Carbonic anhydrase binding ligands, methods of making and using the same

Abstract

This invention belongs to the field of biochemistry and specifically provides a carbonic anhydrase-binding ligand, wherein the ligand includes at least one of euphorbia alcohol-type diterpenes and oleuropein-type diterpenes. The aforementioned carbonic anhydrase-binding ligand possesses antitumor, antiviral, antifungal, anti-inflammatory, edema-treating, glaucoma-treating, Alzheimer's disease-treating, and hyperammonemia-treating effects. Enzymatic experiments show that it is a carbonic anhydrase agonist and also has industrial applications such as carbon dioxide capture and fixation, and accelerated mineralization of biological tissues and corals. This invention also provides a method for preparing the aforementioned carbonic anhydrase-binding ligand using Euphorbia kansui as raw material; the preparation is simple and provides a new avenue for the application of Euphorbia kansui.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a carbonic anhydrase-binding ligand, its preparation method, and its application. Background Technology

[0002] Euphorbia kansui (TNLiou ex), the dried tuberous root of the Euphorbiaceae plant, is a traditional Chinese medicine with the effects of purging water and reducing swelling. It is used clinically, either alone or in combination with other herbs, to treat ascites, edema, and difficulty in urination and defecation. In clinical applications, Euphorbia kansui is often processed, such as by vinegar or tofu preparation, to reduce toxicity and enhance efficacy. The chemical components of Euphorbia kansui include diterpenes (euphorbia alcohol-type diterpenes and oleuropein-type diterpenes), triterpenes, sterols, and flavonoids. Diterpenes and triterpenes are characteristic components of Euphorbia kansui.

[0003] Carbonic anhydrases are a class of metal-containing enzymes widely found in mammals and microorganisms. Taking carbonic anhydrase in humans as an example, this enzyme uses zinc ions as the catalytic center to catalyze the hydration reaction of CO2, and its reaction formula is as follows:

[0004]

[0005] This reaction can increase intracellular electrolyte concentration, thereby reducing water excretion; simultaneously, it increases the acidic microenvironment of the cell. Ligands of carbonic anhydrase have therapeutic effects on tumors, viruses, inflammation, edema, and glaucoma. Research on carbonic anhydrase agonists is currently limited, but their potential pharmacological uses include enhancing memory, treating Alzheimer's disease, treating hyperammonemia, and antifungal activity. Additionally, potential industrial uses include carbon dioxide capture and fixation, and accelerated mineralization of biological tissues and corals. Currently, there are no research reports on the use of Euphorbia kansui extract and its components as carbonic anhydrase binding ligands and agonists, nor are there reports on the application of Euphorbia kansui extract and its components in the above potential uses. Summary of the Invention

[0006] The purpose of this invention is to provide a carbonic anhydrase-binding ligand, wherein the ligand comprises at least one of euphorbia alcohol-type diterpenes and oleuropein-type diterpenes.

[0007] Specifically, the above includes 3-O-(2'E,4'E-decadienoyl)-20-O-acetyl giant euphorbia diterpene alcohol and Euphorbia kansui ester B.

[0008] The present invention also provides a method for preparing the above-mentioned carbonic anhydrase-binding ligand, comprising the following steps: pulverizing Euphorbia kansui medicinal material and extracting it with a polar solvent, evaporating the solvent after extraction to obtain the carbonic anhydrase-binding ligand.

[0009] Specifically, the aforementioned Euphorbia kansui medicinal material includes raw Euphorbia kansui or vinegar-processed Euphorbia kansui.

[0010] Specifically, the above-mentioned vinegar-processed Euphorbia kansui is prepared by the following steps: raw Euphorbia kansui is soaked in acetic acid solution and stir-fried to obtain vinegar-processed Euphorbia kansui.

[0011] Specifically, the mass-to-volume ratio (g / ml) of raw Euphorbia kansui to acetic acid in the acetic acid solution is 1000:(10.5-17.5), the soaking time is 1-8 hours, and the stir-frying conditions are 150-250℃ for 1-25 minutes.

[0012] Specifically, the aforementioned organic solvents include any one of ethanol, dichloromethane, ethyl acetate, or petroleum ether.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] The carbonic anhydrase-binding ligand provided by this invention has anti-tumor, antiviral, antifungal, anti-inflammatory, edema-treating, glaucoma-treating, Alzheimer's disease-treating, and hyperammonemia-treating effects. Enzymatic experiments show that it is a carbonic anhydrase agonist and also has industrial applications such as carbon dioxide capture and fixation, and accelerated mineralization of biological tissues and corals.

[0015] The preparation method provided by this invention uses Euphorbia kansui as raw material, is simple to produce, and SPR analysis shows that the obtained extract binds to carbonic anhydrase. The extract prepared after processing Euphorbia kansui with vinegar has a stronger binding force on carbonic anhydrase. Enzymatic experiments show that the extract is a carbonic anhydrase agonist. This provides a new avenue for the application of Euphorbia kansui.

[0016] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0017] Figure 1 The curves show the SPR curves of extracts R and P from Example 1 binding with carbonic anhydrase.

[0018] Figure 2 The activation curves of carbonic anhydrase by extracts R and P in Example 1 are shown.

[0019] Figure 3 The SPR curves are those of components J-1 and I-21 in the Euphorbia kansui extract of Example 2, which bind to carbonic anhydrase.

[0020] Figure 4 The activation curves of carbonic anhydrase by components J-1 and I-21 in the Euphorbia kansui extract in Example 2 are shown.

[0021] Figure 5 The docking simulation in Example 3 confirmed the binding affinity between oleuropein and euphorbia alcohol diterpenes. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0023] This invention provides a carbonic anhydrase-binding ligand comprising an extract of Euphorbia kansui, the extract comprising at least one of euphorbia alcohol-type diterpenoids and oleuropein-type diterpenoids. Preferably, it comprises 3-O-(2'E,4'E-decadienoyl)-20-O-acetyleuphorbia diterpenol and Euphorbia kansui ester B.

[0024] This invention also provides a method for preparing the above-mentioned carbonic anhydrase-binding ligand, comprising the following steps: pulverizing Euphorbia kansui medicinal material and extracting it with an organic solvent, preferably a moderately polar or low-polarity organic solvent such as ethanol, dichloromethane, ethyl acetate, or petroleum ether; evaporating the solvent after extraction to obtain Euphorbia kansui extract, which is the carbonic anhydrase-binding ligand. The method further includes separating and purifying the extract to obtain the component compounds in the extract.

[0025] The medicinal material mentioned includes raw Euphorbia kansui, vinegar-processed Euphorbia kansui, or other processed Euphorbia kansui products.

[0026] The vinegar-processed Euphorbia kansui is preferably prepared using the following steps: Raw Euphorbia kansui is soaked in an acetic acid solution, with a mass-to-volume ratio (g / ml) of raw Euphorbia kansui to acetic acid in the solution of 1000:(10.5-17.5), for 1-8 hours. Subsequently, it is stir-fried at 150-250℃ for 5-15 minutes to obtain the vinegar-processed Euphorbia kansui. During the vinegar-processing, an acetic acid solution or its equivalents can be used to soak the Euphorbia kansui, such as edible vinegar containing the same amount of acetic acid.

[0027] The effects of the carbonic anhydrase-binding ligand, its preparation method, and its application are studied through specific embodiments below.

[0028] Example 1:

[0029] 1. Vinegar-processed Euphorbia kansui

[0030] 100 kg of raw Euphorbia kansui was soaked in 30 liters of edible vinegar (acetic acid content 3.5%) for 2 hours, and then stir-fried at 180 degrees Celsius for 10 minutes to obtain vinegar-processed Euphorbia kansui.

[0031] 2. Extraction of Euphorbia kansui

[0032] Raw Euphorbia kansui and vinegar-processed Euphorbia kansui were pulverized and extracted separately with 500 ml of ethanol as the extraction solvent. After extraction, the solvent was evaporated to obtain raw Euphorbia kansui extract and vinegar-processed Euphorbia kansui extract, which were named extracts R and P, respectively.

[0033] 3. SPR combined analysis

[0034] Extracts R and P were dissolved in phosphate buffer containing Tween 80 (containing 1% dimethyl sulfoxide) to prepare a series of analytes at various concentrations. Simultaneously, carbonic anhydrase was immobilized on a CM5 chip as a stationary phase for SPR binding analysis. Results are as follows: Figure 1 As shown, extracts R and P are carbonic anhydrase ligands. Based on an average molecular weight of 600, the dissociation constants of extracts R and P with carbonic anhydrase are KD = 1.86 * 10⁻⁶. -4 MS -1 and 3.01*10 -5 MS -1 This indicates that both extracts R and P can bind to carbonic anhydrase, and the extract after vinegar treatment has a stronger binding affinity to carbonic anhydrase.

[0035] 4. Enzyme experiments

[0036] The substrate (4-nitrophenylacetic acid ester) was diluted to 2 mM with phosphate buffer (pH 8.0), and the carbonic anhydrase was diluted to 25 ng / μL with the same buffer. Extracts R and P were prepared in concentration gradients using a buffer containing 1% DMSO. 2 mL of each extract solution, 50 μL of carbonic anhydrase solution, 200 μL of substrate, and 2 mL of buffer solution were added to volumetric tubes. After incubation at room temperature for 15 min, the absorbance at 400 nm was measured using UV spectroscopy. Results are as follows: Figure 2 As shown, calculations revealed that extracts R and P both increased the activity of carbonic anhydrase in hydrolyzing substrates, with their EC50 values ​​being... 50 The values ​​are 1.29*10 -4 MS -1 and 8.13*10 -5 MS -1 This indicates that both extracts R and P can activate carbonic anhydrase, and the extract after vinegar treatment has a stronger carbonic anhydrase activation ability.

[0037] Example 2:

[0038] 1. Vinegar-processed Euphorbia kansui

[0039] 100 kg of raw Euphorbia kansui herbs are soaked in 50 liters of edible vinegar (3% acetic acid) for 2 hours, and then stir-fried at 150 degrees Celsius for 20 minutes to obtain vinegar-processed Euphorbia kansui.

[0040] 2. Extraction of Euphorbia kansui

[0041] Raw Euphorbia kansui and vinegar-processed Euphorbia kansui were pulverized and extracted with 2000 ml of ethyl acetate as the extraction solvent. After extraction, the solvent was evaporated to obtain raw Euphorbia kansui extract and vinegar-processed Euphorbia kansui extract, which were named extracts R and P, respectively.

[0042] 3. Separation and purification

[0043] Extracts R and P were purified by preparative liquid chromatography (C18 column) to obtain Euphorbia kansui ester B and 3-O-(2'E,4'E-decadienoyl)-20-O-acetyl giant euphorbia diterpene alcohol.

[0044] 4. SPR combined analysis

[0045] Euphorbia kansui ester B and 3-O-(2'E,4'E-decadienoyl)-20-O-acetylgalactosidol were dissolved in phosphate buffer containing Tween 80 (containing 2% dimethyl sulfoxide) to prepare a series of concentrations as analytes. Simultaneously, carbonic anhydrase was immobilized on a CM5 chip as a stationary phase for SPR binding analysis. The results are as follows: Figure 3 As shown. Euphorbia kansui ester B (J-1) and 3-O-(2'E,4'E-decadienoyl)-20-O-acetylgalactosidase diterpenol (I-21) are carbonic anhydrase ligands. The dissociation constants of Euphorbia kansui ester B (J-1) and 3-O-(2'E,4'E-decadienoyl)-20-O-acetylgalactosidase diterpenol (I-21) with carbonic anhydrase are KD = 1.48 * 10⁻⁶. -7 MS -1 and 3.52*10 -8 MS -1 This indicates that extracts J-1 and I-21 can both bind to carbonic anhydrase, and I-21, which has a euphorane-type skeleton, has a stronger binding affinity to carbonic anhydrase.

[0046] 5. Enzyme experiments

[0047] The substrate (4-nitrophenylacetic acid ester) was diluted to 2 mM with phosphate buffer (pH 8.0), and the carbonic anhydrase was diluted to 25 ng / μL with the same buffer. Extracts J-1 and I-21 were prepared in concentration gradients using a buffer containing 1% DMSO. 2 mL of each extract solution, 50 μL of carbonic anhydrase solution, 200 μL of substrate, and 2 mL of buffer solution were added to volumetric tubes. After incubation at room temperature for 15 min, the absorbance at 400 nm was measured using UV spectroscopy. Results are as follows: Figure 4 As shown, calculations show that both J-1 and I-21 can increase the activity of carbonic anhydrase in hydrolyzing substrates, and I-21, which has a euphorane-type skeleton, has a stronger activating effect on carbonic anhydrase.

[0048] Example 3:

[0049] 1. Extraction of Euphorbia kansui

[0050] 50 grams of raw Euphorbia kansui was pulverized and extracted with 500 ml of dichloromethane as the extraction solvent. After extraction, the solvent was evaporated to dryness to obtain Euphorbia kansui extract.

[0051] 2. Collect ligand small molecules

[0052] The extract was dissolved in phosphate buffer containing Tween 80 (containing 5% dimethyl sulfoxide), and carbonic anhydrase was immobilized on a CM5 chip as a stationary phase. The extract solution was introduced into an SPR flow cell for thorough binding. The tubing and chip were rinsed with PBST for 30 seconds, and then the ligand molecules were collected using 10 mM pH 2.0 glycine hydrochloride buffer. This process was repeated multiple times. The collected solution was freeze-dried and dissolved in acetonitrile for LC-HR-MS analysis to identify the small ligand components with strong binding to carbonic anhydrase. The results are shown in Table 1. The experimental results confirmed that the strongly binding small molecules were mainly euphorbia alcohol-type diterpenoids from Euphorbia kansui.

[0053] Table 1 Small molecule ligands obtained by SPR fishing

[0054]

[0055]

[0056] Through computer simulation of Doking Figure 5 Combined with other studies, it was confirmed that both oliveone-type and euphorbia alcohol-type diterpenes in Euphorbia kansui are bound to carbonic anhydrase, with euphorbia alcohol-type diterpenes having a stronger binding affinity. Therefore, the extracts containing euphorbia alcohol-type diterpenes have a stronger carbonic anhydrase binding and binding affinity.

[0057] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. Use of 3-O-(2'E,4'E-decadienoyl)-20-O-acetylin-kuringijiol and Tigliasteryl ester B for the preparation of carbonic anhydrase binding ligands, characterized in that: The carbonic anhydrase-binding ligand is a carbonic anhydrase agonist.

2. Use according to claim 1, wherein The preparation method of the carbonic anhydrase-binding ligand includes the following steps: pulverizing Euphorbia kansui medicinal material and extracting it with an organic solvent, then evaporating the solvent after extraction to obtain the carbonic anhydrase-binding ligand.

3. Use according to claim 2, wherein: The medicinal material mentioned includes raw Euphorbia kansui or Euphorbia kansui processed with vinegar.

4. The application as described in claim 2, characterized in that: The organic solvent includes any one of ethanol, dichloromethane, ethyl acetate, or petroleum ether. Applications of 5,3-O-(2'E,4'E-decadienoyl)-20-O-acetylgalactoside and Euphorbia kansui ester B in the preparation of antiviral, antifungal, anti-inflammatory, Alzheimer's disease treatment, and hyperammonemia treatment drugs. Application of 6,3-O-(2'E,4'E-decadienoyl)-20-O-acetyl-gilopyrhein diterpenol and Euphorbia kansui ester B in carbon dioxide capture and fixation. Application of 7.3-O-(2'E,4'E-decadienoyl)-20-O-acetyl-gilopyrhein diterpene alcohol and Euphorbia kansui ester B in accelerating mineralization of biological tissues.

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