CN110857291B - Natural compound for slowing rumen microorganism from decomposing amino acid and urea and application thereof - Google Patents

Abstract

The invention belongs to the technical field of animal feed additives, and particularly relates to a natural compound for reducing ammonia nitrogen generation amount and improving rumen nitrogen utilization rate by slowing down rumen microorganisms to decompose amino acid and urea. The invention mainly relates to an application of a compound shown in the following formula I and pharmaceutically acceptable salts thereof in preparing amino acid deaminase and urease inhibitors,

wherein R is₁、R₂、R₃、R₄、R₅Identical or different, independently of one another, from H or the following radicals: c_1‑12Alkyl radical, C_3‑20Cycloalkyl radical, C_6‑20Aryl, 5-20 membered heteroaryl.

Description

Natural compound for slowing rumen microorganism from decomposing amino acid and urea and application thereof

Technical Field

The invention belongs to the technical field of animal feed additives, and particularly relates to a natural compound for slowing rumen microorganisms from decomposing amino acid and urea and application thereof.

Background

Ruminant rumen microorganisms can degrade part of feed protein or other nitrogen-containing substances into ammonia nitrogen, and degrade the other part of feed protein into amino acids and polypeptides. The microorganism utilizes ammonia nitrogen, amino acid and polypeptide generated by decomposition to synthesize microbial protein again, and the microbial protein is utilized by animals. While the excess production of rumen ammonia nitrogen provides an inorganic nitrogen source for ruminant microorganisms, part of feed high-quality protein is degraded, so that feed protein is wasted, and the protein raw material is the part with the largest price proportion in the feed, so that the protein hydrolysis and the amino acid degradation in rumen show considerable economic loss in ruminant production. In addition, too fast decomposition of urea reduces its utilization rate, and too fast ammonia production is asynchronous with energy, reducing ammonia utilization. The generation of excessive ammonia nitrogen is easy to cause environmental pollution caused by high urinary nitrogen emission, and meanwhile, the excessive ammonia enters blood through the rumen wall, so that the ammonia stress of animal organisms and even ammonia poisoning are easily caused, and the animal health is harmed.

Compared with the traditional feed additive, the natural extract feed additive has the advantages of naturalness, multiple functions, sustainability and the like, has the characteristics of multiple components and multiple functions, and has a plurality of advantages and characteristics which are the core for replacing the traditional feed additive. The natural extract feed additive can better accord with the rule that the functions of animal organisms are mutually coordinated and integrally unified, the action is more comprehensive, and simultaneously, the multi-component synergistic effect ensures that the content of a certain specific component is lower, the toxicity (metabolic toxicity, teratogenicity, carcinogenicity and mutation capacity) of the feed additive is further weakened or eliminated after processing and processing, no obvious side effect is generated on animals, the drug resistance is not easy to generate, and the feed additive is suitable for long-term use. The active ingredients of the natural extract are natural products, most of the active ingredients are inherent ingredients of the feed, can be degraded by animals in the process of exerting the adjusting effect, have less residual quantity in animal products, do not bring harm to the ecological environment and have good safety. Natural, organic and residue-free are the development directions of future feed additives. The natural feed additive has wide market prospect as a green feed additive.

However, how to use natural compounds to solve the above technical problems is a difficult point and a focus of the current research.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a compound shown as the following formula I or a pharmaceutically acceptable salt thereof:

wherein R is₁、R₂、R₃、R₄、R₅Identical or different, independently of one another, from H or the following radicals: c_1-12Alkyl radical, C_3-20Cycloalkyl radical, C_6-20Aryl, 5-20 membered heteroaryl.

According to an embodiment of the invention, R in the compound of formula I₂、R₃、R₄、R₅Selected from H, R₁Is selected from C_1-3An alkyl group.

As an example, the compound of formula I is selected from biochanin a represented by the following formula:

the invention also provides a pharmaceutical composition which comprises the compound shown in the formula I and pharmaceutically acceptable auxiliary materials.

According to an embodiment of the invention, the pharmaceutical composition is an amino acid deaminase and/or urease inhibitor, e.g. a rumen microbial amino acid deaminase and/or urease inhibitor.

According to the invention, the mass fraction of the compound shown in the formula I in the pharmaceutical composition is 1-99%, preferably 10-90%, and further preferably 20-80%.

Optionally, the pharmaceutical composition may further comprise one or more other active ingredients.

According to the invention, the further active ingredient is selected from other rumen microorganisms amino acid deaminase and/or urease inhibitors, or compounds which enhance the inhibitory activity of the compounds of formula I, such as acetohydroxamic acid.

The invention also provides application of the compound shown in the formula I or pharmaceutically acceptable salt thereof in preparing a medicament.

Preferably, the medicament has an activity of inhibiting rumen microbial amino acid deaminase and/or urease.

Preferably, the medicament is for inhibiting the degradation rate of amino acids, for example for slowing the decomposition of amino acids by rumen microorganisms.

Preferably, the medicament is for inhibiting the production of ammonia nitrogen in the rumen.

Preferably, the medicament is for use in avoiding ruminal ammonia poisoning in ruminants.

Preferably, the medicament is for increasing the efficiency of rumen nitrogen utilization.

Preferably, the medicament is for promoting rumen fermentation.

According to the present invention, the inhibitory activity of the drug against urease, such as rumen microbial urease, was 340.82 μmol/L as the IC50 value.

According to the invention, the medicine can promote rumen fermentation, and the gas production is increased by 1.5-2.5%.

According to the invention, the medicine can inhibit the generation of ammonia nitrogen in rumen, and the inhibition rate is 21-28%.

According to the present invention, the drug can inhibit the degradation rate of amino acids (such as valine and/or lysine) by 12% to 18%.

The present invention also provides a method of inhibiting microbial amino acid deaminase and/or urease for non-therapeutic purposes, such as a method of inhibiting rumen microbial amino acid deaminase and/or urease activity, comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof to a ruminant animal.

The present invention also provides a method of inhibiting microbial amino acid deaminase and/or urease for non-therapeutic purposes, such as a method of inhibiting rumen microbial amino acid deaminase and/or urease activity, comprising administering the pharmaceutical composition to a ruminant.

Advantageous effects

The inventor of the application unexpectedly finds that the compound shown in the formula I can simultaneously slow down the decomposition speed of amino acid and urea by rumen microorganisms, thereby avoiding feed waste and rumen ammonia poisoning of ruminants and improving the utilization efficiency of rumen nitrogen.

Definition and description of terms

Unless defined otherwise below, all terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. If there are multiple definitions of terms herein, the following definitions apply.

The term "C_1-12Alkyl is understood to mean a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C_1-10An alkyl group. "C_1-10Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group or a 1, 2-dimethylbutyl group. In particular, the radicals have 1,2, 3, 4, 5, 6 carbon atoms ("C)_1-6Alkyl groups) such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly groups having 1,2 or 3 carbon atoms ("C)_1-3Alkyl groups) such as methyl, ethyl, n-propyl or isopropyl.

The term "C_3-20Cycloalkyl is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C_3-10Cycloalkyl groups ". The term "C_3-10Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Said C is_3-10Cycloalkyl groups may be monocyclic hydrocarbon groups, e.g. cyclopropyl, cyclobutylA cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl group, or a bicyclic hydrocarbon group such as a decalin ring.

The term "C_6-20Aryl "is understood to preferably mean a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6 to 20 carbon atoms, preferably" C_6-14Aryl ". The term "C_6-14Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C_6-14Aryl group "), in particular a ring having 6 carbon atoms (" C₆Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C₉Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C₁₀Aryl radicals), such as tetralinyl, dihydronaphthyl or naphthyl, or rings having 13 carbon atoms ("C₁₃Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C)₁₄Aryl), such as anthracenyl.

The term "5-20 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: having 5 to 20 ring atoms and comprising 1 to 5 heteroatoms independently selected from N, O, S, such as "5-14 membered heteroaryl".

The term "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: which has 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contains 1 to 5, preferably 1 to 3 heteroatoms selected independently of one another from N, O, S and, in addition, can be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or azocinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.

Unless otherwise indicated, heterocyclyl, heteroaryl or heteroarylene include all possible isomeric forms thereof, e.g., positional isomers thereof. Thus, for some illustrative, non-limiting examples, pyridyl or pyridinylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl, and pyridinylene-4-yl; thienyl or thienylene includes thien-2-yl, thien-3-yl and thien-3-yl.

The term "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

According to the invention, examples of said amino acids may be valine and/or lysine.

Drawings

FIG. 1 is a graph showing the effect of biochanin A on rumen gas production during passage.

FIG. 2 shows the effect of biochanin A on ammonia nitrogen yield during passage.

FIG. 3 is a graph of the effect of biochanin A on total amino acid degradation rate at different time points.

FIG. 4 is a graph of the effect of biochanin A on valine and lysine degradation rates at various time points.

Detailed Description

The compounds of the general formula and the preparation and use thereof according to the present invention will be described in further detail with reference to the following examples. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

1. Laboratory apparatus

Amino acid analyzer (Hitachi L-8900)

Enzyme mark instrument (Thermo Electron Varioskan Flash G-282)

Barometer (ConST 211)

2. Primary reagent

(1)50mmol/L urea buffer solution: 0.3g of urea was weighed, dissolved in deionized water and brought to a volume of 100 mL.

(2) Phenol-sodium nitroprusside solution: 10g of phenol and 50mg of sodium nitroprusside (also known as sodium nitroferricyanide) are weighed out, dissolved in deionized water and brought to a volume of 1L. Placing into brown bottle, and storing at 4 deg.C for one month.

(3) Alkaline sodium hypochlorite solution: 5g of NaOH and 8.4mL of NaClO solution were dissolved in deionized water and brought to 1L. Placing into brown bottle, and storing at 4 deg.C for one month.

(4)10mmol/L NH₄Standard solution of Cl: scale 0.5349NH₄And dissolving Cl in deionized water and fixing the volume to 1L.

(5)50mmol/L HEPES buffer solution (pH 7.5): 1.19g of sodium 4-hydroxyethyl piperazine sulfonate (HEPES) was weighed, dissolved in deionized water and made to volume of 100mL, and the pH was adjusted to 7.5 with NaOH.

Example 1 Effect of biochanin A on decomposition of amino acids and Urea by rumen microorganisms

The present embodiment adopts the following steps:

step one, collecting rumen fluid: under anaerobic conditions, fistula Holstein cow rumen fluid (550kg) before early feeding is collected, and the nutrient components for feeding the cow (17.3% of alfalfa hay, 18.7% of corn silage, 11.3% of soybean meal, 4.2% of rapeseed meal, 2.1% of cottonseed meal, 2.1% of puffed soybean, 4.2% of beet pulp, 10.4% of all-cotton seeds, 25.6% of corn, 0.5% of salt and 3.6% of premix) are collected. The rumen fluid is filtered by four layers of gauze to remove solid feed particles, then is filled into a bottle containing anaerobic preservation fluid, and is placed into an ice box to be rapidly taken back to a laboratory for preservation at-80 ℃.

Step two, preparation of a culture medium: anaerobic reaction according to Table 1 belowOxygen medium composition table, preparation of medium, CO₂After anaerobic aeration is carried out until the color is colorless, the pH value is adjusted to be 6.8, and the mixture is subpackaged into 10mL of Hantage anaerobic tubes in an anaerobic incubator. Sterilizing at 121 deg.C for 15 min.

TABLE 1 anaerobic culture Medium ingredient Table

Solution 1

Trace elements

Step three, preparing a chickpea essence A solution: dissolving in DMSO to prepare 30mg/mL chickpea extract A stock solution.

In an anaerobic incubator, 2mL of chickpea A solution (30mg/mL) is mixed with 9mL of anaerobic diluent to complete the preparation of a chickpea A working solution (6mg/mL), and the solution is placed for 1h to remove oxygen. At the time of inoculation, the working solution of biochanin A (6mg/mL) was aspirated by a syringe, filtered through a 0.22 μm filter, and injected into an anaerobic tube containing a culture medium.

Step four, enrichment culture: in an anaerobic incubator, 200. mu.l rumen fluid and 50. mu.l chickpea A solution (6mg/mL) were mixed and inoculated into anaerobic medium (10mL), while 50. mu.l DMSO (corresponding concentration) was set as a control group, each group was replicated in triplicate for a total of 6 tubes per generation. The anaerobic culture tube was placed in an incubator at 39 ℃ for 24 hours as the first generation. And (3) sucking 200 mu l of culture, mixing with 50 mu l of biochanin A or DMSO solution, inoculating to a new anaerobic culture medium, sequentially culturing, carrying out passage, and transferring to the fourth generation for 24 hours.

After 24h of incubation for one to four passages, the gas production was recorded while 5mL of sample was collected, centrifuged at 12000g for 10min at 4 ℃ and the supernatant was transferred to a new centrifuge tube (1.2mL of supernatant +0.12mL of 25% metaphosphoric acid (ammoniacal nitrogen); 1.5mL of supernatant was 2 (amino acids)). And (4) storing the supernatant at-20 ℃, and using the supernatant for ammonia nitrogen analysis.

After 24h of passage 4 culture, the cultures of both treatment groups were inoculated into a new flask as a fifth culture. The flasks contained 80mL of medium and were inoculated with 1.5mL of the culture of the fourth two treatment groups and 400. mu.l of 0, 6mg/mL biochanin A solution, respectively. Three replicates of each treatment were performed. After incubation for 0, 12, 16, 24h, respectively, 5mL samples were collected, centrifuged at 12000g at 4 ℃ for 10min, and the supernatant was transferred to a new centrifuge tube for amino acid composition analysis.

And step five, respectively testing the influence of the biochanin A on rumen gas production in passage, the influence of the biochanin A on ammonia nitrogen yield in passage, the influence of the biochanin A on total amino acid degradation rate at different time points and the influence of the biochanin A on valine and lysine degradation rate at different time points.

From fig. 1, it can be seen that when rumen microorganisms are cultured to 3 rd and 4 th generations, the biochanin a can significantly improve the gas production, and the gas production is increased by 1.5% -2.5% compared with the control group.

As can be seen from figure 2, when rumen microorganisms are cultured to the 2 nd, 3 rd and 4 th generations, the biochanin A can obviously reduce the generation amount of ammonia nitrogen, and the inhibition rate is 21% -28%.

From fig. 3, it can be seen that the rate of amino acid degradation increases gradually and slowly during the rumen microbial culture process, and therefore, the degradation rate of amino acids can be significantly reduced by the chickpea extract a, and the inhibition rate is 12% -18%. It can be seen from FIG. 4 that the degradation rates of valine and lysine were mainly reduced.

Example 2 Cicer arietin A inhibition of rumen microbial urease Activity assay

The method comprises the following steps: the rumen fluid was centrifuged (12000 Xg, 20min, 4 ℃), the pellet was washed twice with 50mmol/L HEPES buffer, the cells were resuspended in 1mL of 50mmol/L cold HEPES buffer, the rumen cells were sonicated on ice (40% intensity, three times 30S each time), centrifuged (12000 Xg, 10min, 4 ℃), the supernatant was collected as the enzyme solution, and the enzyme solution was taken to measure the protein concentration.

Step two: preparing a chickpea extract A solution, wherein the solvent is methanol, the final concentration is 20mg/mL, mixing the centrifuged supernatant with the chickpea extract A solution according to the following table 2,

TABLE 2

Step three: add 50mmol/L urea buffer 500. mu.l to make the final volume 1mL, and incubate at 37 ℃ for 20 min.

Step four: adding 1.5mL phenol-sodium nitroprusside solution and 1.5mL alkaline sodium hypochlorite solution, mixing well, incubating at 37 deg.C for 30min, and measuring absorbance at 625nm with spectrophotometer.

Step five: preparation of Standard working curves

(1)1mmol/L NH₄Cl standard working curve solution: adding 10mmol/L NH₄The Cl standard solution was diluted 10-fold with water.

(2) NH was diluted in gradient according to Table 3 below₄Cl standard working solution.

TABLE 3

Add 1.5mL phenol-sodium nitroprusside solution and 1.5mL alkaline sodium hypochlorite solution to each tube, mix well, incubate for 30min at 37 ℃, determine absorbance at 625nm with spectrophotometer, make standard working curve.

Step six: analysis of results

Definition of urease activity: nanomole of ammonia produced per minute per milligram of enzyme protein [ n mol/(min. mg) ]

Step seven: IC50 value calculation

According to different chickpea extract A concentration conditions, fitting a straight line on urease activity, and calculating the chickpea extract A concentration when the enzyme activity is 50% as an IC50 value by taking the chickpea extract A not added as 100%. The experimental result shows that the biochanin A can inhibit the urease activity through calculation, and the IC50 value of the biochanin A is 340.82 mu mol/L.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. Use of biochanin a or a pharmaceutically acceptable salt thereof for the manufacture of a medicament having inhibitory activity against rumen microbial amino acid deaminase and/or urease.

2. Use according to claim 1, wherein the medicament is for inhibiting the degradation rate of amino acids, or for inhibiting the production of ammonia nitrogen in the rumen, or for avoiding ruminal ammonia poisoning in ruminants, or for increasing the efficiency of ruminal nitrogen utilization, or for promoting ruminal fermentation.

3. The use according to claim 2, wherein the medicament is for inhibiting the degradation rate of valine and/or lysine.

4. The use of claim 3, wherein the medicament promotes rumen fermentation with a 1.5% -2.5% increase in gas production; or the medicament inhibits the generation of ammonia nitrogen in rumen with an inhibition rate of 21-28%; or the drug inhibits the degradation rate of amino acid, and the inhibition rate is 12% -18%.

5. A method of inhibiting rumen microbial amino acid deaminase and/or urease activity for non-disease treatment purposes comprising administering biochanin a or a pharmaceutically acceptable salt thereof to a ruminant.

Images