Method for rapidly screening active pharmaceutical ingredients based on competitive ultrafiltration technology

Abstract

The invention belongs to the field of traditional Chinese medicine natural active ingredient analysis and medicine discovery, and particularly relates to a method for rapidly screening medicine active ingredients based on a competitive ultrafiltration technology. Based on the principle of'target site occupation competition ', the replacement effect of a high-activity inhibitor on a low-activity inhibitor is utilized to directionally capture a potent inhibitor. In the incubation process, the small molecule compound is combined with an enzyme active center to form an enzyme-ligand compound. Most of the enzyme-ligand compound forming processes are reversible, and molecules with relatively strong activity have relatively strong binding force with an enzyme active center, so that molecules with relatively weak activity can be replaced, and a new enzyme-ligand compound is formed. Based on this, a competitive ultrafiltration technology is proposed.

Description

Technical Field

[0001] The present invention belongs to the field of analysis of natural active ingredients of traditional Chinese medicine and drug discovery, and specifically relates to a method for rapidly screening active ingredients of drugs based on competitive ultrafiltration technology. Background Art

[0002] Ultrafiltration technology provides a reliable approach for the rapid discovery of bioactive molecules in complex matrices, such as Chinese herbal medicines. This technique exploits the specific binding properties between a small molecule ligand and a target protein, forming an enzyme-ligand complex. Under high-speed centrifugation, unbound compounds pass through the ultrafiltration membrane, while the enzyme-ligand complex is retained in the ultrafiltration tube. Dissociation of the enzyme-ligand complex using a specific solvent releases the ligand, which has an affinity for the enzyme.

[0003] However, due to the diverse components in complex matrices such as traditional Chinese medicine, multiple molecules compete for the active cavity of the enzyme, and ultrafiltration technology is difficult to quickly identify active molecules with strong binding ability to the enzyme.

[0004] During the application of ultrafiltration technology, it was found that the active molecules captured by ultrafiltration technology generally had weak activity, making it difficult to discover higher-quality active molecules. In order to better discover more active molecules, competitive ultrafiltration technology was proposed. Summary of the Invention

[0005] Competitive ultrafiltration technology, based on the principle of "target occupancy competition," exploits the displacement effect of highly active inhibitors on less active inhibitors to selectively capture potent inhibitors. During the incubation process, small molecules bind to the enzyme's active site to form an enzyme-ligand complex. The formation of enzyme-ligand complexes is largely reversible. More active molecules bind more strongly to the enzyme's active site, displacing less active molecules to form new enzyme-ligand complexes, thereby capturing higher-quality active molecules. Based on this principle, competitive ultrafiltration technology was proposed.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for rapidly screening active ingredients based on competitive ultrafiltration technology, which utilizes the displacement effect of high-activity inhibitors on low-activity inhibitors to directionally capture potent inhibitors.

[0008] As a specific embodiment of the present invention, the active ingredient is a protein enzyme inhibitor.

[0009] As a specific embodiment of the present invention, the active ingredient is derived from traditional Chinese medicine.

[0010] As a specific embodiment of the present invention, the following steps are specifically included:

[0011] (1) Using proteinase inhibitors as competitive inhibitors, neuraminidase was first incubated with rutin to form a neuraminidase-rutin complex;

[0012] (2) adding a Chinese herbal medicine extract solution for incubation, centrifuging after incubation to remove excess protein enzyme inhibitors and Chinese herbal medicine extracts, collecting the enzyme-ligand complex, dissociating it with an organic solvent, and collecting and identifying the small molecule ligand.

[0013] As a specific embodiment of the present invention, the proteinase inhibitor in step (1) is a natural inhibitor of neuraminidase.

[0014] As a specific embodiment of the present invention, the natural neuraminidase inhibitor is at least one of rutin, apigenin and luteolin.

[0015] Rutin is in a dynamic binding process with the neuraminidase NA active pocket. When compounds with stronger binding affinity to NA are present, they can displace rutin to form new complexes, thereby selectively capturing compounds with stronger binding affinity to NA. Compared to existing ultrafiltration technologies, competitive ultrafiltration can capture small molecule active compounds with stronger binding affinity to the enzyme.

[0016] As a specific embodiment of the present invention, the preparation method of the Chinese medicine extract solution in step (2) is as follows:

[0017] Weigh Chinese medicinal materials, extract with alcohol, and filter; concentrate the extract under reduced pressure, evaporate to dryness in a water bath; add alcohol to dissolve, and prepare a Chinese medicinal extract solution.

[0018] As a specific embodiment of the present invention, the alcohol is ethanol or methanol.

[0019] In a second aspect, the present invention provides an active ingredient of Flos Lonicerae screened by the above method, wherein the active ingredient is a neuraminidase inhibitor.

[0020] As a specific embodiment of the present invention, the active ingredients of honeysuckle include apigenin-7-O-glucoside, chlorogenic acid, and luteolin.

[0021] Beneficial effects of the present invention

[0022] Competitive affinity ultrafiltration was used to rapidly identify NA inhibitors in honeysuckle, a traditional Chinese medicine for clearing heat and detoxification. This method is simple, rapid, and suitable for industrial application.

[0023] Three NA inhibitors were identified in honeysuckle, namely chlorogenic acid, apigenin-7-O-glucoside, and luteolin. These components have different degrees of NA inhibition. Among them, luteolin and apigenin-7-O-glucoside have the most significant inhibitory effects, with their IC 50The values ​​were 44.98 μM and 40.71 μM, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the technical route of Example 1; wherein ① adding sample 1 and enzyme; ② first incubation; ③ adding sample 2; ④ second incubation; ⑤ ultrafiltration; ⑥ dissociation; ⑦ collection; ⑧ structure identification;

[0025] Figure 2 This is the liquid phase detection result (254nm) of the competitive ultrafiltrate of honeysuckle prepared in Example 1. DETAILED DESCRIPTION

[0026] Example 1

[0027] Screening of neuraminidase active components from honeysuckle:

[0028] (1) Weigh 50 g of honeysuckle and ultrasonically extract with methanol for 30 min at 70 W for three times. Filter. Concentrate the extract under reduced pressure and evaporate to dryness in a 40°C water bath. Dissolve 100 mg of the extract in 10 mL of methanol to prepare a honeysuckle extract solution for later use.

[0029] (2) NA powder (1.0 mg, 39.7 U / mg) was dissolved in 550 μL of MES buffer (0.64 g MES, 44 mg anhydrous calcium chloride dissolved in 100 mL pure water) at pH 6.5 to prepare a MES-NA solution.

[0030] (3) Add 25 μL of 0.5 mM rutin solution and 275 μL of MES-NA solution to two 0.5 mL ultrafiltration tubes. Add 25 μL of 0.5 mM rutin solution and 275 μL of MES solution to two other ultrafiltration tubes as blank controls. Incubate at 37°C for 30 min.

[0031] (4) Add 5 μL of honeysuckle extract solution to the two incubated ultrafiltration tubes of the experimental group and the two blank group ultrafiltration tubes, respectively, and incubate for 30 minutes.

[0032] (5) The incubated mixture was centrifuged at 15,000 r for 10 min, and the lower filtrate was discarded; 200 μL of 8% DMSO was added, and the mixture was centrifuged at 15,000 r for 5 min, and the lower filtrate was discarded; 200 μL of 8% DMSO was added, and the mixture was centrifuged at 15,000 r for 5 min, and the lower filtrate was discarded; 200 μL of chromatographic methanol was added, and the mixture was allowed to stand at room temperature for 15 min, and the mixture was centrifuged at 15,000 r for 5 min, and the lower filtrate was retained; 150 μL of chromatographic methanol was added, and the mixture was centrifuged at 15,000 r for 5 min, and the lower filtrate was retained as the collected active small molecule ligand. Finally, the structure of the small molecule ligand compound was rapidly identified using liquid chromatography-mass spectrometry. The results were as follows: Formulas I-III.

[0033]

[0034]

[0035] Implementation effect examples

[0036] Detection of the inhibitory effect of compounds identified in honeysuckle on NA

[0037] Detection method:

[0038] The inhibitory effect of the compounds on NA was determined using a neuraminidase detection kit. The procedure was performed according to the kit instructions, and the fluorescence intensity of the reaction system was measured using 322 nm and 450 nm as the excitation and emission wavelengths. Each well was repeated 3 times, and a dose-effect curve was drawn based on the inhibition rate of each concentration, and the average 50% inhibitory concentration (IC) of the compound on NA was calculated. 50 ).

[0039] The test results are shown in Table 1:

[0040] Table 1 IC values ​​of compounds identified from honeysuckle for NA inhibition 50 value

[0041]

[0042] As can be seen from Table 1, the active ingredients screened by the method of the present application all have good inhibitory effects on NA.

[0043] Example 2

[0044] Screening of neuraminidase active components from Polygonum cuspidatum:

[0045] The specific steps are the same as in Example 1. The active ingredients screened are as follows:

[0046]

[0047] The inhibitory effect of the compounds identified in Polygonum cuspidatum on NA was detected using the same detection method as in Example 1.

[0048] Table 2 IC values ​​of compounds identified from Polygonum cuspidatum for inhibition of NA 50 value

[0049]

[0050] Although the above describes the specific implementation scheme of the present invention, it does not limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A method for rapid screening of active ingredients based on competitive ultrafiltration technology, characterized in that: The displacement effect of high-activity inhibitors on low-activity inhibitors is utilized to capture potent inhibitors in a targeted manner.

2. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 1, characterized in that: The active ingredient is a protein enzyme inhibitor.

3. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 2, characterized in that: The active ingredient is derived from traditional Chinese medicine.

4. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 2 or 3, characterized in that: The specific steps include: (1) Using proteinase inhibitors as competitive inhibitors, neuraminidase was first incubated with rutin to form a neuraminidase-rutin complex; (2) adding a Chinese herbal medicine extract solution for incubation, centrifuging after incubation to remove excess protein enzyme inhibitors and Chinese herbal medicine extracts, collecting the enzyme-ligand complex, dissociating it with an organic solvent, and collecting and identifying the small molecule ligand.

5. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 4, characterized in that: The proteinase inhibitor in step (1) is a natural inhibitor of neuraminidase.

6. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 4, characterized in that: The natural neuraminidase inhibitor is at least one of rutin, apigenin and luteolin.

7. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to any one of claims 4 to 6, characterized in that: The preparation method of the Chinese medicine extract solution in step (2) is as follows: Weigh Chinese medicinal materials, extract with alcohol, and filter; concentrate the extract under reduced pressure, evaporate to dryness in a water bath; add alcohol to dissolve, and prepare a Chinese medicinal extract solution.

8. The method for rapid screening of active ingredients based on competitive ultrafiltration technology according to claim 7, characterized in that: The alcohol is ethanol or methanol.

9. The active ingredient of Flos Lonicerae screened by the method according to any one of claims 1 to 8, characterized in that: The active ingredient is a neuraminidase inhibitor.

10. The active ingredient of Flos Lonicerae according to claim 9, characterized in that The active ingredients of honeysuckle include apigenin-7-O-glucoside, chlorogenic acid and luteolin.

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