The use of β-glucuronidase in the preparation of an antidote for the prevention and / or treatment of zearalenone poisoning, and an antidote for the prevention and / or treatment of zearalenone poisoning.

Abstract

This invention provides the use of β-glucuronidase in the preparation of an antidote for the prevention and / or treatment of zearalenone poisoning, and an antidote for the prevention and / or treatment of zearalenone poisoning, belonging to the field of feed additives. This invention, for the first time from the perspective of in vivo exposure control in animals, proposes a method to reduce the overall ZEN exposure level by inhibiting the activity of intestinal bacterial β-glucuronidase. The technical path is clear and highly operable. This is achieved through in vivo toxicokinetic parameters (AUC, C...). max Changes in (and / or MRT) objectively reflect a reduction in ZEN exposure levels in vivo, and the technical effects are quantifiable and verifiable. The β-glucuronidase inhibitors used in this invention are widely available, particularly suitable for the field of feed additives, and have promising application prospects.

Description

Technical Field

[0001] This invention relates to the use of β-glucuronidase inhibitors in the preparation of antidotes for the prevention and / or treatment of zearalenone poisoning, and to an antidote for the prevention and / or treatment of zearalenone poisoning, belonging to the field of feed additives. Background Technology

[0002] ZEN (Zearalenone) is a common estrogen-like fungal toxin produced by Fusarium fungi, which widely contaminates feed ingredients such as corn and its by-products. After entering the animal body, ZEN can bind to estrogen receptors, inducing reproductive disorders, reproductive organ abnormalities, and decreased production performance, and has become one of the important risk factors restricting the healthy breeding of livestock and poultry and feed safety.

[0003] Current strategies for controlling ZEN contamination mainly include raw material detoxification, the addition of physical or chemical adsorbents, and biodegradation. Adsorbents primarily reduce ZEN absorption in the digestive tract through physical adsorption; however, their adsorption efficiency is easily affected by factors such as feed composition and pH conditions, and their effectiveness is limited for ZEN that has already been absorbed or metabolized. While biodegradation methods have shown some potential under in vitro conditions, their stability and efficacy in animals remain highly uncertain.

[0004] Existing literature reports that some compounds can inhibit β-glucuronidase activity under in vitro conditions. However, related studies mainly focus on their enzyme inhibitory properties or the structure-activity relationship between the compounds and β-glucuronidase, and have not yet conducted in vivo animal experiments to verify this. In other words, it is unclear whether these compounds can effectively inhibit β-glucuronidase activity derived from gut microbiota under actual intake conditions. Furthermore, some studies have indicated that inhibiting β-glucuronidase activity can significantly alleviate irinotecan-induced colitis, but the inhibitors used in these studies are mostly inorganic compounds, and their safety and long-term application potential still require further evaluation.

[0005] Therefore, there is an urgent need to provide a method that can effectively reduce ZEN exposure levels at the in vivo level, with a clear technical path and verifiable effects, to make up for the shortcomings of existing technologies. Summary of the Invention

[0006] The present invention relates to the use of β-glucuronidase inhibitors in the preparation of antidotes for the prevention and / or treatment of zearalenone poisoning, and to an antidote for the prevention and / or treatment of zearalenone poisoning.

[0007] This invention provides the use of β-glucuronidase inhibitors in the preparation of antidotes for the prevention and / or treatment of zearalenone poisoning.

[0008] The aforementioned antidote is used to reduce the level of zearalenone exposure in animals.

[0009] The antidote mentioned above reduces the absorption and accumulation of zearalenone in animals.

[0010] The antidote is used to alleviate the reproductive toxicity induced by zearalenone.

[0011] The β-glucuronidase inhibitors include one or a mixture of two or more of the following: epigallocatechin gallate (EGCG), gallatechin gallate (GCG), catechin gallate (CG), epicatechingallate (ECG), 3,4,5-tricaffeoylquinic acid (345), and delphinidin (DP).

[0012] Preferably, the β-glucuronidase inhibitor is EGCG, GCG, or 3,4,5-tricaffeoylquinic acid.

[0013] The present invention provides an antidote for the prevention and / or treatment of zearalenone poisoning, which comprises a β-glucuronidase inhibitor.

[0014] The β-glucuronidase inhibitors include one or a mixture of two or more of EGCG, GCG, CG, ECG, 3,4,5-tricaffeoylquinic acid, and DP.

[0015] Preferably, the β-glucuronidase inhibitor is EGCG, GCG, or 3,4,5-tricaffeoylquinic acid.

[0016] The present invention also provides the use of the aforementioned antidote in the preparation of feed additives or veterinary drugs for the prevention and / or treatment of zearalenone poisoning.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] This invention is the first to propose a method for reducing the overall ZEN exposure level by inhibiting the activity of β-glucuronidase in intestinal bacteria from the perspective of exposure control in animals. The technical path is clear and highly operable.

[0019] Based on in vivo toxicokinetic parameters (AUC, C) maxChanges in (and / or MRT) objectively reflect the reduction in ZEN exposure levels in vivo, and the technical effects are quantifiable and verifiable.

[0020] The β-glucuronidase inhibitor used in this invention is widely available and is particularly suitable for the field of feed additives, showing promising application prospects. Attached Figure Description

[0021] Figure 1 The effect of six natural plant active ingredient inhibitors on the EcGUS-mediated ZEN-GlcA hydrolysis reaction is shown in the figure; a: ZEN production, b: ZEN-GlcA retention.

[0022] Figure 2 The mean plasma concentration-time curves of ZEN in each group after oral gavage with ultrapure water / EGCG; the CK group was administered ultrapure water orally, and the EG group was administered EGCG orally.

[0023] Figure 3 Figure showing the effect of different concentrations of EGCG on β-glucuronidase in rat intestinal bacteria;

[0024] Figure 4 Figure showing the effect of EGCG intervention on ZEN-induced reproductive toxicity in rats;

[0025] Figure 5 The structure-activity relationship and key residue diagrams of EcGUS with substrates PNPG and EGCG based on molecular simulation are shown. a: PNPG, b: EGCG. Detailed Implementation

[0026] Example 1: Detection of the inhibitory effect of flavonoids on β-glucuronidase based on the ZEN-GlcA hydrolysis inhibition system

[0027] To preliminarily verify whether flavonoids EGCG, GCG, CG, ECG, 3,4,5-tricaffeoylquinic acid, and DP, which have inhibitory effects on β-glucuronidase, can act as effective inhibitors of β-glucuronidase to block ZEN-GlcA hydrolysis, a β-glucuronidase-mediated ZEN-GlcA hydrolysis inhibition assay was conducted. Prior to this assay, an in vitro two-phase incubation system was established using liver microsomes, and the optimal protein concentration, incubation time, and incubation termination conditions were screened. The ZEN-GlcA content was detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to screen for the optimal two-phase incubation system. The total reaction system (200 μL) consisted of: Tris-HCl buffer (109 μL, 100 M), liver microsomes (10 μL, 0.5 mg / mL), ZEN standard solution (1 μL, 1 μM), D-gluconic acid-1,4-lactone (DSL) (20 μL, 5 mM), promethazine (40 μL, 50 μg / mL), and uridine diphosphate glucuronide (20 μL, 5 mM). Subsequently, based on this, EcGUS was used as the enzyme source (EcGUS is a typical representative of β-glucuronidase, and due to its well-defined structure and function, it has been widely used in in vitro enzyme inhibition and kinetic studies), EGCG, GCG, CG, ECG, 3,4,5-tricaffeoylquinic acid, and DP were used as inhibitors, and D-gluconic acid-1,4-lactone was used as a positive control. 10% DMSO was used as a negative control, and ZEN-GlcA was used as the substrate. The amount of ZEN generated was detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Figure 1 a) and the retention of ZEN-GlcA ( Figure 1 (b) To evaluate the inhibitory activity against EcGUS. The total reaction system (100 μL) consisted of: phosphate buffer (70 μL, 0.1 M sodium diphosphate, pH 6.8), EcGUS solution (10 μL, 20 μg / mL EcGUS), EGCG (10 μL, 1 mM), and substrate (10 μL); the inhibitory activity against EcGUS was calculated as 1 - (amount of ZEN generated / amount of ZEN-GlcA retained).

[0028] As shown in the table, after two-phase incubation, most of ZEN was converted into ZEN-GlcA. Furthermore, in the subsequent EcGUS-mediated ZEN-GlcA hydrolysis inhibition assay, EGCG, GCG, and 3,4,5-tricaffeoylquinic acid all showed excellent inhibitory ability against EcGUS (>80%), and their inhibitory effect was comparable to that of the positive inhibitor DSL.

[0029] Table 1. Inhibitory effects of six natural active inhibitors on EcGUS-mediated ZEN-GlcA hydrolysis

[0030] Sample Name ZEN peak area ZEN-G peak area Inhibitory activity Two-phase incubation - P 779148 250266212 - P-EGCG 1522795 12020198 0.873314 P-GCG 2245589 13085995 0.828398 P-DSL 2282457 12900232 0.823069 P-345 2321770 11913183 0.805109 P-CG 6379976 9099426 0.29886 P-ECG 7141795 4715506 -0.51453 P-DP 12921036 2399538 -4.3848 P-10%DMSO 19187176 740454 -24.9127

[0031] Example 2: Effects of epigallocatechin gallate (EGCG) on ZEN exposure levels and enterohepatic circulation

[0032] Healthy female SD rats were selected as model animals to establish a ZEN exposure model. The experimental animals were randomly divided into a control group and an EGCG treatment group. The control group received ZEN exposure treatment, while the EGCG treatment group received epigallocatechin gallate under the same conditions, with an EGCG dose of 100 mg / kg body weight. Blood samples were collected at different time points after administration, and the concentrations of ZEN and its metabolites in plasma were determined using liquid chromatography-mass spectrometry (LC-MS). The area under the concentration-time curve (AUC) and peak plasma concentration (Cmax) were calculated. max The mean residence time (MRT) and mean residence time were also calculated. The toxicokinetic parameters of ZEN in each group were calculated using PhoenixWinnonlin 8.1 software, and the results are shown in the table. The mean plasma concentration-time curves of ZEN in each group after oral gavage with ultrapure water (CK group) / EGCG (EG group) are shown below. Figure 2 As shown.

[0033] The table shows that the half-life of ZEN in the CK group was as high as 18.87 ± 5.18 h, while early intervention with EGCG reduced it from 18.87 ± 5.18 h to 10.28 ± 9.01 h, significantly shortening the residual amount of ZEN in the body. After treatment with EGCG, C max Significant changes also occurred in the C group of EG. max The concentration of ZEN in the plasma was reduced by 2.5 times compared to the control group (CK). This demonstrates that EGCG can effectively reduce the concentration and duration of ZEN exposure in vivo. Comparing the AUC of the mean ZEN duration curves across the groups revealed that EGCG intervention reduced the AUC by 1.19 times compared to the CK group, significantly reducing in vivo ZEN exposure. Combining pharmacokinetic parameters and mean duration curves, the mean duration curve of ZEN in the plasma of the CK group after a single oral administration of ZEN exhibited a typical "bimodal" phenomenon, which is considered a sign of enterohepatic circulation. However, when EGCG was administered beforehand, the "bimodal" phenomenon caused by enterohepatic circulation was reduced.

[0034] Table 2. Toxicokinetic parameters of single-dose ZEN in different groups

[0035] Group CK group EGCG Group <![CDATA[λ z (1 / h)]]> 0.04±0.01 0.10±0.07 <![CDATA[t 1 / 2 (h)]]> 18.87±5.18 10.28±5.31 <![CDATA[T max (h)]]> 0.77±0.20 0.89±0.20 <![CDATA[C max (ng / L)]]> 234977.08±54545.36 93801.49±66173.09 <![CDATA[AUC 0-t (h*ng / L)]]> 764902.10±199215.08 643384.44±14501.84 <![CDATA[AUC 0-∞ (h*ng / L)]]> 1777123.93±533964.25 1129062.07±134958.89 Cl / F (L / h / kg) 29.77±8.22 44.70±5.18 <![CDATA[MRT 0-t (h)]]> 8.37±3.20 6.54±3.37 <![CDATA[MRT 0-∞ (h)]]> 25.85±11.24 14.01±9.67

[0036] EGCG is a multi-target, multi-mechanism compound. In addition to being a β-glucuronidase inhibitor, it also has inhibitory or activating effects on various other enzymes and signaling pathways. It has biological activities in many aspects, including anti-tumor, anti-fibrosis, neuroprotection, metabolic regulation, and cardiovascular protection. This experiment demonstrates that epigallocatechin gallate (EGCG) has a significant effect on the in vivo exposure level of ZEN and enterohepatic circulation, and can be used to prevent and / or treat zearalenone poisoning.

[0037] Example 3: Effects of different concentrations of EGCG on the activity of β-glucuronidase in rat intestinal bacteria

[0038] The above findings indicate that EGCG, as a highly effective inhibitor of EcGUS, can significantly inhibit the hydrolysis of the substrate ZEN-GlcA. To further verify the inhibitory effect of EGCG on intestinal bacterial β-glucuronidase and to screen for suitable feeding concentrations, in vivo experiments were conducted. The results are as follows: Figure 3 a, Figure 3 As shown in b, the CK-N group did not exhibit fluorescence because they did not receive oral fluorescein. In the CK group, after continuous gavage administration of ultrapure water for 7 days, the intestinal bacteria's β-glucuronidase hydrolyzed fluorescein, resulting in strong fluorescence. However, gavage administration of different concentrations of EGCG inhibited the intestinal bacteria's β-glucuronidase, significantly reducing fluorescence (P < 0.05). A moderate dose (100 mg / kg BW) of EGCG showed the best inhibitory effect. Furthermore, after continuous gavage administration of antibiotics for 7 days, the intestinal bacteria were eliminated, hence no fluorescence was observed in the intestines. Other results are as follows: Figure 3 As shown in c, the activity of β-glucuronidase in intestinal bacteria in different groups of intestinal contents was detected. It was also found that EGCG has a significant inhibitory effect on β-glucuronidase in intestinal bacteria, and 100 mg / kg BW EGCG is the most suitable addition dose.

[0039] Example 4: The alleviating effect of EGCG on ZEN-induced reproductive toxicity in rats

[0040] Based on the ZEN exposure model described above, the effects of EGCG on reproductive toxicity were further evaluated. Changes in reproductive toxicity were assessed by measuring the uterine index and related estrogen-like effect indices in the animals. The results showed that compared with the ZEN exposure group, the EGCG treatment group exhibited a significantly lower uterine index and a markedly weakened estrogen-like effect, indicating that reducing ZEN exposure levels in vivo can effectively alleviate its induced reproductive toxicity.

[0041] To verify that EGCG can improve ZEN-induced reproductive toxicity in rats, a rat efficacy study was conducted. Results are as follows: Figure 4 As shown in a and b, due to daily oral ZEN intake, the CK group showed significant differences compared to the NC group on days 7 and 11 (P < 0.05). ZEN intake also had a certain effect on rat body weight; the CK group showed almost no significant change in body weight throughout the experiment, while the NC and Ve groups, due to the absence of ZEN intervention, showed a gradual increase in body weight each day. In addition, the ovarian coefficient and uterine coefficient were statistically analyzed, and the results are as follows: Figure 4 As shown in Figures c and d, there was no significant difference in ovarian coefficient among the groups. The uterine coefficient in the CK group was significantly higher than that in the NC group (P < 0.05), indicating that the ZEN-induced estrogen effect model was successfully established. The EG and AB groups showed a significant decrease compared to the CK group (P < 0.05), indicating that intervention with EGCG and antibiotics can significantly improve ZEN-induced reproductive toxicity. In addition, the uterine horn width, uterine body width, and uterine length of rats in each group were measured, and the results are as follows: Figure 4 As shown in Figures e, f, and g, the uterine horn width and uterine width of the CK group rats changed significantly due to ZEN induction (P < 0.05), while the estrogen effect induced by ZEN was improved to some extent with the intervention of EGCG.

[0042] Example 5: Molecular docking of EGCG and EcGUS

[0043] To further investigate the structure-activity relationship between flavonoids and intestinal bacterial β-glucuronidase, molecular docking simulations were performed using EcGUS as the receptor and different flavonoids as ligands, employing Discovery Studio software. Figure 5 As shown in figure a, the substrate PNPG (blue) binds to residues such as GLU413, ASP163, and HIS330, which is equivalent to binding to the catalytic active site of EcGUS. EGCG ( Figure 5 b) It mainly binds to key amino acids such as GLU413, GLU504, ASP163, and LEU361 through interactions such as hydrogen bonds, Pi-anions, C-H bonds, Pi-alkyl bonds, and van der Waals forces, thereby binding to the catalytic site of EcGUS and exerting a competitive inhibitory effect.

Claims

1. Use of a β-glucuronidase inhibitor in the manufacture of a detoxifying agent for preventing or / and treating zearalenone intoxication. The β-glucuronidase inhibitor is EGCG, GCG or 3,4,5-tricaffeoylquinic acid.

2. Use according to claim 1, characterized in that: The detoxifying agent is used to reduce the level of zearalenone exposure in an animal.

3. Use according to claim 2, characterized in that: The detoxifying agent is used to reduce the absorption and accumulation of zearalenone in an animal.

4. Use according to claim 1 or 2, characterized in that: The detoxifying agent is used to alleviate zearalenone-induced reproductive toxicity.

5. Use according to any one of claims 1 to 3, characterized in that: The detoxifying agent is used in a feed additive or a veterinary drug.

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