Carbonic anhydrase binding ligand as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly provides a carbonic anhydrase binding ligand which comprises at least one of euphorbia alcohol type diterpene and olivone type diterpene. The carbonic anhydrase binding ligand has the effects of resisting tumors, viruses, fungi and inflammation, treating edema, glaucoma, senile dementia, hyperammonemia and the like. An enzymology experiment shows that the compound is a carbonic anhydrase agonist and also has industrial applications of capturing and fixing carbon dioxide, accelerating mineralization of biological tissues and corals and the like. The invention also provides a preparation method of the carbonic anhydrase binding ligand, which takes euphorbia kansui as a raw material, is simple to prepare and provides a new way for application of euphorbia kansui.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a carbonic anhydrase binding ligand and a preparation method and application thereof. Background Art

[0002] Kansui is the dried root of Euphorbia kansui TNLiou ex, a plant of the Euphorbiaceae family. It is a traditional Chinese medicinal material with the effects of purging water and removing swelling, reducing swelling and dispersing knots. It is used in the clinic to treat ascites, edema, and constipation, etc. in a single or combined form. In clinical applications, Kansui is often processed, such as by vinegar or tofu, to reduce toxicity and improve efficacy. The chemical components in Kansui include: diterpenes (euphorbiol-type diterpenes and olive ketone-type diterpenes), triterpenes, sterols, flavonoids and other chemical components, among which diterpenes and triterpenes are the characteristic components in Kansui.

[0003] Carbonic anhydrase is a type of metal-containing enzyme widely found in mammals and microorganisms. Taking the carbonic anhydrase in the human body as an example, the enzyme uses zinc ions as the catalytic center to catalyze the hydration reaction of CO2. The reaction formula is as follows:

[0004]

[0005] This reaction can increase the intracellular electrolyte concentration, thereby reducing the discharge of water; at the same time, it increases the acidic microenvironment of the cell. The ligands of carbonic anhydrase have the effects of treating tumors, anti-virus, anti-inflammatory, treating edema and glaucoma. There are currently few studies on carbonic anhydrase agonists, and their potential pharmacological uses include: enhancing memory, treating Alzheimer's disease, treating hyperammonemia, and anti-fungal. In addition, their potential industrial uses include: carbon dioxide capture and fixation, accelerated mineralization of biological tissues and corals, etc. At present, there are no reports on the research of Gan Sui extracts and components contained in the extracts as carbonic anhydrase binding ligands and agonists, nor are there any reports on the application of Gan Sui extracts and components contained in the extracts in the above potential uses. Summary of the invention

[0006] The invention aims to provide a carbonic anhydrase binding ligand, wherein the ligand comprises at least one of euphorbiacetyl alcohol type diterpenes and olive ketone type diterpenes.

[0007] Specifically, the above-mentioned includes 3-O-(2'E, 4'E-decadienoyl)-20-O-acetylingenol and gansuline ester B.

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

[0009] Specifically, the above-mentioned Gan Sui medicinal material includes raw Gan Sui or vinegar-processed Gan Sui.

[0010] Specifically, the vinegar-processed gansui is prepared by the following steps: adding raw gansui into an acetic acid solution to soak and stir-fry to obtain the vinegar-processed gansui.

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

[0012] Specifically, the organic solvent includes 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 the present invention has the effects of anti-tumor, anti-virus, anti-fungal, anti-inflammatory, edema treatment, glaucoma treatment, Alzheimer's treatment, hyperammonemia treatment, etc. Enzyme experiments show that it is a carbonic anhydrase agonist, and also has industrial uses such as carbon dioxide capture and fixation, and accelerated mineralization of biological tissues and corals.

[0015] The preparation method provided by the invention uses the gansui medicinal material as a raw material, is simple to prepare, and SPR analysis shows that the obtained extract binds to carbonic anhydrase. The extract prepared after the gansui vinegar treatment has a stronger binding force to carbonic anhydrase, and enzymatic experiments show that the extract is a carbonic anhydrase agonist, providing a new approach for the application of gansui.

[0016] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 1 is the SPR curve of extracts R and P combined with carbonic anhydrase in Example 1.

[0018] Figure 2 This is the activation curve of extracts R and P in Example 1 on carbonic anhydrase.

[0019] Figure 3 This is the SPR curve of the components J-1 and I-21 in the Kansui extract in Example 2 binding to carbonic anhydrase.

[0020] Figure 4 This is the activation curve of components J-1 and I-21 in the Kansui extract in Example 2 on carbonic anhydrase.

[0021] Figure 5 The docking simulation in Example 3 confirmed the binding ability of olive ketone type and euphorbiacetyl alcohol type diterpenes. DETAILED DESCRIPTION

[0022] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art 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 limited by the attached claims and their equivalents.

[0023] The present invention provides a carbonic anhydrase binding ligand, wherein the ligand comprises a gansui extract, and the gansui extract comprises at least one of an euphorbiacetyl alcohol type diterpene and an oleuropein type diterpene, preferably comprising 3-O-(2'E, 4'E-decadienoyl)-20-O-acetyl ingenol and gansui terpene ester B.

[0024] The present invention also provides a method for preparing the carbonic anhydrase binding ligand, comprising the following steps: crushing the kansui medicinal material and extracting it with an organic solvent, wherein the organic solvent is preferably a medium-polarity or low-polarity organic solvent such as ethanol, dichloromethane, ethyl acetate, petroleum ether, etc., and evaporating the solvent after extraction to obtain a kansui extract, i.e., a carbonic anhydrase binding ligand. The method also includes separating and purifying the extract to obtain component compounds in the extract.

[0025] The gansui medicinal materials include raw gansui, vinegar-processed gansui or other gansui processed products.

[0026] The vinegar-processed gansui is preferably prepared by the following steps: adding raw gansui to an acetic acid solution for soaking, the mass volume ratio (g / ml) of raw gansui to acetic acid in the acetic acid solution is 1000:(10.5-17.5), soaking time is 1-8 hours, and then stir-frying at 150-250° C. for 5-15 minutes to obtain the vinegar-processed gansui. During vinegar processing, the gansui can be soaked with acetic acid solution and its equivalent, such as edible vinegar containing the same amount of acetic acid.

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

[0028] Embodiment 1:

[0029] 1. Vinegar-processed kansui

[0030] Add 100 kg of raw gansui medicinal materials into 30 liters of edible vinegar (acetic acid content 3.5%) and soak them fully for 2 hours, then stir-fry at 180 degrees Celsius for 10 minutes to make vinegar-processed gansui.

[0031] 2. Extraction of Kansui

[0032] After the raw gansui and vinegar-processed gansui were crushed, they were extracted with 500 ml of ethanol as the extraction solvent, and the solvent was evaporated after extraction to obtain the raw gansui extract and the vinegar-processed gansui extract, which were named extracts R and P, respectively.

[0033] 3. SPR binding analysis

[0034] Extracts R and P were dissolved in phosphate buffered saline (containing 1% dimethyl sulfoxide) containing Tween 80, and a series of concentrations were prepared as analytes. Carbonic anhydrase was immobilized on a CM5 chip as a stationary phase, and SPR binding analysis was performed. The results are shown in Figure 1 As shown, extracts R and P are carbonic anhydrase ligands. Calculated based on an average molecular weight of 600, the dissociation constants of extracts R and P with carbonic anhydrase are KD=1.86*10 -4 MS -1 and 3.01*10 -5 MS -1 , indicating that both extracts R and P can bind to carbonic anhydrase, and the extract after vinegar treatment has a stronger binding ability with carbonic anhydrase.

[0035] 4. Enzyme experiments

[0036] The substrate (4-nitrophenyl acetate) was diluted to 2mM with a phosphate buffer at pH 8.0, and the carbonic anhydrase was diluted to 25ng / μL with the same buffer. The concentration gradient of extracts R and P was prepared with a buffer containing 1% DMSO. 2ml of extract solutions of different concentrations, 50μL of carbonic anhydrase solution, 200μL of substrate and 2mL of buffer solution were added to the volumetric tube, and incubated at room temperature for 15min, and then the absorbance at 400nm was measured by UV spectroscopy. The results are shown in Figure 2. Figure 2 As shown in the figure, it was calculated that extracts R and P could increase the activity of carbonic anhydrase in hydrolyzing substrates, and their EC 50 The values ​​are 1.29*10 -4 MS -1 and 8.13*10 -5 MS -1 The results showed that both extracts R and P could activate carbonic anhydrase, and the extracts treated with vinegar had stronger carbonic anhydrase activation ability.

[0037] Embodiment 2:

[0038] 1. Vinegar-processed kansui

[0039] Add 100 kg of raw gansui medicinal materials into 50 liters of edible vinegar (3% acetic acid content) and fully soak them for 2 hours, then stir-fry at 150 degrees Celsius for 20 minutes to obtain vinegar-processed gansui.

[0040] 2. Extraction of Kansui

[0041] After the raw kansui and the vinegar-processed kansui were crushed, they were extracted with 2000 ml of ethyl acetate as the extraction solvent, and after extraction, the solvent was evaporated to obtain the raw kansui extract and the vinegar-processed 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) as separation medium to obtain gansuiditerpene ester B and 3-O-(2'E,4'E-decadienoyl)-20-O-acetyl ingenol.

[0044] 4. SPR binding analysis

[0045] In phosphate buffered saline (containing 2% dimethyl sulfoxide) containing Tween 80, gansuline ester B and 3-O-(2'E,4'E-decadienoyl)-20-O-acetyl ingenol were dissolved and a series of concentrations were prepared as analytes. At the same time, carbonic anhydrase was fixed on a CM5 chip as a stationary phase, and SPR binding analysis was performed. The results are shown in FIG. Figure 3 As shown. Gansuide terpene ester B (J-1) and 3-O-(2'E, 4'E-decadienoyl)-20-O-acetyl-ingenol (I-21) are carbonic anhydrase ligands. The dissociation constants of Gansuide terpene ester B (J-1) and 3-O-(2'E, 4'E-decadienoyl)-20-O-acetyl-ingenol (I-21) with carbonic anhydrase are KD=1.48*10 -7 MS -1 and 3.52*10 -8 MS -1 , indicating that extracts J-1 and I-21 can both bind to carbonic anhydrase, and I-21 with a euphorbane-type skeleton has a stronger binding ability with carbonic anhydrase.

[0046] 5. Enzyme experiments

[0047] The substrate (4-nitrophenyl acetate) was diluted to 2mM with a phosphate buffer at pH 8.0, and the carbonic anhydrase was diluted to 25ng / μL with the same buffer salt. The concentration gradient solutions of extracts J-1 and I-21 were prepared with a buffer salt containing 1% DMSO. 2ml of extract solutions of different concentrations, 50μL of carbonic anhydrase solution, 200μL of substrate and 2mL of buffer solution were added to the volumetric tube, and the absorbance at 400nm was measured by ultraviolet spectroscopy after incubation at room temperature for 15min. The results are shown in Figure 2. Figure 4 As shown, it was calculated that both J-1 and I-21 could increase the activity of carbonic anhydrase in hydrolyzing substrates, and I-21 with a euphorbane-type skeleton had a stronger activation effect on carbonic anhydrase.

[0048] Embodiment 3:

[0049] 1. Extraction of Kansui

[0050] 50 grams of raw kansui were crushed, and then extracted with 500 milliliters of dichloromethane as an extraction solvent. After the extraction, the solvent was evaporated to obtain the kansui extract.

[0051] 2. Collect ligand small molecules

[0052] The extract was dissolved in phosphate buffered saline (containing 5% dimethyl sulfoxide) containing Tween 80, and carbonic anhydrase was fixed on a CM5 chip as a stationary phase. The extract solution was introduced into the SPR flow cell for full binding, and the pipeline and chip were rinsed with PBST for 30 seconds, and then the ligand small molecules were collected with 10mM pH2.0 glycine hydrochloride buffer. The above steps were repeatedly collected for many times, and the collected solution was freeze-dried and dissolved with acetonitrile for LC-HR-MS analysis to determine the small molecule ligand components with strong binding to carbonic anhydrase. The results are shown in Table 1. The experimental results confirmed that the small molecules with strong binding were mainly the euphorbiacetol-type diterpene components in Gan Sui.

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

[0054]

[0055]

[0056] Through computer simulation of Docking ( Figure 5 ) combined with the results of the present study confirmed that both the olive ketone-type and euphorbiacetic alcohol-type diterpenes in Gansui bind to carbonic anhydrase, and the euphorbiacetic alcohol-type diterpenes have a stronger binding force. Therefore, the component extracts containing euphorbiacetic alcohol-type diterpenes have stronger carbonic anhydrase binding and binding effects.

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

Claims

1. A carbonic anhydrase binding ligand, characterized in that: The ligand includes at least one of euphorbiacetyl alcohol-type diterpenes and olive ketone-type diterpenes.

2. The carbonic anhydrase binding ligand according to claim 1, characterized in that: The ligands include 3-O-(2'E, 4'E-decadienoyl)-20-O-acetylingenol and gansuline ester B.

3. The method for preparing a carbonic anhydrase binding ligand according to any one of claims 1 or 2, characterized in that: The following steps are involved: The kansui medicinal material is crushed and then extracted with an organic solvent, and the solvent is evaporated after the extraction to obtain the carbonic anhydrase binding ligand.

4. The method for preparing a carbonic anhydrase binding ligand according to claim 3, characterized in that: The gansui medicinal material includes raw gansui or vinegar-processed gansui.

5. The method for preparing a carbonic anhydrase binding ligand according to claim 4, characterized in that: The vinegar-processed gansui is prepared by the following steps: adding raw gansui into an acetic acid solution for soaking and stir-frying to obtain the vinegar-processed gansui.

6. The method for preparing a carbonic anhydrase binding ligand according to claim 5, characterized in that: The mass volume ratio of the raw kansui to the acetic acid in the acetic acid solution is 1000g:(10.5-17.5)ml, the soaking time is 1-8 hours, and the stir-frying condition is 150-250°C for 1-25 minutes.

7. The method for preparing a carbonic anhydrase binding ligand according to claim 3, characterized in that: The organic solvent includes any one of ethanol, dichloromethane, ethyl acetate or petroleum ether.

8. Use of the carbonic anhydrase binding ligand as claimed in any one of claims 1 or 2 in the preparation of anti-tumor, anti-viral, anti-fungal, anti-inflammatory, edema treatment, glaucoma treatment, Alzheimer's treatment, and hyperammonemia treatment drugs.

9. Use of the carbonic anhydrase binding ligand according to any one of claims 1 or 2 in carbon dioxide capture and fixation.

10. Use of the carbonic anhydrase binding ligand according to any one of claims 1 or 2 in accelerating mineralization of biological tissues.

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