Use of chlorogenic acid as a chitinase inhibitor

Abstract

This invention relates to the field of chitinase inhibitor technology, and discloses the application of chlorogenic acid as a chitinase inhibitor. This invention is the first to discover and demonstrate that chlorogenic acid can act as an effective chitinase inhibitor, inhibiting the activity of endogenous chitinases in fungi and bacteria, thereby suppressing fungal and bacterial growth and weakening pathogenicity. It has been verified that chlorogenic acid inhibits the IC50 of chitinases derived from *Streptomyces griseus*. 50 The IC50 value for inhibiting chitinase from Verticillium dahliae was 1.095 μg / μL. 50 The concentration was 0.754 μg / μL, demonstrating the significant chitinase inhibitory activity of chlorogenic acid. Furthermore, this invention also confirmed that chlorogenic acid can interact with human chitinase CHIT1 and chitinase-like protein CHI3L1, inhibiting their function. Therefore, chlorogenic acid can provide a new strategy for the intervention of chronic diseases such as chronic inflammation, fibrosis, metabolic disorders, and tumors.

Description

Technical Field

[0001] This invention relates to the field of chitinase inhibitor technology, and more particularly to the application of chlorogenic acid as a chitinase inhibitor, and the application of chlorogenic acid in the prevention and control of chitinase-producing fungi and bacteria. Background Technology

[0002] Fungi are pathogens in humans, and fungal diseases are among the most destructive biological stresses in global agricultural production. For example, *Candida albicans* is an opportunistic pathogen in humans, causing thrush, vaginitis, and esophageal candidiasis; in severe cases, it can lead to invasive candidemia and disseminated visceral infections. *Botrytis cinerea* primarily causes gray mold disease in strawberries, grapes, and tomatoes; in a small number of immunocompromised individuals, it may cause rare allergic pneumonia or occupational pulmonary fungal disease.

[0003] Chitinases are the core enzyme system for fungal growth and development. Fungi synthesize their own chitinases to regulate reversible cell wall remodeling. This remodeling process mainly involves the localized hydrolysis of chitin at hyphal tips, septa, and infection sites, providing the necessary physical space and mechanical elasticity for hyphal extension, branching, spore germination, and hyphal morphology transformation. For example, in *Candida albicans*, hyphal formation is highly dependent on the localized enzymatic hydrolysis of the cell wall by endogenous chitinases; similarly, *Botrytis cinerea* requires chitinase-mediated cell wall plastic deformation to complete the development of infection pegs when penetrating plant epidermis. Therefore, endogenous chitinases in fungi are key regulatory nodes determining their pathogenicity. Inhibiting endogenous chitinases in fungi can effectively disrupt fungal pathogenicity. Chitinase inhibitors, as developmental inhibitors, play an important role in the control of harmful fungi and pests.

[0004] In the prior art, Chinese patent application CN108467395A discloses a chitinase inhibitor and its application, which found that the 1- and 3-position derivatives of 2-amino-pyridopiperidine exhibit certain inhibitory activity against chitinases in humans, Onchocerciasis, Nematodea elegans, Asian corn borer, Aspergillus fumigatus, and Serratia marcescens. However, developing more chitinase inhibitor products will not only be helpful for in-depth research on the structure and function of chitinases, but also provide new directions for the control of pathogenic fungi and pests. Summary of the Invention

[0005] To address the shortcomings of existing technologies, and more specifically, to provide an active substance that effectively inhibits endogenous chitinases in fungi or bacteria, this invention provides the application of chlorogenic acid as a chitinase inhibitor.

[0006] The specific technical solution of this invention is as follows:

[0007] In a first aspect, the present invention provides the application of chlorogenic acid in the preparation of chitinase inhibitors.

[0008] Furthermore, when the chlorogenic acid inhibits chitinase in Streptomyces griseus, the concentration of the chlorogenic acid in the reaction system is not less than 1.09 μg / μL.

[0009] Furthermore, when the chlorogenic acid inhibits chitinase in Verticillium dahliae, the concentration of the chlorogenic acid in the reaction system is not less than 0.75 μg / μL.

[0010] In a second aspect, the present invention provides the use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting chitinase.

[0011] Furthermore, pharmaceutically acceptable salts of chlorogenic acid include sodium chlorogenicate, potassium chlorogenicate, calcium chlorogenicate, magnesium chlorogenicate, and ammonium chlorogenicate.

[0012] Furthermore, the drug for inhibiting chitinase is used to prevent, alleviate, and / or treat diseases associated with inflammatory responses, tissue remodeling, or fibrosis resulting from elevated CHIT1 activity, elevated CHI3L1 expression, elevated chitinase or chitinase-like protein expression; said diseases are selected from any one or more of the following: infectious diseases, inflammatory or fibrotic diseases of the respiratory system, inflammatory or fibrotic diseases of the liver, inflammatory or neurodegenerative diseases of the neuron, autoimmune diseases, inflammatory bowel disease, metabolic inflammatory diseases, renal fibrosis-related diseases, cardiovascular inflammatory diseases, and tumors.

[0013] Furthermore, the drug for inhibiting chitinase is used to treat any of the following diseases:

[0014] Bacterial infections, fungal infections, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, sarcoidosis, alcoholic liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, chronic hepatitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autoimmune diseases, multiple sclerosis, rheumatoid arthritis, atopic dermatitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, diabetic nephropathy, chronic kidney disease, atherosclerosis, coronary artery disease, breast cancer, colorectal cancer, glioma, glioblastoma, gastric cancer, lung cancer, hepatocellular carcinoma, prostate cancer, cervical cancer, ovarian cancer, pancreatic cancer, melanoma, and solid tumors with high CHI3L1 expression.

[0015] Thirdly, this invention provides the application of chlorogenic acid in the control of agricultural pests.

[0016] Furthermore, the agricultural pests include arthropods containing chitinase, nematodes, bacteria, and fungi.

[0017] Compared with the prior art, the present invention has the following technical effects:

[0018] This invention is the first to discover that chlorogenic acid has the activity to inhibit chitinases and applies it to the inhibition of fungal or bacterial chitinases. It confirms that chlorogenic acid has a significant inhibitory effect on a variety of important chitinase-producing fungi and bacteria, including bacterial chitinases from *Bacillus thuringiensis*, *Streptomyces griseus*, fungal chitinases from *Aspergillus niger*, and fungal chitinases from *Verticillium dahliae*. Furthermore, it demonstrates that chlorogenic acid can interact with human chitinase CHIT1 and the chitinase-like protein CHI3L1, inhibiting their function. Therefore, chlorogenic acid can provide a new strategy for the intervention of chronic diseases such as chronic inflammation, fibrosis, metabolic disorders, and tumors by inhibiting CHIT1 enzyme activity and regulating CHI3L1-related pathological functions. Specifically, chlorogenic acid inhibits chitinases from *Streptomyces griseus* at an IC50 value. 50 The concentration was 1.095 μg / μL, and the IC50 for inhibiting chitinase from Streptomyces griseus was 0.754 μg / μL. Attached Figure Description

[0019] Figure 1 Scatter plot showing the molecular docking results of chlorogenic acid with chitinases from Bacillus thuringiensis, Aspergillus niger, barley seeds, and humans.

[0020] Figure 2 This is the substrate-binding pocket for chlorogenic acid to bind to chitinase 6IGY derived from Aspergillus niger.

[0021] Figure 3 This is the chemical structural formula of chlorogenic acid.

[0022] Figure 4 The results show the inhibitory effect of chlorogenic acid on chitinase activity derived from Streptomyces griseus.

[0023] Figure 5 The results show the inhibitory effect of chlorogenic acid on chitinase activity derived from Verticillium dahliae. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0025] In one embodiment, chlorogenic acid was discovered for the first time to inhibit chitinase activity, and its application in inhibiting chitinases of fungal or bacterial origin was demonstrated, providing the use of chlorogenic acid in the preparation of chitinase inhibitors. This embodiment found that chlorogenic acid has significant inhibitory effects on a variety of important chitinase-producing fungi and bacteria.

[0026] In one embodiment, chlorogenic acid was demonstrated to have a significant inhibitory effect on a variety of important chitinase-producing fungi and bacteria, including bacterial chitinases from Bacillus thuringiensis, bacterial chitinases from Streptomyces griseus, fungal chitinases from Aspergillus niger, and fungal chitinases from Verticillium dahliae. Experimental verification showed that chlorogenic acid inhibited chitinases from Streptomyces griseus by an IC50 value. 50 The concentration of chlorogenic acid in the reaction system is 1.095 μg / μL; the IC50 of chlorogenic acid inhibiting chitinase from *Streptomyces griseus* is 0.754 μg / μL. When inhibiting chitinase from *Streptomyces griseus*, the concentration of chlorogenic acid in the reaction system is not less than 1.09 μg / μL, which can inhibit the activity of 50% of chitinase from *Streptomyces griseus*. The concentration of chlorogenic acid in the reaction system is not less than 0.75 μg / μL, which can inhibit the activity of 50% of chitinase from *Verticillium dahliae*.

[0027] In one embodiment, chlorogenic acid was molecularly docked with chitinase from Bacillus thuringiensis (PDB ID: 6BT9), chitinase ChiB from Aspergillus niger (PDB ID: 6IGY), plant chitinase from barley seeds (PDB ID: 1CNS), and the catalytic domain of human acidic mammalian chitinase (PDB ID: 3FXY). The results showed that chlorogenic acid, specifically (1S,3R,4R,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]-1,4,5-trihydroxycyclohexane-1-carboxylic acid, exhibited a strong binding affinity for the aforementioned chitinases. Figure 1 As shown and Figure 2 visible. Figure 1In the diagram, the yellow dots represent Bisdionin F, a chitinase inhibitor; compound B42 represents chlorogenic acid; 6BT9 represents chitinase from Bacillus thuringiensis; 6IGY represents chitinase from Aspergillus niger; 3FXY represents human acidic mammalian chitinase; and 1CNS represents plant chitinase from barley seeds. Figure 1 It is evident that chlorogenic acid exhibits a significantly enhanced binding affinity for various chitinases compared to Bisdionin F, while the binding affinity for other compounds, such as B8, is relatively weak. Figure 2 The conformation of chlorogenic acid bound to the substrate-binding pocket of chitinase ChiB (PDB ID: 6IGY) derived from Aspergillus niger is shown.

[0028] In one embodiment, chlorogenic acid was experimentally verified to strongly bind to CHIT1 and CHI3L1 and inhibit their function. CHIT1 is a human-derived chitinase, namely chitotriosidase, and CHI3L1 is chitinase-3-like protein-1, which has a pocket structure for binding chitin.

[0029] Thus, the above embodiments provide the use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting chitinase.

[0030] Through the above examples illustrating the uses of chlorogenic acid, those skilled in the art will understand that, by inhibiting chitinase, chlorogenic acid can be used to prepare drugs for treating bacterial and fungal infections. The efficacy of these drugs primarily benefits from the inhibitory effect of chlorogenic acid on endogenous chitinases in bacteria and fungi. Through the above examples illustrating the uses of chlorogenic acid, those skilled in the art will also understand that chlorogenic acid can be used to prepare drugs for treating CHIT1 and CHI3L1 activation-related diseases. The efficacy of these drugs primarily benefits from the inhibitory effect of chlorogenic acid on human chitinases. For example, a review by Zhao et al. indicates that abnormally high expression of CHI3L1 is associated with 18 types of cancer and 26 other non-cancerous diseases. High levels of CHIT1 are associated not only with fibrosis but also with inflammation. High levels of CHI3L1 contribute to many inflammatory and immune diseases (such as pulmonary fibrosis, alcoholic liver fibrosis, multiple sclerosis, and Alzheimer's disease). For details, see the literature: Zhao, T., et al. "Chitinase-3 like-protein-1 function and its role in diseases." Signal Transduction and Targeted Therapy, vol. 5, 14 Sep. 2020, article no. 201.

[0031] It is evident that chlorogenic acid can inhibit chitinases, including those from fungi, bacteria, and various human sources, specifically including the inhibition of CHIT1 and CHI3L1. Those skilled in the art will understand that, due to the aforementioned inhibitory effects, chlorogenic acid can be used to prepare pharmaceuticals for the treatment of any of the following diseases:

[0032] Bacterial infections, fungal infections, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, sarcoidosis, alcoholic liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, chronic hepatitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autoimmune diseases, multiple sclerosis, rheumatoid arthritis, atopic dermatitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, diabetic nephropathy, chronic kidney disease, atherosclerosis, coronary artery disease, breast cancer, colorectal cancer, glioma, glioblastoma, gastric cancer, lung cancer, hepatocellular carcinoma, prostate cancer, cervical cancer, ovarian cancer, pancreatic cancer, melanoma, and solid tumors with high CHI3L1 expression.

[0033] Furthermore, those skilled in the art will recognize that pharmaceutically acceptable salts of chlorogenic acid should possess the chlorogenic acid uses demonstrated in the above examples. Pharmaceutically acceptable salts of chlorogenic acid include, for example, sodium chlorogenicate, potassium chlorogenicate, calcium chlorogenicate, magnesium chlorogenicate, and ammonium chlorogenicate.

[0034] In one embodiment, the application of chlorogenic acid in the control of agricultural pests is provided. Further, the agricultural pests include chitinase-containing arthropods, nematodes, bacteria, and fungi.

[0035] The following specific embodiments, using the inhibitory effects of bacterial (Streptomyces griseus) and fungal (Verticillium dahliae) chitinases as examples, demonstrate the inhibitory effect of chlorogenic acid on chitinase, thus illustrating the invention more clearly. The chemical structural formula of chlorogenic acid is as follows: Figure 3 As shown.

[0036] Example 1: Preparation and application of chlorogenic acid

[0037] Chlorogenic acid (CGA; Sangon Biotech (Shanghai) Co., Ltd., catalog number: A423287-0100) was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentrated stock solution, which was then sterilized by filtration through a 0.22 μm filter membrane. In all experimental treatments, the chlorogenic acid stock solution was added to the culture medium to achieve the specified final concentration. To eliminate any potential solvent-related artifacts, an equal volume of DMSO was added to the control group to ensure that the final concentration of DMSO in all treatment and control samples was consistent.

[0038] Subsequent embodiments will use this embodiment for the preparation and application of chlorogenic acid.

[0039] Example 2: Detection of the inhibitory activity of chlorogenic acid on chitinase from Streptomyces griseus

[0040] To verify the inhibitory effect of chlorogenic acid on chitinase activity, this example used a commercial chitinase derived from the pathogenic bacterium *Streptomyces griseus* as the test enzyme, and determined the enzyme activity using a chitinase kit (Aidison, Jiangsu, China, catalog number: ADS-W-TDX017-48, microplate method). The specific steps are as follows:

[0041] Chitinase (Maclean's, catalog number: C923201) derived from *Streptomyces griseus* was dissolved and diluted with phosphate-buffered saline (PBS) to obtain the test enzyme solution. The test enzyme solution was then mixed with chlorogenic acid solutions of different concentrations to achieve a final chlorogenic acid concentration ranging from 0 to 7 mmol / L. The reaction system with a final chlorogenic acid concentration of 0 mmol / L served as a control group without inhibitors. After mixing, the mixture was pre-incubated at 37°C for 2 hours to ensure sufficient contact between chlorogenic acid and chitinase, thereby maximizing its inhibitory effect on chitinase.

[0042] After pre-incubation, each group was treated with the same concentration of colloidal chitin as substrate, and the enzymatic reaction was carried out for 1 hour. After the reaction, following the chitinase kit instructions, potassium ferricyanide was added to initiate a colorimetric reaction. The mass of chitin hydrolyzed by the enzyme in each reaction system with different final concentrations of chlorogenic acid was measured to determine the relative activity of chitinase in each group. The enzyme activity of the inhibitor-free control group was taken as 100%. The chitinase activity results for each group with different final concentrations of chlorogenic acid are shown below. Figure 4 As shown.

[0043] Figure 4 The results of the assay show the inhibitory effects of different concentrations of chlorogenic acid on the activity of chitinases derived from *Streptomyces griseus*. Figure 4 It is evident that the chitinase activity decreases with increasing chlorogenic acid concentration; the IC50 of chlorogenic acid inhibiting chitinase from Streptomyces griseus is 0.754 μg / μL; when the chlorogenic acid concentration is above 2.480 μg / μL, the activity of chitinase from Streptomyces griseus is 100% inhibited.

[0044] Example 3: Detection of the inhibitory activity of chlorogenic acid on chitinases derived from Verticillium dahliae

[0045] To verify the inhibitory effect of chlorogenic acid on chitinase activity, this example used crude chitinase derived from the plant pathogen *Verticillium dahliae* as the test enzyme, and determined the enzyme activity using a chitinase kit (Aidison, Jiangsu, China, catalog number: ADS-W-TDX017-48, microplate method). The specific steps are as follows:

[0046] To extract endogenous chitinase from Verticillium dahliae, the fungus was cultured in CDB medium for 3 days. The cells were then collected by centrifugation at 8000 rpm, frozen in liquid nitrogen, and then lysed using a grinder. Chitinase was extracted using the extraction buffer provided with a commercial kit (Aidison, Jiangsu, China, catalog number: ADS-W-TDX017-48). The supernatant was collected after centrifugation and used as the enzyme to be tested.

[0047] The enzyme solution to be tested was mixed with chlorogenic acid solutions of different concentrations to achieve a final concentration of chlorogenic acid ranging from 0 to 7 mmol / L in the reaction system. The reaction system with a final chlorogenic acid concentration of 0 mmol / L served as a control group without inhibitors. After mixing, the mixture was pre-incubated at 37 °C for 2 hours to ensure sufficient contact between chlorogenic acid and chitinase, thereby maximizing its inhibitory effect on chitinase.

[0048] After pre-incubation, each group was treated with the same concentration of colloidal chitin as substrate, and the enzymatic reaction was carried out for 1.5 hours. After the reaction, following the chitinase kit instructions, potassium ferricyanide was added to initiate a colorimetric reaction. The mass of chitin hydrolyzed by the enzyme in each reaction system with different final concentrations of chlorogenic acid was measured to determine the relative activity of chitinase in each group. The enzyme activity of the inhibitor-free control group was taken as 100%. The chitinase activity results for each group with different final concentrations of chlorogenic acid are shown below. Figure 5 As shown.

[0049] Depend on Figure 5 The results of the assay show the inhibitory effects of different concentrations of chlorogenic acid on the activity of chitinases derived from Verticillium dahliae. Figure 5 It is evident that the chitinase activity decreases with increasing chlorogenic acid concentration. The IC50 of chlorogenic acid for chitinase activity from Verticillium dahliae is 1.095 μg / μL. Compared with chitinase from Streptomyces griseus, the inhibitory effect of chlorogenic acid on the former increases more significantly with increasing concentration. When the chlorogenic acid concentration reaches 2.480 μg / μL, the chitinase activity from Verticillium dahliae is completely inhibited.

[0050] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of chlorogenic acid in the preparation of chitinase inhibitors.

2. The application as described in claim 1, characterized in that: When the chlorogenic acid inhibits chitinase in Streptomyces griseus, the concentration of the chlorogenic acid in the reaction system is not less than 1.09 μg / μL.

3. The application as described in claim 1, characterized in that: When chlorogenic acid inhibits chitinase in Verticillium dahliae, the concentration of chlorogenic acid in the reaction system is not less than 0.75 μg / μL.

4. Use of chlorogenic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for inhibiting chitinase.

5. The application as described in claim 4, characterized in that: Pharmaceutically acceptable salts of chlorogenic acid include sodium chlorogenicate, potassium chlorogenicate, calcium chlorogenicate, magnesium chlorogenicate, and ammonium chlorogenicate.

6. The application as described in claim 4, characterized in that: The drug used to inhibit chitinase is used to treat any of the following diseases: Bacterial infections, fungal infections, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, sarcoidosis, alcoholic liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, chronic hepatitis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, autoimmune diseases, multiple sclerosis, rheumatoid arthritis, atopic dermatitis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, diabetes, diabetic nephropathy, chronic kidney disease, atherosclerosis, coronary artery disease, breast cancer, colorectal cancer, glioma, glioblastoma, gastric cancer, lung cancer, hepatocellular carcinoma, prostate cancer, cervical cancer, ovarian cancer, pancreatic cancer, melanoma, and solid tumors with high CHI3L1 expression.

7. Application of chlorogenic acid in the control of agricultural pests.

8. The application as described in claim 7, characterized in that: The agricultural pests include arthropods containing chitinase, nematodes, bacteria, and fungi.

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