A ligand-based carbonic anhydrase cross-linked aggregate, and a preparation method and application thereof

Abstract

The application discloses a preparation method of ligand-based carbonic anhydrase cross-linking aggregate, which comprises the following steps: (1) mixing and incubating a carbonic anhydrase solution and a ligand solution to obtain a first mixed solution, wherein the ligand is a carbonic anhydrase inhibitor; (2) adding a precipitant to the first mixed solution to perform a precipitation reaction, and obtaining a second mixed solution; (3) adding a cross-linking agent to the second mixed solution to perform a cross-linking reaction, and obtaining a third mixed solution; (4) centrifuging the third mixed solution to obtain a first precipitate, eluting and washing the first precipitate, centrifuging again to obtain a second precipitate, and the second precipitate is the ligand-based carbonic anhydrase cross-linking aggregate. By adding the carbonic anhydrase inhibitor, the active sites of the carbonic anhydrase can be effectively combined, which can prevent the cross-linking agent from reacting with the residues at the positions, after the cross-linking is completed, the ligand molecules on the aggregate are eluted through the elution and washing steps, the binding sites of the carbonic anhydrase inhibitor are exposed, and the screening of the carbonic anhydrase inhibitor can be realized.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a ligand-based carbonic anhydrase cross-linked aggregate, its preparation method, and its application. Background Technology

[0002] Carbonic anhydrase (CA) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, playing a crucial role in many physiological and pathological processes. Carbonic anhydrase inhibitors (CAIs) are used to treat various diseases such as glaucoma, retinopathy, cardiogenic edema, and acute mountain sickness. However, the clinical use of CAIs (acetazolamide, bullinamide, dzoxol, etc.) is often accompanied by side effects, limiting their long-term use. Therefore, the screening and development of CAIs is of great significance.

[0003] Traditional Chinese medicine contains abundant active ingredients, which are important resources for CAI screening and development. The discovery of active ingredients in traditional Chinese medicine is based on activity screening models, mainly cell and animal models. Although this method is effective, it has problems such as long modeling time and high cost.

[0004] In recent years, the "ligand fishing" method, which integrates separation and analysis, has been widely used in the screening of active substances in traditional Chinese medicine. Ligand fishing is a method based on the identification of interacting ligands and receptors through intermolecular affinity, and then combined with modern analytical techniques to screen and identify bioactive molecules that bind to proteins. Common ligand fishing methods include affinity ultrafiltration, affinity magnetization, cell membrane chromatography, and bioreactor methods. These methods require the immobilization of enzymes using carriers, such as magnetic nanoparticles, carbon nanotubes, and hollow fibers. For example, Chinese patent document CN104404024A discloses an immobilized carbonic anhydrase and its preparation method. This immobilized carbonic anhydrase includes magnetic nanoparticles, an aldehyde-rich composite material, and carbonic anhydrase; the aldehyde-rich composite material coats the surface of the magnetic nanoparticles to form an aldehyde-rich shell, and the surface aldehyde groups of the aldehyde-rich shell are covalently bonded to the carbonic anhydrase. While these carriers facilitate the isolation and reuse of enzymes, the introduction of such inactive carriers often leads to higher costs, decreased enzyme activity, and additional physical and chemical modification steps.

[0005] Carrier-free immobilization technology, which forms enzyme aggregates and uses cross-linking agents to immobilize them, is expected to become an alternative to carrier immobilization technology for ligand fishing due to its advantages of low cost and high enzyme activity. For example, Chinese patent document CN119265170A discloses a cross-linked carbonic anhydrase, its preparation method, and its application. The preparation method of the cross-linked carbonic anhydrase includes the following steps: S1, mixing a carbonic anhydrase solution and a precipitant to carry out a precipitation reaction, obtaining a first mixture; the precipitant is ethanol or acetonitrile; S2, adding a cross-linking agent to the first mixture and carrying out a cross-linking reaction, obtaining a second mixture; S3, centrifuging the second mixture to obtain a precipitate, which is the cross-linked carbonic anhydrase. However, in the process of preparing enzyme aggregates by cross-linking, the cross-linking agent often damages the internal structure of the protein, causing a loss of enzyme activity. This greatly reduces the enzyme's adsorption efficiency and is not conducive to the screening of potential active ingredients.

[0006] Therefore, there is an urgent need to find a new method for preparing cross-linked aggregates to solve the problems of enzyme activity decline and enzyme aggregate instability caused by excessive cross-linking agent and excessively long cross-linking time in the existing technology. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing carbonic anhydrase cross-linked aggregates based on ligands. By adding a carbonic anhydrase inhibitor (i.e., a ligand), the ligand can effectively bind to the active site of carbonic anhydrase, which prevents the cross-linking agent from reacting with the residues at this location. After cross-linking is completed, the ligand molecules on the aggregates are eluted through a washing step to expose the binding sites of the carbonic anhydrase inhibitor, thereby enabling the screening of carbonic anhydrase inhibitors.

[0008] A method for preparing ligand-based carbonic anhydrase cross-linked aggregates includes the following steps: (1) The carbonic anhydrase solution and the ligand solution are mixed and incubated to obtain a first mixture, wherein the ligand is a carbonic anhydrase inhibitor; (2) Add a precipitant to the first mixture and mix to carry out a precipitation reaction to obtain the second mixture; (3) Add a crosslinking agent to the second mixture and mix to carry out a crosslinking reaction to obtain a third mixture; (4) Centrifuge the third mixture to obtain the first precipitate, and use an eluent and a buffer to elute and wash it respectively. Centrifuge again to obtain the second precipitate, which is the carbonic anhydrase cross-linked aggregate based on the ligand.

[0009] In traditional cross-linked aggregate preparation processes, the cross-linking agent not only cross-links carbonic anhydrases to form aggregates, but also reacts within the carbonic anhydrases (especially at the active site), causing carbonic anhydrase inactivation and reducing the catch efficiency of the aggregates. In this invention, by adding a carbonic anhydrase inhibitor (i.e., a ligand), the ligand can effectively bind to the active site of carbonic anhydrase, preventing the cross-linking agent from reacting with residues at these sites, thereby protecting the active site and carbonic anhydrase activity, and thus improving the catch efficiency of the carbonic anhydrase cross-linked aggregates. After cross-linking, the ligand molecules on the aggregates are eluted, exposing the binding sites of the carbonic anhydrase inhibitor, enabling the screening of carbonic anhydrase inhibitors.

[0010] Preferably, in step (1), the ligands are salvianolic acid C and shikonin.

[0011] Preferably, in step (1), the molar ratio of carbonic anhydrase to ligand in the first mixture is 1~10:0.06~600.

[0012] More preferably, the ligand is salvianolic acid C, and the mass ratio of carbonic anhydrase to ligand is 1~2:0.6~60.

[0013] Preferably, in step (2), the precipitant is ammonium sulfate, ethanol, or acetonitrile.

[0014] More preferably, the precipitant is ammonium sulfate.

[0015] Preferably, in step (2), the volume ratio of the first mixture to the precipitant is 1~1000:90~9000.

[0016] Preferably, in step (2), the precipitation reaction is carried out at a temperature of 2~10 ℃ for 0.5~10 h.

[0017] Preferably, in step (3), the crosslinking agent is glutaraldehyde, genipin, tannic acid, etc.

[0018] More preferably, the crosslinking agent is glutaraldehyde.

[0019] Preferably, in step (3), the molar ratio of the crosslinking agent to the carbonic anhydrase is 1~10:60~6000.

[0020] Preferably, in step (3), the temperature of the crosslinking reaction is 5~45 ℃ and the time is 1~3 h.

[0021] More preferably, the crosslinking reaction is carried out at a temperature of 20-30 °C for 2-2.5 h.

[0022] Preferably, in step (4), the centrifugation conditions are a centrifugal force of 1000~20000 g and a centrifugation time of 5~30 min.

[0023] Preferably, in step (4), the elution time is 1~60 min and the elution temperature is 30~50 ℃.

[0024] Preferably, in step (4), the eluent is methanol and the buffer is 3-morpholinopropanesulfonic acid (MOPS).

[0025] The present invention also provides ligand-based carbonic anhydrase cross-linked aggregates prepared by the above method.

[0026] The present invention also provides the application of the above-mentioned ligand-based carbonic anhydrase cross-linked aggregates in the screening of carbonic anhydrase inhibitors.

[0027] Preferably, the application involves screening carbonic anhydrase inhibitors from traditional Chinese medicine extracts, wherein the traditional Chinese medicine extract is ginkgo biloba extract.

[0028] This invention provides a method for screening carbonic anhydrase inhibitors from Ginkgo biloba extract, comprising the following steps: S1. Preparation of sample eluent: The above-mentioned ligand-based carbonic anhydrase cross-linked aggregates are mixed with Ginkgo biloba extract solution and incubated to obtain an incubation solution. The incubation solution is centrifuged and washed to obtain a precipitate. An elution solution is added to the precipitate for elution, and the supernatant is collected to obtain the sample eluent. S2. Preparation of negative control group sample eluent: After heating and inactivating the above-mentioned ligand-based carbonic anhydrase cross-linked aggregates, mix them with Ginkgo biloba extract solution and incubate to obtain negative control group incubation solution. Repeat the centrifugation, washing and elution steps in S1, and collect the supernatant to obtain negative control standard eluent. S3. Structural Identification: The eluents of the samples from steps S1 and S2 and the eluent of the negative control group were analyzed by HPLC-MS. The peak areas of each peak in the total ion chromatograms of the two groups of samples were compared. If the peak area difference between the peaks in the total ion chromatogram is greater than 15%, then the peak areas of the extracted ion chromatograms corresponding to the mass-to-charge ratio shown in the first-order mass spectrum should be further compared. If the peak area difference of the extracted ion map corresponding to the mass-to-charge ratio is greater than 15%, then further UPLC-QTOF analysis should be performed to confirm the structure.

[0029] Preferably, in steps S1 and S2, the preparation method of the ginkgo leaf extract includes the following steps: weighing ginkgo leaves, adding 70% ethanol to swell for 2 h, heating the mixture under reflux for 1.5 h, filtering and collecting the first decoction; adding 70% ethanol to the filter residue, heating under reflux for 1 h, filtering and collecting the second decoction and combining it with the first decoction; concentrating the combined extract by rotary evaporation, and freeze-drying to obtain the ginkgo leaf extract.

[0030] Preferably, in steps S1 and S2, the ginkgo leaf extract solution is a MOPS solution of ginkgo leaf extract, and the concentration of the ginkgo leaf extract is 0.5~20 mg / mL.

[0031] Preferably, in steps S1 and S2, the incubation temperature is 15~45 ℃ and the incubation time is 0.5~5 h.

[0032] Preferably, in steps S1 and S2, the desorption liquid is methanol, and the desorption time is 0.5 to 5 hours.

[0033] Preferably, in step S2, the conditions for heat deactivation are: heating temperature of 90~150 ℃ and heating time of 0.5~5 h.

[0034] Preferably, in step S3, the HPLC-MS conditions are as follows: The chromatographic conditions were as follows: Column: Agilent ZORBAX SB Aq column (4.6 × 100 mm, 5 μm); Mobile phase: Mobile phase A was 0.05–0.15% formic acid aqueous solution, and mobile phase B was acetonitrile; Elution method: Gradient elution, the gradient elution process was set as shown in Table 1, the flow rate was 0.6–1.0 mL / min, the column temperature was 30–40 ℃, and the injection volume was 4–6 μL. Table 1: Setting parameters for gradient elution process The mass spectrometry conditions were as follows: the ion source was an ESI ion source, negative ion mode scanning, and the ion scan range was m / z 100~1000; the fragmentation voltage was stepped, m / z 75~90 V, m / z 250~100 V, m / z 450~110 V, m / z 750~120 V, m / z 1000~130 V; the scan / dwell time was 1147 ms; the dryer flow rate was 13.0 L / min, the dryer temperature was 325 ℃; the nebulizer gas pressure was 55.0 psi; the capillary voltage was -3.5 kV; the capillary current was 5 nA, and the chamber current was 0.04 μA.

[0035] Preferably, in step S3, the conditions for UPLC-QTOF are: The chromatographic conditions are the same as those for HPLC-MS. The mass spectrometry conditions were as follows: the ion source was an ESI ion source, and negative ion mode was used for scanning; the nebulizing gas was nitrogen, with Curtain gas at 35 psi; Gas 1 at 55 psi; Gas 2 at 55 psi; ISVF at -4.5 kV; CE at -10 V; DP at -100 V; TEM at 550 ℃; and the ion scan range was m / z 100~1500.

[0036] The present invention also provides a carbonic anhydrase inhibitor obtained by the above method, wherein the carbonic anhydrase inhibitor is at least one of ginkgo biloba flavonoids, ginkgolides biloba flavonoids, ginkgo biloba flavonoids, isoginkgo biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C13:0, and ginkgolic acid C17:1.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The carbonic anhydrase cross-linked aggregate based on ligands provided by the present invention has advantages such as good stability and high fishing efficiency compared with cross-linked carbonic anhydrase without additives.

[0038] (2) The method for screening carbonic anhydrase inhibitors from Ginkgo biloba extract based on the carbonic anhydrase cross-linked aggregates of the present invention can quickly and accurately capture the active components in Ginkgo biloba extract that can inhibit carbonic anhydrase, and identify them with the assistance of HPLC-MS technology. It has higher sensitivity, especially for lipid-soluble components such as ginkgolic acid compounds.

[0039] (3) The present invention uses a method for screening carbonic anhydrase inhibitors from Ginkgo biloba extract based on ligand-based carbonic anhydrase cross-linked aggregates. For the first time, it was discovered that Ginkgo biloba flavonoids, Pine flavonoids, Ginkgo biloba flavonoids, Isoginkgo biloba flavonoids, Ginkgo biloba acid C15:1, Ginkgo biloba acid C13:0, and Ginkgo biloba acid C17:1 can inhibit carbonic anhydrase, providing a basis for the development of novel carbonic anhydrase inhibitors. Attached Figure Description

[0040] Figure 1 The peak area of ​​acetazolamide eluted from the carbonic anhydrase crosslinked aggregates of Examples 1-6 and Comparative Examples 1-4 is shown.

[0041] Figure 2The image shows a comparison of the extraction ion chromatograms of the eluents (i.e., the negative control group) after acetazolamide was incubated with ligand-based carbonic anhydrase cross-linked aggregates, ligand-free carbonic anhydrase cross-linked aggregates, and inactivated carbonic anhydrase cross-linked aggregates, respectively. In the image, a represents the signal of the desorption solution after incubation with ligand-based carbonic anhydrase cross-linked aggregates, b represents the signal of the desorption solution after incubation with ligand-free carbonic anhydrase cross-linked aggregates, and c represents the signal of the desorption solution after incubation with inactivated carbonic anhydrase cross-linked aggregates.

[0042] Figure 3 The peak areas of the extraction ion chromatograms of various potential carbonic anhydrase inhibitors in the eluent after incubation of Ginkgo biloba extract with ligand-based carbonic anhydrase cross-linked aggregates, ligand-free carbonic anhydrase cross-linked aggregates, and inactivated carbonic anhydrase cross-linked aggregates were compared.

[0043] Figure 4 The diagram shows the extracted ion sequence of potential carbonic anhydrase inhibitors, where a represents the signal of the desorption solution after incubation with ligand-based carbonic anhydrase cross-linked aggregates, b represents the signal of the desorption solution after incubation with ligand-free carbonic anhydrase cross-linked aggregates, and c represents the signal of the desorption solution after incubation with inactivated cross-linked carbonic anhydrase. A to F are, in order, ginkgo biloba flavonoids, ginkgo biloba flavonoids (① in B), isoginkgo biloba flavonoids (② in B), ginkgolides biloba flavonoids, ginkgolic acid C13:1, ginkgolic acid C15:1, and ginkgolic acid C17:1.

[0044] Figure 5 The results of the carbonic anhydrase inhibitory activity assay for potential carbonic anhydrase inhibitors are shown. In this figure, A represents the inhibition rate of carbonic anhydrase by ginkgo biloba flavonoids, ginkgo biloba flavonoids, isoginkgo biloba flavonoids, ginkgolides, ginkgolic acid C13:1, ginkgolic acid C15:1, and ginkgolic acid C17:1 at 50 μM. B to H represent the inhibition rates of carbonic anhydrase by different concentrations of ginkgo biloba flavonoids, ginkgo biloba flavonoids, isoginkgo biloba flavonoids, ginkgolides, ginkgolic acid C15:1, ginkgolic acid C15:1, and ginkgolic acid C17:1, respectively.

[0045] Figure 6 The chemical structures of ginkgo biflavonoids, ginkgolic biflavonoids, isoginkgo biflavonoids, ginkgolic biflavonoids, ginkgolic acid C13:1, ginkgolic acid C15:1, and ginkgolic acid C17:1 are described.

[0046] Figure 7 The rate of pH recovery in HK-2 cells after acidification under treatment with acetazolamide, ginsenosides, and ginkgo biloba extract C15:1 was measured. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0048] All raw materials used in this invention are commercially available.

[0049] Example 1: Preparation of carbonic anhydrase cross-linked aggregates based on salvianolic acid C (1) Mix 90 μL of carbonic anhydrase solution (carbonic anhydrase dissolved in MOPS, concentration of 1.1 mg / mL) and 10 μL of salvianolic acid C solution (dissolved in MOPS, 500 μM), and shake at 25 °C for 5 min to obtain the first mixture. (2) Add 900 μL of saturated ammonium sulfate solution to the first mixture, shake and mix at 4 °C for 4 h to obtain the second mixture; (3) Add glutaraldehyde to the second mixture, with a final concentration of 2 nM, and shake at 25 °C for 2.5 h to carry out the cross-linking reaction to obtain the third mixture; (4) Centrifuge the third mixture at a relative centrifugal force of 18000 g for 10 min to obtain the first precipitate. Wash it with methanol at 45 °C for 5 min and centrifuge to remove the supernatant. Repeat this process three times. Then wash the precipitate with MOPS three times and centrifuge it again at 18000 g for 10 min to obtain the second precipitate, which is the carbonic anhydrase cross-linked aggregate based on salvianolic acid C.

[0050] Example 2 The preparation method is the same as in Example 1, except that the concentration of salvianolic acid C is 250 μM.

[0051] Example 3 The preparation method is the same as in Example 1, except that the concentration of salvianolic acid C is 125 μM.

[0052] Example 4 The preparation method is the same as in Example 1, except that the concentration of salvianolic acid C is replaced with an equivalent concentration of lithospermic acid solution.

[0053] Example 5 The preparation method is the same as in Example 1, except that the concentration of shikonin is 250 μM.

[0054] Example 6 The preparation method is the same as in Example 1, except that the concentration of shikonin is 125 μM.

[0055] Comparative Example 1 The preparation method is the same as in Example 1, except that the concentration of tanshinone C is replaced with an equal concentration of tanshinone solution.

[0056] Comparative Example 2 The preparation method was the same as that of Comparative Example 1, except that the concentration of tanshinone was 250 μM.

[0057] Comparative Example 3 The preparation method was the same as that of Comparative Example 1, except that the concentration of tanshinone was 125 μM.

[0058] Comparative Example 4 The preparation method is the same as in Example 1, except that the concentration of salvianolic acid C is replaced with blank MOPS buffer.

[0059] In the above examples and comparative examples, tanshinone C and shikonin are carbonic anhydrase inhibitors (ligands), while tanshinone does not have inhibitory activity and cannot bind to the active site.

[0060] Sample Analysis Fishing efficiency was obtained based on ligand-based carbonic anhydrase cross-linked aggregates: Fishing efficiency was obtained by analyzing the peak area of ​​the positive drug acetazolamide after binding and elution of the carbonic anhydrase cross-linked aggregates. The specific steps are as follows: (1) Mix 0.1 mg of the carbonic anhydrase cross-linked aggregate prepared in the example or comparative example with 1 mL of 0.5 mg / mL acetazolamide solution (MOPS solution of acetazolamide) and incubate at 40 °C for 1 h to obtain the incubation solution; (2) Centrifuge the incubation solution for 10 min, wash the precipitate with MOPS, repeat 3 times to obtain the precipitate; (3) Add 400 μL of methanol to the precipitate for desorption, shake for 1 h and centrifuge, and collect the supernatant as the eluent for the positive control group; (4) Quantitative analysis: The eluent was injected into an HPLC-MS for analysis under the following conditions: The chromatographic conditions were as follows: the column was an Agilent ZORBAX SB Aq column (4.6 × 100 mm, 5 μm); the mobile phase was 0.1% formic acid aqueous solution for mobile phase A and acetonitrile for mobile phase B; the elution method was gradient elution, and the gradient elution process was set as shown in Table 2; the flow rate was 0.8 mL / min; the column temperature was 35 ℃; and the injection volume was 5 μL. Table 2: Gradient elution conditions for HPLC-MS The mass spectrometry conditions were as follows: ESI ion source, positive ion mode, ion scan range m / z 200~500; fragmentation voltage 120 V; scan / dwell time 723 ms; dryer flow rate 13.0 L / min, dryer temperature 325 ℃; nebulizer gas pressure 55.0 psi; capillary voltage 3.5 kV; capillary current 5 nA; chamber current 0.04 μA.

[0061] The peak areas of the extracted ion maps with an m / z of 223.2 were compared among the different examples and comparative samples. The results are as follows: Figure 1 As shown, the extracted ion chromatogram of Example 1 is as follows. Figure 2 As shown.

[0062] The results of Examples 1 and 4 and Comparative Examples 1 and 4 show that the adsorption of acetazolamide by the enzyme cross-linked aggregates only increases when a ligand is added and the ligand is a carbonic anhydrase ligand (inhibitor).

[0063] The results of the measurements in Examples 1-6 show that the adsorption efficiencies of enzyme cross-linked aggregates prepared by adding different concentrations of ligands (inhibitors) are different; only when 500, 250 μM salvianolic acid C and 500 μM shikonin are added is the adsorption efficiency of enzyme cross-linked aggregates the highest.

[0064] The extracted ion map of Example 1 is shown in [reference needed]. Figure 2 The results showed that the peak signal and area of ​​acetazolamide (m / z 223.2) were significantly improved after the addition of the ligand compared with the group without ligand. Furthermore, the signal difference between this group and the negative control group was further amplified, indicating that the introduction of the ligand effectively improved the binding efficiency and significantly enhanced the sensitivity of peak area-based screening.

[0065] The above results indicate that carbonic anhydrase cross-linked aggregates prepared by introducing ligands salvianolic acid C and shikonin (both carbonic anhydrase inhibitors) can bind the positive drug acetazolamide more efficiently than carbonic anhydrase cross-linked aggregates prepared by adding non-ligand compounds (Comparative Examples 1-3) and without adding ligands (Comparative Example 4). This demonstrates that introducing ligands can improve the efficiency of carbonic anhydrase cross-linked aggregates.

[0066] Application example: A method for screening carbonic anhydrase inhibitors from Ginkgo biloba extract, comprising the following steps: (1) Preparation of Ginkgo biloba extract solution: Weigh 100g of dried Ginkgo biloba powder, add 800 mL of 70% (v / v) ethanol, and pre-soak at 25°C for 2 hours. Then heat to boiling in a water bath, reflux and extract for 2 hours, filter and collect the first extract; add 800 mL of 70% ethanol to the residue again, repeat the above reflux extraction process and filter. Combine the two extracts, remove ethanol by rotary evaporation under reduced pressure, and freeze-dry the resulting concentrate to obtain Ginkgo biloba extract powder. Take an appropriate amount of powder and dissolve it in a small amount of DMSO, then dilute it 100 times with MOPS buffer to obtain Ginkgo biloba extract solution.

[0067] (2) Preparation of sample eluent: 0.1 mg of carbonic anhydrase cross-linked aggregate based on salvianolic acid C prepared in Example 1 or carbonic anhydrase cross-linked aggregate without ligand introduction prepared in Comparative Example 4 was mixed with 1 mL of 10 mg / mL Ginkgo biloba extract solution (MOPS solution of Ginkgo biloba extract) and incubated at 40 °C for 1 h to obtain incubation solution; the incubation solution was centrifuged for 10 min and the precipitate was washed 3 times with MOPS solution to obtain precipitate; 400 μL of methanol was added to the precipitate for desorption, shaken for 1 h, centrifuged, and the supernatant was collected as sample eluent; (3) Preparation of negative control eluent: 0.1 mg of carbonic anhydrase cross-linked aggregate based on salvianolic acid C prepared in Example 1 was placed in an oil bath and heated at 120 °C for 2 h to inactivate it. The inactivated cross-linked carbonic anhydrase was mixed with 1 mL of 10 mg / mL Ginkgo biloba extract solution and incubated at 40 °C for 1 h to obtain negative control incubation solution. The negative control incubation solution was subjected to the same centrifugation and elution steps as in step (2), and the supernatant was collected as negative control eluent. (4) Structural identification: The eluent of the sample and the eluent of the negative control group were analyzed by HPLC-MS. The HPLC-MS conditions were as follows: The chromatographic conditions were as follows: the column was an Agilent ZORBAX SB Aq column (4.6 × 100 mm, 5 μm); the mobile phase was: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; the elution method was gradient elution, and the gradient elution process was set as in Table 1; the flow rate was 0.8 mL / min; the column temperature was 35 ℃; and the injection volume was 5 μL.

[0068] The mass spectrometry conditions were as follows: the ion source was an ESI ion source, and the scanning mode was negative ion mode, with an ion scan range of m / z 100~1000; the fragmentation voltage was stepped, 75 m / z-90 V, 250 m / z-100 V, 450 m / z-110 V, 750 m / z-120 V, and 1000 m / z-130 V; the scan / dwell time was 1147 ms; the dryer flow rate was 13.0 L / min, and the dryer temperature was 325 ℃; the nebulizer gas pressure was 55.0 psi; the capillary voltage was -3.5 kV; the capillary current was 5 nA, and the chamber current was 0.04 μA.

[0069] For peaks with peak area changes greater than 10%, the mass-to-charge ratio in their primary mass spectra was examined. Seven potential carbonic anhydrase inhibitors with corresponding peak area changes greater than 15% in their extracted ion spectra were screened. The results are as follows: Figure 3 , Figure 4 As shown, UPLC-QTOF analysis was then performed to confirm the structure; The conditions for UPLC-QTOF: The chromatographic conditions are as follows: the same as the HPLC-MS chromatographic conditions described above; The mass spectrometry conditions were as follows: the ion source was an ESI ion source, and negative ion mode was used for scanning; the nebulizer gas was nitrogen, with Curtain gas at 35 psi; Gas 1 at 50 psi; Gas 2 at 50 psi; ISVF at -4.5 kV; CE at -10 V; DP at -100 V; TEM at 550 ℃; and the ion scan range was m / z 100~1500.

[0070] Seven potential carbonic anhydrase inhibitors were identified: ginkgo biloba flavonoids, ginkgolic acid flavonoids, isoginkgo biloba flavonoids, ginkgolic acid flavonoids, ginkgolic acid C13:0, ginkgolic acid C15:1, and ginkgolic acid C17:1. Figure 3 , Figure 4 As shown in A~F, the addition of ligands significantly improved the adsorption efficiency of enzyme cross-linked aggregates, with the three ginkgo biloba acid compounds showing the most significant improvement; without the addition of ligands, they could not be identified as potential carbonic anhydrase inhibitors.

[0071] Validation of the activity of carbonic anhydrase inhibitors Ginkgo biloba flavonoids, ginkgo biloba flavonoids, isoginkgo biloba flavonoids, pine flavonoids, ginkgolic acid C13:0, ginkgolic acid C15:1, and ginkgolic acid C17:1 were prepared into different concentration gradients using MOPS buffer containing DMSO in the range of 0.5-100 μM as analyte solutions. 40 μL of each analyte solution was added to a 96-well plate and mixed with 50 μL of PNPA solution (6 mM). Then, 10 μL of carbonic anhydrase solution (100 nM) was added to initiate the reaction. The plate was incubated at 37 ℃ and 600 r / min for 20 min using a thermostat. The absorbance of each well was then measured at 405 nm using a microplate reader, and the absorbance values ​​were recorded. The inhibition rate of the analyte was calculated according to formula ②.

[0072] in, The absorbance represents the absorbance of the well containing the analyte solution and the carbonic anhydrase solution; This represents the absorbance of the wells when the carbonic anhydrase solution was replaced with MOPS buffer; This represents the absorbance of the well when the analyte solution was replaced with MOPS buffer; This represents the absorbance of the wells when MOPS buffer was used to replace the analyte solution and carbonic anhydrase solution. The results are shown in [Figure Number]. Figure 5 .

[0073] like Figure 3 , Figure 4As shown in A~F, the addition of ligands significantly improved the adsorption efficiency of ligand-based carbonic anhydrase cross-linked aggregates, with the three ginkgolic acid compounds showing the most significant improvement; without the addition of ligands, they could not be identified as potential carbonic anhydrase inhibitors. Figure 5 As can be seen from A to H, the seven potential active ingredients obtained by the method of screening carbonic anhydrase inhibitors from Ginkgo biloba extract using ligand-based carbonic anhydrase cross-linked aggregates all exhibit carbonic anhydrase inhibitory activity, and this is a first-time discovery. These results indicate that the ligand-based carbonic anhydrase cross-linked aggregates provided in this invention can more effectively screen carbonic anhydrase inhibitors from active ingredients of traditional Chinese medicine, greatly improving the sensitivity and efficiency of the method.

[0074] Cellular activity verification of ginkgo biloba acid and ginkgo biflavonoids To verify whether ginkgolic acid and ginkgo biflavonoids can act on intracellular carbonic anhydrase and thus affect corresponding biological functions, an intracellular pH acidification model of HK-2 cells was used for testing. The specific steps are as follows: (1) Digest, resuspend, and count HK-2 cells, and seed them in black transparent 96-well plates at a density of 8,000 to 10,000 cells / well. Incubate at 37 °C and 5% CO2 for 24 h.

[0075] (2) Acetazolamide, Ginkgo biloba extract C15:1 and Jinsong double flavonoids were prepared into 100 μM, 5 μM and 10 μM test samples respectively using culture medium or assay buffer containing 0.1% DMSO.

[0076] (3) Discard the supernatant and add 100 μL of blank culture medium (containing 0.1% DMSO) or the sample to be tested into the well, and incubate in the incubator at 37 ℃ and 5% CO2 for 30 min.

[0077] (4) Discard the supernatant, add 100 μL of blank culture medium (containing 0.1% DMSO) or the sample to be tested to each well containing 5 μM BCECF-AM, and incubate at 37 ℃ and 5% CO2 for 30 min.

[0078] (5) Discard the supernatant, add 100 μL of blank assay buffer (containing 0.1% DMSO) or the sample to be tested to each well, incubate at 37 °C for 5 min, discard the supernatant, add 100 μL of blank assay buffer (containing 0.1% DMSO) or the sample to be tested to each well, incubate at 37 °C for 5 min.

[0079] (6) Discard the supernatant, add 100 μL of blank assay buffer (containing 0.1% DMSO) or the sample to be tested to each well, and quickly place the plate into the microplate reader. Within 3 min, perform fluorescence measurements on the sample in two detection channels: emission wavelength 535 nm, excitation wavelength 505 nm, and emission wavelength 535 nm, excitation wavelength 440 nm. Calculate the ratio of the fluorescence values ​​of the two channels and establish a linear regression curve with the measurement time. The slope obtained is the intracellular pH recovery rate (ΔpHi / Δt).

[0080] This experiment used the pH-sensitive fluorescent probe BCECF-AM to label HK-2 renal tubular epithelial cells, and monitored intracellular pH changes in real time using the dual excitation wavelength (490 / 440nm) ratio method. A model was established using the classic NH4Cl pulse method: NH4Cl loading and elution induced acute acidification in the cells, activating their inherent acid excretion mechanism (mainly dependent on NHE3 and carbonic anhydrase). By comparing the slope of the pH recovery curve after acidification, it was determined whether carbonic anhydrase was inhibited in the cells.

[0081] like Figure 7 As shown, the positive control drug acetazolamide significantly slowed the rate of intracellular pH recovery, confirming the successful establishment of the model. Meanwhile, the treatment groups with ginseng and ginkgolic acid C15:1 also exhibited significant inhibitory effects, leading to a marked decrease in the pHi recovery slope. These results suggest that these two types of compounds have good membrane permeability, enabling them to enter cells and inhibit intracellular carbonic anhydrase activity or related acid excretion mechanisms, thereby blocking the pH recovery process in HK-2 cells.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing ligand-based carbonic anhydrase cross-linked aggregates, characterized in that, Includes the following steps: (1) The carbonic anhydrase solution and the ligand solution are mixed and incubated to obtain a first mixture, wherein the ligand is a carbonic anhydrase inhibitor; (2) Add a precipitant to the first mixture and mix to carry out a precipitation reaction to obtain the second mixture; (3) Add a crosslinking agent to the second mixture and mix to carry out a crosslinking reaction to obtain a third mixture; (4) Centrifuge the third mixture to obtain the first precipitate, and use an eluent and a buffer to elute and wash it respectively. Centrifuge again to obtain the second precipitate, which is the carbonic anhydrase cross-linked aggregate based on the ligand.

2. The method for preparing ligand-based carbonic anhydrase cross-linked aggregates according to claim 1, characterized in that, In step (1), the ligand is a carbonic anhydrase inhibitor or ligand, such as salvianolic acid C, shikonin, etc.

3. The method for preparing ligand-based carbonic anhydrase cross-linked aggregates according to claim 1, characterized in that, In step (1), the molar ratio of carbonic anhydrase to ligand in the first mixture is 1~10:0.06~600.

4. The method for preparing ligand-based carbonic anhydrase cross-linked aggregates according to claim 1, characterized in that, In step (3), the crosslinking agent is glutaraldehyde, genipin, tannic acid, etc.

5. The method for preparing ligand-based carbonic anhydrase cross-linked aggregates according to claim 1, characterized in that, In step (3), the molar ratio of the cross-linking agent and the carbonic anhydrase is 1~10:60~6000.

6. The ligand-based carbonic anhydrase cross-linked aggregate prepared by any one of the preparation methods according to claims 1 to 5.

7. The application of the ligand-based carbonic anhydrase cross-linked aggregates according to claim 6 in the screening of carbonic anhydrase inhibitors.

8. The application according to claim 7, characterized in that, The application described is to screen carbonic anhydrase inhibitors from extracts of traditional Chinese medicine, wherein the extract of traditional Chinese medicine is ginkgo leaf extract.

9. The application according to claim 8, characterized in that, A method for screening carbonic anhydrase inhibitors from Ginkgo biloba extract includes the following steps: S1. Preparation of sample eluent: The ligand-based carbonic anhydrase cross-linked aggregates described in claim 6 are mixed with Ginkgo biloba extract solution and incubated to obtain an incubation solution. The incubation solution is centrifuged and washed to obtain a precipitate. An elution solution is added to the precipitate for elution, and the supernatant is collected to obtain the sample eluent. S2. Preparation of negative control group sample eluent: After heating and inactivating the ligand-based carbonic anhydrase cross-linked aggregates as described in claim 6, mix them with Ginkgo biloba extract solution and incubate to obtain negative control group incubation solution. Repeat the centrifugation, washing and elution steps in S1, and collect the supernatant to obtain negative control eluent. S3. Structural Identification: The eluents of the samples from steps S1 and S2 and the eluent of the negative control group were analyzed by HPLC-MS. The peak areas of each peak in the total ion chromatograms of the two groups of samples were compared. If the peak area difference between the peaks in the total ion chromatogram is greater than 15%, then the peak areas of the extracted ion chromatograms corresponding to the mass-to-charge ratio shown in the first-order mass spectrum should be further compared. If the peak area difference of the extracted ion map corresponding to the mass-to-charge ratio is greater than 15%, further UPLC-QTOF analysis should be performed to confirm the structure of the carbonic anhydrase inhibitor.

10. The application according to claim 9, characterized in that, The carbonic anhydrase inhibitor is at least one of ginkgo biloba flavonoids, ginkgolides biloba flavonoids, ginkgo biloba flavonoids, isoginkgo biloba flavonoids, ginkgolic acid C15:1, ginkgolic acid C13:0, and ginkgolic acid C17:1.

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