Beta-glucuronidase inhibitor composition as well as preparation method and application thereof

Abstract

The invention discloses a beta-glucuronidase inhibitor composition as well as a preparation method and application thereof. The composition is prepared from the following substances: ursolic acid, procyanidine, icariin, curcumin, epigallocatechin gallate and glycyrrhetinic acid. The beta-glucuronidase inhibitor composition has a broad-spectrum and good inhibition effect, the preparation method is simple, large-scale production is easy, and the inhibitor with a natural source is higher in use compliance, lower in toxic and side effects and more suitable for developing intestinal health products and has an application prospect.

Description

Technical Field

[0001] The present invention relates to a beta-glucuronidase inhibitor composition, and also relates to a preparation method and application of the composition. Background Art

[0002] The human gut microbiota is complex and dynamic, coevolving and codeveloping with the host. The gut microbiota catalyzes a variety of metabolic reactions of structurally diverse endogenous / exogenous compounds, among which reduction and hydrolysis dominate. Glucuronidation is the major detoxification pathway in the liver, where UDP-glucuronosyltransferases catalyze the conjugation of hydrophobic xenobiotics and endogenous substances to glucuronic acid, thereby increasing their solubility. The resulting glucuronide enters the intestine, where enteric bacterial β-D-glucuronidase (bGUS) removes the glucuronic acid (GlcA), thereby releasing the original molecule into the intestinal lumen. Therefore, the activity of these enzymes affects the physiological activity and toxicity of various xenobiotics (e.g., irinotecan, nonsteroidal anti-inflammatory drugs, and heterocyclic aromatic amines) and endogenous substances.

[0003] Metabolism mediated by intestinal bacterial β-GUS is the cause of delayed diarrhea in patients with advanced colorectal cancer treated with irinotecan, as well as the cause of enteropathy caused by long-term use of nonsteroidal anti-inflammatory drugs (such as diclofenac, ketoprofen, indomethacin, etc.). In addition, the inactivation of certain foodborne carcinogens, such as heterocyclic aromatic amines, by host UDP-glucuronyl transferase may also be eliminated by intestinal bacterial β-GUS, leading to an increased risk of cancer, especially colon cancer. Studies have also shown that intestinal bacterial β-GUS activity in certain disease states (such as colon cancer) is much higher than in healthy states. Increased intestinal bacterial β-GUS activity increases exposure to toxic compounds in the intestine, leading to adverse effects and serious side effects. Therefore, effective β-GUS inhibitors can regulate intestinal bacterial β-GUS activity, thereby reducing the intestinal toxicity of certain xenobiotics or preventing colon-related diseases.

[0004] However, even enteric bacterial β-GUS expressed by the same strain may differ in structure and function. For example, three GUS proteins (Bu GUS3 to Bu GUS14) were found in the same polysaccharide utilization site of the human intestinal commensal microorganism Bacteroides uniformis. They have similar TIM barrel core fold characteristics, but show different sequence compositions and tertiary and quaternary structures, and have unique structural features within their active sites, resulting in their differential substrate processing capabilities and different inhibition tendencies. The genetic diversity, functional promiscuity and different inhibition tendencies of GUS have brought great challenges to the development of specific or broad-spectrum GUS inhibitors. Natural compounds in medicinal plants, such as quercetin, eugenol and rhamnolide, have EcoGUS inhibitory activity, but the amino acid identity between most intestinal bacterial β-GUS and Escherichia coli GUS is <25%. EcoGUS inhibitors are not sufficient to block the activity of intestinal bacterial β-GUS and have limited effect on alleviating drug-induced intestinal toxicity. Chemical inhibitors or antibiotics have strong inhibitory effects to a certain extent, but they will indiscriminately eliminate a large number of intestinal commensal bacteria, cause a large number of pathogenic species to proliferate, and produce negative effects or toxicity. It is urgent to develop more efficient methods to inhibit β-glucuronidase. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a β-glucuronidase inhibitor composition, and also to provide a method for preparing the composition and its use in intestinal enzyme inhibitors or intestinal injury therapeutic drugs.

[0006] Technical solution: The present invention discloses a β-glucuronidase inhibitor composition, which is composed of the following substances in parts by mass: 1 to 10 parts of ursolic acid, 1 to 10 parts of proanthocyanidins, 1 to 10 parts of icariin, 1 to 10 parts of curcumin, 1 to 10 parts of epigallocatechin gallate, and 1 to 10 parts of glycyrrhetinic acid.

[0007] The composition is composed of the following substances in parts by mass: 1 to 5 parts of ursolic acid, 1 to 5 parts of proanthocyanidins, 1 to 5 parts of icariin, 1 to 5 parts of curcumin, 1 to 5 parts of epigallocatechin gallate, and 1 to 5 parts of glycyrrhetinic acid.

[0008] The composition is composed of the following substances in parts by mass: 5 parts of ursolic acid, 5 parts of proanthocyanidins, 1 part of icariin, 1 part of curcumin, 5 parts of epigallocatechin gallate, and 1 part of glycyrrhetinic acid.

[0009] The preparation method of the β-glucuronidase inhibitor composition is as follows: ursolic acid, proanthocyanidins, icariin, curcumin, epigallocatechin gallate and glycyrrhetinic acid are prepared into solutions with dimethyl sulfoxide respectively, and the diluted solutions are mixed to obtain the composition.

[0010] Wherein, the configuration environment is room temperature, and the mixing is vortexing for 3 to 5 minutes.

[0011] Specifically, at room temperature, 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water; 4.7 mg of proanthocyanidin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water; 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water. M; 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water; 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water; 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL was taken from the 10 mM solution and diluted to 1 mM with water;

[0012] Take 100 μL of each of the prepared ursolic acid solution, proanthocyanidin solution, epigallocatechin gallate solution, icariin solution, curcumin solution, and glycyrrhetinic acid solution, mix the six solutions, and vortex for 5 minutes to obtain a solution of formula 1;

[0013] Take 500 μL of the prepared ursolic acid solution, and take 100 μL of each of the prepared proanthocyanidin solution, epigallocatechin gallate solution, icariin solution, curcumin solution, and glycyrrhetinic acid solution. Mix these six solutions and vortex for 5 minutes to obtain a solution of formula 2.

[0014] Take 500 μL of the prepared proanthocyanidin solution, and take 100 μL of each of the prepared ursolic acid solution, epigallocatechin gallate solution, icariin solution, curcumin solution, and glycyrrhetinic acid solution. Mix these six solutions and vortex for 5 minutes to obtain the solution of formula 3.

[0015] Take 500 μL of the prepared epigallocatechin gallate solution, and take 100 μL of each of the prepared ursolic acid solution, proanthocyanidin solution, icariin solution, curcumin solution, and glycyrrhetinic acid solution. Mix the six solutions and vortex for 5 minutes to obtain a solution of formula 4.

[0016] Take 500 μL of the prepared curcumin solution, and take 100 μL of each of the prepared ursolic acid solution, proanthocyanidin solution, epigallocatechin gallate solution, icariin solution, and glycyrrhetinic acid solution. Mix these six solutions and vortex for 5 minutes to obtain a solution of formula 5.

[0017] Take 500 μL of the prepared icariin solution, and take 100 μL of each of the prepared ursolic acid solution, proanthocyanidin solution, epigallocatechin gallate solution, curcumin solution, and glycyrrhetinic acid solution. Mix the six solutions and vortex for 5 minutes to obtain a solution of formula 6.

[0018] Take 500 μL of the prepared glycyrrhetinic acid solution, and take 100 μL of each of the prepared ursolic acid solution, proanthocyanidin solution, epigallocatechin gallate solution, icariin solution, and curcumin solution. Mix the six solutions and vortex for 5 minutes to obtain the solution of formula 7.

[0019] Take 500 μL each of the prepared ursolic acid solution, proanthocyanidin solution, and epigallocatechin gallate solution, and take 100 μL each of the prepared icariin solution, curcumin solution, and glycyrrhetinic acid solution, mix these six solutions, and vortex for 5 minutes to obtain the solution of formula 8.

[0020] The above-mentioned β-glucuronidase inhibitor composition can also be used in the preparation of intestinal enzyme inhibitors.

[0021] Wherein, the secretion source of the enteric enzyme is Escherichia coli, Streptococcus agalactiae, Staphylococcus pasteurianus, Clostridium perfringens or Ruminococcus.

[0022] The above-mentioned β-glucuronidase inhibitor composition can also be used in the preparation of drugs for treating intestinal damage.

[0023] Principle of the invention: The β-glucuronidase inhibitor composition of the present invention comprises polyphenolic compounds, diketone compounds, glycoside compounds, and terpenoid compounds, involving a wide range of natural compounds. After combination, the composition brings about a synergistic effect, wherein each compound focuses on inhibiting different GUS enzymes, and through combination, it makes up for the broad-spectrum and efficient inhibition effect that a single compound cannot achieve. Ursolic acid can increase the number of beneficial bacteria in the intestine and has excellent antioxidant and anti-inflammatory properties. Proanthocyanidins can effectively improve ulcerative colitis by regulating intestinal flora and restore intestinal Th17 / Treg homeostasis in a microbiota-dependent manner. Icariin regulates the redox balance of intestinal epithelial cells by inhibiting the expression of p38 MAPK, thereby improving the destruction of intestinal barrier function. Curcumin can reduce the development of colorectal cancer by regulating intestinal flora homeostasis and intestinal metabolites. Epigallocatechin gallate can stimulate the growth of beneficial intestinal bacteria and inhibit pathogenic bacteria. Glycyrrhetinic acid may regulate blood sugar by inhibiting α-glucosidase and regulating intestinal microbial short-chain fatty acids. In summary, the composition composed of the above-mentioned natural products shows beneficial effects on the intestine. In addition to acting as a GUS enzyme inhibitor in the intestine, it can also regulate intestinal flora, improve inflammation, and regulate the intestinal barrier.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The β-glucuronidase inhibitor composition of the present invention has a broader spectrum and better inhibitory effect. The inhibitory activity of the composition against EcoGUS is 3 times that of the control group, the inhibitory activity against CpGUS is 4.6 times that of the control group, the inhibitory activity against SagaGUS is 3.9 times that of the control group, the inhibitory activity against SpasGUS is 5.4 times that of the control group, and the inhibitory activity against RgGUS is 1.2 times that of the control group; (2) The preparation method is simple, and only requires simple preparation and quantitative mixing to obtain the composition, without the need for extraction and purification; (3) Inhibitors of natural origin have higher compliance in use and lower toxic and side effects, and are more suitable for the development of intestinal health products, and have application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The inhibition rates of five GUS enzymes in Examples 1 to 8 of the present invention;

[0026] Figure 2 is the half inhibition rate of the control group (A) and the composition of Example 1 of the present invention (B) on Escherichia coli EcoGUS;

[0027] Figure 3 is the half inhibition rate of the control group (A) and the composition of Example 1 of the present invention (B) against CpGUS of Clostridium perfringens;

[0028] Figure 4is the half inhibition rate of the control group (A) and the composition (B) of Example 1 of the present invention on Streptococcus agalactiae SagaGUS;

[0029] Figure 5 is the half inhibition rate of the control group (A) and the composition (B) of Example 1 of the present invention on Staphylococcus pasteurianus SpasGUS;

[0030] Figure 6 It is the half inhibition rate of the control group (A) and the composition of Example 1 of the present invention (B) on Ruminococcus RgGUS. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0032] Example 1

[0033] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 1 part of proanthocyanidins, 1 part of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0034] The preparation method is:

[0035] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0036] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0037] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0038] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0039] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0040] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0041] (7) Take 100 μL of each of the solutions in steps (1), (2), (3), (4), (5), and (6) above and mix them to obtain a mixed solution of formula 1.

[0042] Example 2

[0043] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 5 parts of ursolic acid, 1 part of proanthocyanidins, 1 part of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0044] The preparation method is:

[0045] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0046] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0047] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0048] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0049] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0050] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0051] (7) Take 500 μL of the solution in step (1) and 100 μL of each of the solutions in steps (2), (3), (4), (5), and (6) and mix them to obtain a mixed solution of formula 2.

[0052] Example 3

[0053] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 5 parts of proanthocyanidins, 1 part of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0054] The preparation method is:

[0055] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0056] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0057] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0058] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0059] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0060] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0061] (7) Take 500 μL of the solution in step (2) and 100 μL of each of the solutions in steps (1), (3), (4), (5), and (6) and mix them to obtain a mixed solution of formula 3.

[0062] Example 4

[0063] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 1 part of proanthocyanidins, 5 parts of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0064] The preparation method is:

[0065] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0066] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0067] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0068] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0069] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0070] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0071] (7) Take 500 μL of the solution in step (3) and 100 μL of each of the solutions in steps (1), (2), (4), (5), and (6) and mix them to obtain a mixed solution of formula 4.

[0072] Example 5

[0073] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 1 part of proanthocyanidins, 1 part of epigallocatechin gallate, 5 parts of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0074] The preparation method is:

[0075] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0076] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0077] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0078] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0079] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0080] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0081] (7) Take 500 μL of the solution in step (4) and 100 μL of each of the solutions in steps (1), (2), (3), (5), and (6) and mix them to obtain a mixed solution of formula 5.

[0082] Example 6

[0083] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 1 part of proanthocyanidins, 1 part of epigallocatechin gallate, 1 part of curcumin, 5 parts of icariin and 1 part of glycyrrhetinic acid.

[0084] The preparation method is:

[0085] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0086] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0087] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0088] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0089] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0090] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0091] (7) Take 500 μL of the solution in step (5) and 100 μL of each of the solutions in steps (1), (2), (3), (4), and (6) and mix them to obtain a mixed solution of formula 6.

[0092] Example 7

[0093] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 1 part of ursolic acid, 1 part of proanthocyanidins, 1 part of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 5 parts of glycyrrhetinic acid.

[0094] The preparation method is:

[0095] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0096] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0097] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0098] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0099] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0100] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0101] (7) Take 500 μL of the solution in step (6) and 100 μL of the solutions in steps (1), (2), (3), (4), and (5) and mix them to obtain a mixed solution of formula 7.

[0102] Example 8

[0103] The β-glucuronidase inhibitor composition of the present invention is composed of the following substances in parts by mass: 5 parts of ursolic acid, 5 parts of proanthocyanidins, 5 parts of epigallocatechin gallate, 1 part of curcumin, 1 part of icariin, and 1 part of glycyrrhetinic acid.

[0104] The preparation method is:

[0105] (1) 6.8 mg of ursolic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then 200 μL of the 10 mM solution was diluted with water to 1 mM.

[0106] (2) 4.6 mg of epigallocatechin gallate was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0107] (3) Take 4.7 mg of proanthocyanidins and add 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. Then take 200 μL of the 10 mM solution and dilute it to 1 mM with water;

[0108] (4) 3.7 mg of curcumin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0109] (5) 6.8 mg of icariin was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution, and then 200 μL of the 10 mM solution was diluted with water to 1 mM;

[0110] (6) 4.7 mg of glycyrrhetinic acid was added to 1 mL of dimethyl sulfoxide (DMSO) solution to prepare a 10 mM solution. 200 μL of the 10 mM solution was then diluted with water to 1 mM.

[0111] (7) Take 500 μL of each of the solutions in steps (1), (2), and (3) and 100 μL of each of the solutions in steps (4), (5), and (6) and mix them to obtain a mixed solution of formula 8.

[0112] The screening method of the β-glucuronidase inhibitor composition of the present invention specifically comprises the following steps:

[0113] Verify the inhibitory effect of single compound inhibitors on GUSs:

[0114] The following method was used to detect the inhibitory effects of 30 single compounds on five enteric enzymes: Escherichia coli EcoGUS, Streptococcus agalactiae SagaGUS or Staphylococcus pasteurianus SpasGUS, Clostridium perfringens CpGUS, and Ruminococcus aureus RgGUS. The specific results are shown in Table 1.

[0115] (1) taking a plant-derived monomer compound and preparing a solution with a concentration of 1000 μM;

[0116] (2) Prepare the reaction system, which consists of 10 μL PNPG (final concentration 300 μM), 10 μL GUS, 10 μL test compound (final concentration 100 μM) and 0.1 M phosphate buffer (pH 7.4), with a total volume of 100 μL;

[0117] (3) After pre-incubation at 37°C for 5 minutes, PNPG was added to initiate the reaction, which continued for 15 minutes in the absence or presence of test compounds.

[0118] (4) After the reaction was completed, 10% Na2CO3 was used to terminate the reaction, and the reaction product p-nitrophenol (PNP) was detected at 405 nm using a Thermo Scientific Multiskan GO full wavelength reader / ultra-micro spectrophotometer; the residual activity was calculated using the following formula (1):

[0119] Inhibition%=[(Ac-Ac0)-(Ae-Ae0)] / (Ac-Ac0) (1)

[0120] Wherein, Ae: the reaction active group in the presence of the test compound; Ae0: the reaction inactive group in the presence of the test compound; Ac: the reaction active group in the absence of the test compound; Ac0: the reaction inactive group in the absence of the test compound.

[0121] (5) Further combine the effective inhibitors obtained by screening, increase the proportion of each component, and screen according to steps (2), (3), and (4). Finally, select the top three formulations of each GUS inhibitory composition according to the inhibition rate, and then summarize and select the three compounds with the highest frequency in the top three inhibitory formulations of the five GUS enzymes, increase their proportions, and obtain the final composition formulation.

[0122] Verification of the inhibitory effect of the highly effective β-glucuronidase inhibitor composition of the present invention on the activity of the intestinal enzyme GUSs:

[0123] The detection steps for the intestinal enzyme GUSs are as follows: prepare a reaction system consisting of 10 μL PNPG (final concentration of 300 μM), 10 μL GUS, 10 μL of solution of formula 8 / different concentration dilutions (final concentrations of 100 μM, 50 μM, 20 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.01 μM, 0.001 μM) and 0.1 M phosphate buffer (pH 7.4), with a total volume of 100 μL. After pre-incubation at 37°C for 5 minutes, PNPG was added to start the reaction, which lasted for 15 minutes. After the reaction was completed, 10% Na2CO3 was used to terminate the reaction, followed by high-speed centrifugation at 10,000 rpm for 10 minutes, and absorbance was detected using an enzyme reader at a wavelength of 405 nm. The relative activity was calculated using the following formula (1):

[0124] Relativeactivity%=(Ae-Ae0) / (Ac-Ac0) (1)

[0125] Wherein, Ae: the reaction active group in the presence of the test compound; Ae0: the reaction inactive group in the presence of the test compound; Ac: the reaction active group in the absence of the test compound; Ac0: the reaction inactive group in the absence of the test compound.

[0126] The control group was: p-nitrophenyl-β-D-pyranoglucopyranoside, a commercially available GUS enzyme inhibitor. Figures 2 to 6 As shown, the IC values ​​of the combined compounds for five enteric enzymes: Escherichia coli EcoGUS, Streptococcus agalactiae SagaGUS or Staphylococcus pasteurianus SpasGUS, Clostridium perfringens CpGUS, Ruminococcus pyogenes RgGUS 50 (half-maximal inhibitory concentration) were lower than that of the control group, that is, only a lower compound concentration was needed to achieve a half-maximal inhibitory effect. The inhibitory activity of the composition on EcoGUS was 3 times that of the control group, the inhibitory activity on CpGUS was 4.6 times that of the control group, the inhibitory activity on SagaGUS was 3.9 times that of the control group, the inhibitory activity on SpasGUS was 5.4 times that of the control group, and the inhibitory activity on RgGUS was 1.2 times that of the control group, all of which indicated that the composition had a broader-spectrum and more efficient effect on GUS.

[0127] Table 1. GUSs inhibition effects of monomeric compounds derived from medicinal materials with the same medicinal and edible properties

[0128]

[0129]

[0130] It can be seen from Table 1 that the inhibition rates of the five GUS enzymes by the thirty natural compounds are significantly different, but the inhibition effect of a single natural compound is limited; and the β-glucuronidase inhibitor composition of the present invention, comprising polyphenolic compounds, diketone compounds, glycoside compounds, and terpenoid compounds, greatly improves the inhibition rate of the five common GUS enzymes in the intestine, achieving a synergistic effect, and after adjusting the proportion, it is found that the inhibition rate of the composition at a concentration of 100 μM for the five GUS enzymes reaches 90%, making up for the effect of broad-spectrum and high-efficiency inhibition that a single compound cannot achieve, and only a lower compound concentration is required to achieve a half inhibition effect. At the same time, as a compound of natural origin, the composition of the present invention, in addition to its role as a GUS enzyme inhibitor, its own biological activity also promotes intestinal health; in addition to playing the role of a GUS enzyme inhibitor in the intestine, it can also regulate intestinal flora, improve inflammation, and regulate intestinal barriers.

Claims

1. A β-glucuronidase inhibitor composition, characterized in that: The composition is composed of the following substances in parts by mass: 1-10 parts of ursolic acid, 1-10 parts of proanthocyanidins, 1-10 parts of icariin, 1-10 parts of curcumin, 1-10 parts of epigallocatechin gallate, and 1-10 parts of glycyrrhetinic acid.

2. The composition according to claim 1, characterized in that The composition is composed of the following substances in parts by mass: 1 to 5 parts of ursolic acid, 1 to 5 parts of proanthocyanidins, 1 to 5 parts of icariin, 1 to 5 parts of curcumin, 1 to 5 parts of epigallocatechin gallate, and 1 to 5 parts of glycyrrhetinic acid.

3. The composition according to claim 1, characterized in that The composition is composed of the following substances in parts by mass: 5 parts of ursolic acid, 5 parts of proanthocyanidins, 1 part of icariin, 1 part of curcumin, 5 parts of epigallocatechin gallate, and 1 part of glycyrrhetinic acid.

4. A method for preparing the β-glucuronidase inhibitor composition according to claim 1, characterized in that: The preparation method comprises the following steps: taking ursolic acid, proanthocyanidin, icariin, curcumin, epigallocatechin gallate and glycyrrhetinic acid, respectively preparing solutions with dimethyl sulfoxide, and mixing the diluted solutions to obtain the product.

5. The preparation method according to claim 4, characterized in that: The preparation environment is room temperature, and the mixing is vortexed for 3 to 5 minutes.

6. Use of the β-glucuronidase inhibitor composition according to claim 1 in the preparation of an intestinal enzyme inhibitor.

7. The use according to claim 6, characterized in that: The secretion source of the enteric enzyme is Escherichia coli, Streptococcus agalactiae, Staphylococcus pasteurianus, Clostridium perfringens or Ruminococcus.

8. Use of the β-glucuronidase inhibitor composition according to claim 1 in the preparation of a medicament for treating intestinal damage.

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