity binding effect of active small molecules and enzyme and the magnetic separation characteristic of magnetic MOF immobilized alpha-glucosidase, and structural identification of an active compound is carried out by applying an ultra-high performance liquid chromatography-time-of-flight mass spectrometry technology. And finally, verifying the in-vitro alpha-glucosidase inhibitory activity of the screened active compound, and simulating the binding information of the two compounds by means of a molecular docking technology. The method provided by the invention has the characteristics of rapidness, high efficiency, accuracy, easiness in separation from a reaction system and reutilization of the target receptor immobilized alpha-glucosidase, is suitable for screening the alpha-glucosidase inhibitor from complex systems such as traditional Chinese medicines and natural products, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of drug screening technology, specifically to a method for rapidly screening and identifying α-glucosidase inhibitors in stevia. Background Technology

[0002] Alpha-glucosidase, a key enzyme in carbohydrate metabolism, hydrolyzes carbohydrates into glucose, leading to elevated postprandial blood glucose levels and making it an important target for the treatment of type 2 diabetes mellitus (T2DM). Inhibiting alpha-glucosidase activity can slow the rate of glucose production, effectively reducing postprandial blood glucose fluctuations and decreasing the risk of complications. Currently, commonly used hypoglycemic drugs (such as acarbose, metformin, and repaglinide) are mostly chemically synthesized, easily causing adverse reactions such as liver and kidney damage, and gastrointestinal discomfort (e.g., bloating and diarrhea). Therefore, developing highly effective and low-toxicity alpha-glucosidase inhibitors derived from traditional Chinese medicine and natural products has become a research hotspot and key challenge in the prevention and treatment of T2DM.

[0003] Stevia ( Stevia rebanudiana Stevia is a perennial medicinal herb belonging to the genus Stevia of the Asteraceae family. Originating in eastern Paraguay, South America, it is now widely cultivated in China, Japan, Brazil, and other regions. Stevia is low in calories and 250-300 times sweeter than sucrose, and has been used as a sugar substitute and herb in the region for hundreds of years. Modern pharmacology has confirmed that stevia is rich in steviol glycosides, flavonoids, polyphenols, carotenoids, ascorbic acid, amino acids, and other plant active ingredients. It is a rich source of secondary metabolites and has the advantages of being all-natural, high in sweetness, low in calories, chemically stable, safe, and low in toxicity. It not only has significant therapeutic effects on cancer, hypertension, and tooth decay, but also possesses properties similar to insulin, insulin-stimulating agents, and glucagon inhibitors, effectively lowering blood sugar. Therefore, stevia is a potential source of α-glucosidase inhibitors. However, stevia contains a wide variety of chemical components with complex structures, and traditional drug screening processes suffer from drawbacks such as complexity, time consumption, labor intensity, high cost, and low throughput, making it difficult to meet the high-efficiency requirements of new drug development. Therefore, developing a rapid, efficient, and low-cost screening method for α-glucosidase inhibitors for screening active ingredients in complex systems is of great significance and value for clarifying the hypoglycemic active substances in stevia and their application potential in the prevention and treatment of diabetes.

[0004] In recent years, enzyme inhibitor screening methods based on immobilized enzyme technology combined with liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) have attracted much attention due to their ability to address the core pain points of traditional free enzyme screening. These methods offer advantages such as low cost, good thermal stability, easy separation from the reaction system, and reusability. Furthermore, this technology achieves efficient screening through a dual mechanism of "targeted anchoring and affinity fishing"—anchoring key disease-regulating enzymes to a carrier material and utilizing the specific interaction between the enzyme's active site and the inhibitor to "precisely fish" for active small molecules from complex matrices of traditional Chinese medicine (such as stevia), effectively avoiding interference from inactive components. Simultaneously, ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS) can acquire chromatographic retention times and mass-to-charge ratio information of active compounds, ultimately achieving an integrated technical process of "directionally capturing active ingredients from complex mixtures—rapid separation—precise identification." This technology is characterized by its sensitivity, speed, simple process, low sample consumption, and high throughput, making it suitable for efficiently screening bioactive components from complex extracts of traditional Chinese medicine and natural products.

[0005] Addressing the three core challenges in screening natural enzyme inhibitors from complex systems of traditional Chinese medicine (TCM)—1. Strong matrix interference (e.g., the active ingredients in TCM extracts are complex and diverse, and inactive components easily mask the signals of active ingredients); 2. Difficulty in capturing active ingredients (target ingredient content is often <0.1%, and traditional methods easily miss them); 3. Difficulty in elucidating the mechanism of action (only activity can be detected, but the component structure and enzyme binding mechanism cannot be correlated)—this invention provides a method for screening and analyzing α-glucosidase inhibitors in stevia using a combination of magnetic MOF immobilized enzyme technology and ultra-high performance liquid chromatography-time-of-flight mass spectrometry. This method offers advantages such as high speed, simple operation, high throughput, and low sample consumption. Summary of the Invention

[0006] This invention discloses a method for rapidly screening and identifying α-glucosidase inhibitors from stevia using magnetic MOF immobilized enzyme technology coupled with ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS). First, compounds with α-glucosidase inhibitory activity are screened by utilizing the affinity between active small molecules and the enzyme. Second, the screened α-glucosidase inhibitors are structurally analyzed and identified using UHPLC-TOF-MS. Finally, the in vitro α-glucosidase inhibitory activity of the screened active compounds is verified, and the interaction between the active small molecules and α-glucosidase is investigated. Attached Figure Description

[0007] Figure 1 Inhibitory activity of stevia extract against α-glucosidase.

[0008] Figure 2 Comparison of total ion chromatograms of stevia extract and stevia extract incubated with immobilized α-glucosidase.

[0009] Figure 3 Mass spectrum of isochlorogenic acid A obtained through screening.

[0010] Figure 4 In vitro inhibitory activity of isochlorogenic acid A against α-glucosidase.

[0011] Figure 5 Molecular docking predicts the binding site of isochlorogenic acid A to α-glucosidase.

[0012] Figure 6 Fluorescence spectroscopy was used to study the mechanism of action of isochlorogenic acid A and α-glucosidase.

[0013] Specific technical solutions Preparation of crude stevia extract: Take a certain amount of dried stevia leaves, grind them with liquid nitrogen, and then extract them with 30% to 80% methanol-water solution by ultrasonic extraction 2 to 3 times, 1 to 3 hours each time. Filter, combine the filtrates, concentrate, and dry to obtain crude stevia extract.

[0014] Preparation of α-glucosidase immobilized by magnetic MOF: Dopamine hydrochloride and polyethyleneimine were dissolved in Tris buffer at pH 7.5–10.5, Fe3O4 magnetic nanoparticles were added, and the mixture was sonicated for 10–60 min, then shaken in a water bath at 20–50 °C for 2–12 h. After washing several times with Tris buffer, zinc acetate was added to achieve a final concentration of 0.1–1 M, and the mixture was shaken in a water bath for 10–60 min. After washing with ultrapure water, 1–10 U·mL⁻¹ was added. -1 α-glucosidase and 0.1–1 M imidazole-2-carboxaldehyde were mixed in a water bath at 20–50 °C and shaken for 1–6 h. Then, 5–10 mL of N,N-dimethylformamide was added, the mixture was allowed to stand, and washed with ultrapure water to obtain magnetically immobilized α-glucosidase in a MOF. This immobilized α-glucosidase can be rapidly separated from the reaction system under the action of an external magnetic field.

[0015] Screening and identification of α-glucosidase inhibitors in stevia: Stevia crude extract samples were pre-incubated with magnetically immobilized α-glucosidase (MOF) to ensure the active components in stevia bind affinityfully to the active site of α-glucosidase. Using an external magnetic field, the MOF-immobilized α-glucosidase was separated from the reaction system, and compounds that did not bind to α-glucosidase or had weak binding affinity were washed away using phosphate buffer. The MOF-immobilized α-glucosidase was then incubated a second time with a specific concentration of organic solvent (methanol-water) to dissociate compounds with α-glucosidase inhibitory activity. The dissociated solution was collected, concentrated using a nitrogen evaporator, dried, reconstituted, and filtered before analysis using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS).

[0016] Chromatographic conditions: Column: Agilent SB C18 (2.1×100 mm, 1.8 μm); Mobile phase: 0.1% formic acid-water (A) and 0.1% formic acid-methanol (B); Column temperature: 25–35℃; Gradient elution program: 0–60 min, 98%A–0%A; Flow rate: 0.5–1 mL / min; Injection volume: 5–10 μL.

[0017] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI) source in negative ion mode; Fragment ion scan range: 50–1200 Da; Capillary voltage: 1.5–2.5 kV; Desolvation gas temperature: 400℃; Desolvation gas flow rate: 500–600 L / h; Cone gas flow rate: 40–50 L / h.

[0018] To verify the inhibitory activity of the screened active compounds against α-glucosidase, this invention employs an in vitro α-glucosidase inhibitory activity detection method.

[0019] To further investigate the interaction mechanism between the screened compounds and α-glucosidase, molecular docking was performed using Autodock Vina software to simulate the binding sites and binding energies of the active small molecules with α-glucosidase.

[0020] The advantages of this invention are as follows: 1. The method for screening α-glucosidase inhibitors by combining magnetic MOF immobilized enzyme technology with ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF-MS / MS) is simple, rapid, efficient, high-throughput, requires minimal sample consumption, and allows for repeated use of the receptor. It is suitable for screening enzyme inhibitors from complex systems of various traditional Chinese medicines and natural product extracts. 2. Utilizing UHPLC-TOF-MS / MS to obtain retention time and fragmentation information of active compounds allows for accurate separation and identification of active compounds, offering advantages such as speed, low cost, high efficiency, and minimal sample consumption. 3. Compared to chemically synthesized inhibitors, the screened α-glucosidase inhibitors are of natural origin, offering advantages such as good efficacy, environmental friendliness, and low toxicity.

[0021] The scope of protection of this invention is not limited to the screening of enzyme inhibitors in Chinese herbal extracts and natural active ingredients. Any technical solution derived by those skilled in the art through logical analysis, deduction and experimentation based on the ideas of this invention shall fall within the scope of protection of the claims.

Claims

1. A method for rapid screening and identification of α-glucosidase inhibitors in stevia, characterized in that, Includes the following steps: (1) Screening and identification of enzyme inhibitors by combining magnetic MOF immobilized α-glucosidase technology with ultra-high performance liquid chromatography-time-of-flight mass spectrometry. (2) Verification of the in vitro α-glucosidase inhibitory activity of the screened active small molecules and molecular docking simulation of the interaction between the active ingredients and α-glucosidase.

2. The method for rapid screening and analysis of α-glucosidase inhibitors in stevia as described in claim 1, characterized in that, In step (1), the magnetic MOF immobilized α-glucosidase used has superparamagnetism and can be separated by an external magnetic field within 60 s, which can be used for magnetic separation and recovery in practical applications.

3. The method for rapid screening and analysis of α-glucosidase inhibitors in stevia as described in claim 1, characterized in that, In step (1), the enzyme inhibitor screening method specifically includes the following steps: pre-incubating the magnetic MOF-immobilized α-glucosidase with a complex solution of crude stevia extract to ensure that compounds with α-glucosidase inhibitory activity bind to the active site of the enzyme with affinity; separating the magnetic MOF-immobilized α-glucosidase from the reaction system under an external magnetic field, and washing away inactive compounds or compounds with weak binding ability on the surface with phosphate buffer; incubating the magnetic MOF-immobilized α-glucosidase a second time with a certain concentration of organic solvent to release the active compounds; collecting the filtrate, drying it with a nitrogen blower, reconstituted it, and passing it through a membrane for analysis by ultra-high performance liquid chromatography-time-of-flight mass spectrometry.

4. The method as described in claim 2, characterized in that, The preparation method of stevia crude extract is as follows: stevia leaves are ground and pulverized using liquid nitrogen, and extracted with 30% to 80% methanol-water solution by ultrasonic extraction 2 to 3 times, 1 to 3 hours each time. The extract is then filtered, the filtrates are combined, concentrated, and dried to obtain stevia crude extract.

5. The method as described in claim 2, characterized in that, The reconstitution system for stevia crude extract consisted of 10%–90% organic solvent (methanol / acetonitrile). The pre-incubation time for magnetic MOF-immobilized α-glucosidase with the stevia crude extract reconstitution solution was 10–60 min at 4–60 °C, repeated 1–3 times. For washing away inactive compounds and weakly binding compounds, a phosphate buffer solution with a concentration of 20–100 mM and a pH of 6.0–8.0 was used, repeated 1–3 times. For the second incubation, a methanol-water solvent with a concentration of 20%–90% was used, with an incubation time of 10–60 min at 4–60 °C, repeated 1–3 times. Nitrogen blowing was performed at 20–60 °C for 20–90 min. For reconstitution after nitrogen blowing, a 20%–70% methanol-water solvent was used, and the filter membrane had a pore size of 0.2 μm.

6. The method as described in claim 2, characterized in that, The samples used were extracts of traditional Chinese medicine or chemical components in traditional Chinese medicine, including but not limited to stevia.

7. The method as described in claim 1, characterized in that, It also includes in vitro α-glucosidase inhibitory activity verification of the screened active compounds.

8. The method as described in claim 1, characterized in that, It also includes molecular docking using Autodock Vina software to simulate the binding sites of screened compounds with α-glucosidase.

9. The method as described in claim 2, characterized in that, Compounds such as chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, quercetin, cynarin, quercetin-3-glucoside-7-rhamnoside, neochlorogenic acid, cryptochlorogenic acid, luteolin, apigenin-7-O-glucoside, kaempferol-3-O-rhamnoside, 3,4,5-tricaffeoylquinic acid, rebaudioside A, and dukloroside A have the potential to be developed into α-glucosidases.

10. The method as described in claim 1, characterized in that, The key enzymes used to regulate the disease include, but are not limited to, α-glucosidase.

Images