Use of arecoline alkaloids for the preparation of urease inhibitors

Abstract

The application belongs to the technical field of plant alkaloid application, and particularly relates to application of areca alkaloid in preparation of urease inhibitor. The application discloses application of areca alkaloid in preparation of urease inhibitor, wherein the areca alkaloid comprises at least one component: (1) norarecoline; (2) norarecolin; (3) arecoline; (4) arecolin. The areca alkaloid disclosed by the application has the functions of inhibiting urease activity, reducing urea decomposition rate and regulating rumen nitrogen metabolism of ruminants; when norarecoline: arecolin: norarecolin: arecoline = 2:3:2:6, the inhibiting effect on rumen urease is better, and is equivalent to the effect of norarecoline and norarecolin; the stability and tolerance of areca total alkaloid in the rumen are better than those of positive drug acetyloxime acid.

Description

Technical Field

[0001] The invention belongs to the technical field of plant alkaloid application, and particularly relates to the application of areca alkaloids in the preparation of urease inhibitors. Background Art

[0002] Ruminants can use urea as a protein feed to replace part of the soybean meal in their diet. Urea is broken down into ammonia by microbial urease in the rumen, which is then used to synthesize microbial protein. However, due to the excessive catalytic effect of urease, the rate of urea decomposition is four times faster than the rate at which microorganisms can utilize it. This produces large amounts of ammonia that enter the bloodstream, which can easily cause ammonia poisoning and nitrogen loss in ruminants, impacting animal health and ecological safety, and limiting the large-scale use of urea in ruminant production. Therefore, developing urease inhibitors to improve urea utilization is of great significance for alleviating the shortage of protein feed resources in my country, reducing breeding costs, and protecting the ecological environment.

[0003] Existing slow-release urea products and technologies, such as coated urea, urea lick blocks, starch-gelatinized urea, and chemically synthesized urea derivatives, require significant investment in equipment and capital. These high costs and complex processes have limited their widespread adoption. Urease inhibitors, on the other hand, can be added directly to feed at a low cost, making them the most convenient and direct method for urea utilization.

[0004] Most urease inhibitors currently available on the market are chemical-based, such as acetohydroxamic acid (AHA) and hydroquinone. AHA is a traditional urease inhibitor widely used in the livestock industry due to its potent inhibitory effect on ruminal microbial urease. It was first used in the 1990s in the UK and the US for ruminant nutrition in cattle, sheep, and other ruminants to improve the rumen ammonia environment, increase protein conversion, increase roughage intake, reduce blood ammonia concentrations, alleviate ammonia stress, and enhance immunity. However, it has poor stability in the rumen and poses health risks to animals. Rumen microbes quickly adapt to this compound, and after one week of feeding, rumen ammonia levels return to pre-experimental levels. Hydroquinone is more effective than AHA as a urease inhibitor, but its price is eight times that of AHA, making it unsuitable for commercial use. Therefore, research into identifying safe, effective, and non-polluting urease inhibitors from natural sources has attracted considerable attention.

[0005] Areca nut is the dried, mature fruit of the palm plant Areca catechu Linnaeus. It primarily contains alkaloids, flavonoids, condensed tannins, steroids, and fatty acids. Alkaloids are important bioactive substances in betel nut, with properties such as promoting gastrointestinal digestion, anthelmintic, antibacterial, and antidepressant effects. These alkaloids primarily include arecoline, arecoline, norarecoline, and norarecoline. Patents CN201610924728 and CN2110923557.2 mention the use of epiberberine and sanguinarine in the preparation of urease inhibitors. However, prior art does not mention the use of areca alkaloids as urease inhibitors. This patent discusses the composition of the total areca alkaloids, which are arecoline, arecoline, norarecoline, and norarecoline. The inhibitory effects and nitrogen utilization efficiency of different areca alkaloid ratios will be explored. Summary of the Invention

[0006] In order to solve the deficiencies in the prior art, the present invention provides the following technical solutions:

[0007] Use of betel nut alkaloids in the preparation of urease inhibitors, wherein the betel nut alkaloids comprise at least one of the following components:

[0008] (1) Norarecoline;

[0009] (2) norarecoline;

[0010] (3) Arecoline;

[0011] (4) Arecaline.

[0012] Preferably, the areca alkaloid comprises norarecoline; preferably, the concentration of norarecoline is greater than or equal to 28.3 ug / ml.

[0013] Preferably, the areca alkaloid comprises norarecoline; preferably, the concentration of the norarecoline is greater than or equal to 26.58 ug / ml.

[0014] Preferably, the areca alkaloid comprises arecoline; preferably, the concentration of arecoline is greater than or equal to 77.34 ug / ml.

[0015] Preferably, the betel nut alkaloid comprises arecoline; and the concentration of the arecoline is greater than or equal to 55.75 ug / ml.

[0016] Preferably, the total alkaloids of areca catenin include demethylarecoline, arecoline, norarecoline and arecoline. More preferably, the mass ratio of demethylarecoline: arecoline: norarecoline: arecoline = (1-6): (1-5): (1-3): (1-8). Optimally, the mass ratio of demethylarecoline: arecoline: norarecoline: arecoline = 2:3:2:6.

[0017] Preferably, the concentration of the total areca alkaloids is greater than or equal to 35.00 ug / ml.

[0018] Preferably, the urease is rumen bacterial urease.

[0019] Beneficial effects of the present invention:

[0020] 1. Areca alkaloids have the function of inhibiting urease activity, reducing the rate of urea decomposition, and regulating rumen nitrogen metabolism in ruminants.

[0021] 2. Compared with monomers of arecoline and arecoline, the total alkaloids of arecoline (demethylarecoline: arecoline: norarecoline: arecoline = 2:3:2:6) have a better inhibitory effect on rumen urease, which is comparable to the effect of norarecoline and norarecoline.

[0022] 3. The stability and tolerance of total alkaloids of areca in the rumen are better than those of the positive drug acetohydroxamic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 IC50 value curve of urease inhibition candidate compounds against rumen urease;

[0024] Figure 2 Kinetic curves of rumen urease of candidate compounds;

[0025] Figure 3 Schematic diagram of the binding of arecoline to rumen urease;

[0026] Figure 4 Schematic diagram of the binding of arecoline to rumen urease;

[0027] Figure 5 Schematic diagram of the binding of norarecoline to rumen urease;

[0028] Figure 6 Schematic diagram of the binding of norarecanidine to rumen urease;

[0029] Figure 7 Rumen degradation rates of acetohydroxamic acid and total areca alkaloids at 48 h (results are expressed as mean ± SEM. Asterisk (*) indicates significant differences between degradation rates (P < 0.05);

[0030] Figure 8 Effects of acetylhydroxamic acid and total alkaloids of areca on urea content in the in vitro anaerobic subculture system of rumen microorganisms (a total of 4 generations) (each point represents the average of 3 observations. Acetylhydroxamic acid was the positive control, and the control blank was added with only methanol solution. Panel A is the first generation, Panel B is the second generation, Panel C is the third generation, and Panel D is the fourth generation. Asterisks (*) indicate significant differences between the control group and the compound group (acetylhydroxamic acid and total alkaloids of areca) (P < 0.05);

[0031] Figure 9 Effect of acetylhomoxylic acid and areca alkaloids on ammonia nitrogen (NH3-N) content in anaerobic rumen microorganism culture system (Each point represents the average of 3 observations. Acetylhomoxylic acid is the positive control, and the control group only adds methanol solution. Figure A is the first generation, Figure B is the second generation, Figure C is the third generation, and Figure D is the fourth generation. The asterisk (*) indicates that the difference between the control group and the compound group (acetylhomoxylic acid and areca alkaloids) is significant (P<0.05). DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0033] Test 1: Effect and mechanism of areca alkaloids on inhibition of rumen urease activity

[0034] Example 1: Determination of the half-inhibitory concentration (IC50) value of areca alkaloids on rumen urease

[0035] 1. Test materials

[0036] Arecoline, arecaidine, nornicotine, nornicodine, and acetylhomoxylic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of >98%; urea, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) powder, and potassium sodium tartrate tetrahydrate were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd., with an analytical purity; sodium hydroxide was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., with an analytical purity; Nash reagent was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with an analytical purity; and ultrapure water was used in the test.

[0037] Areca alkaloids 1: nornicodine: arecaidine: nornicotine: arecoline = 2:3:2:6 (mass ratio);

[0038] Areca alkaloids 2: nornicodine: arecaidine: nornicotine: arecoline = 6:5:3:8 (mass ratio);

[0039] Areca alkaloids 3: nornicodine: arecaidine: nornicotine: arecoline = 1:1:1:1 (mass ratio).

[0040] 2. Test method

[0041] 2.1 Solution preparation

[0042] 1) HEPES buffer (50mM HEPES, pH7.5): Accurately weigh HEPES powder 1.192g, in 100mL volumetric flask, constant volume with ultrapure water, adjust pH to 7.5 with sodium hydroxide, store at 4℃.

[0043] 2) Urea buffer (100mM urea, 50mM HEPES): Accurately weigh urea 0.6g, HEPES powder 1.192g, in 100mL volumetric flask, constant volume with ultrapure water, then put into brown bottle, store at 4℃.

[0044] 2.2 Extraction of rumen bacterial urease

[0045] Select 3 fistulated Xiangxi yellow cattle, collect 1000mL of rumen fluid, filter through 4 layers of sterile gauze, and divide the filtrate (rumen fluid) into 50mL sterile centrifuge tubes and store in liquid nitrogen. After thawing with running water, centrifuge the rumen fluid at 300xg for 5min, collect the supernatant into a new centrifuge tube, centrifuge at 12000xg for 10min, discard the supernatant, resuspend the precipitate with 25mL HEPES buffer, centrifuge at 12000xg for 10min, discard the supernatant, resuspend the precipitate with 25mL HEPES buffer, place it on ice, and crush it with an ultrasonic cell crusher (150W, ultrasonic for 5s, stop for 5s, ultrasonic for 10min), centrifuge at 12000xg for 10min, collect the supernatant, which is the rumen bacterial urease. The centrifugation process is at 4℃.

[0046] 2.3 Urease inhibitory small molecule compound inhibits urease activity test

[0047] In this test, the activity of urease was determined by the Nash reagent method. Add 40ul of urease solution to a 96-well plate and mix with 40ul of a series of concentrations of candidate compound solution, then add 100ul of urea solution, incubate at 37℃ in the dark for 30min, then add 10ul of potassium sodium tartrate solution and 10ul of Nash reagent solution, mix well, and stand at room temperature for 10min. Measure the OD value at 420nm by enzyme marker, and calculate the enzyme activity by concentration-residual enzyme activity curve to obtain the corresponding half inhibitory concentration IC50. The reaction system of the blank control does not add compound solution, and the negative control reaction system adds HEPSE buffer without dissolved urea. Measure in triplicate. The inhibition rate is calculated by the following formula.

[0048] Inhibition rate % = 100-[(measured sample absorbance-negative control absorbance) / (blank control absorbance-negative control absorbance)]*100

[0049] 3. Test results

[0050] IC50 linear fitting was performed in Graphpad prism, and the results were as follows Figure 1As shown, the IC50 values of acetylhydroxamic acid, arecoline 1, arecoline 2, arecoline 3, arecoline, norarecolin, norarecoline and arecolin are 15.77 ug / ml, 35.00 ug / ml, 70.20 ug / ml, 61.97 ug / ml, 77.34 ug / ml, 28.3 ug / ml, 26.58 ug / ml, 55.75 ug / ml, respectively.

[0051] It can be seen that arecoline, arecoline, norarecolin, norarecoline and arecolin have inhibitory effect on rumen urease, and the inhibitory effect of arecoline 1 is better than that of arecoline, arecolin and arecoline 2, 3, and is equivalent to that of norarecolin and norarecoline.

[0052] Example 2: Enzyme kinetics study of arecoline alkaloids

[0053] 1. Test materials

[0054] The water used in the experiment is double distilled water. The reagents used in the experiment are all analytical pure.

[0055] The specific reagents and their preparation methods are as follows:

[0056] HEPES buffer solution (11.92 mg / ml): accurately weigh 1.192 g of 4-hydroxyethyl piperazine sulfonic acid sodium powder, dissolve in deionized water and make up to 100 mL, and adjust the pH to 7.5 with NaOH.

[0057] Urea buffer (8.5716 mg / ml): weigh 0.85716 g of urea, dissolve in HEPES buffer solution (11.92 mg / ml) and make up to 100 mL.

[0058] NH4Cl standard solution (0.5349 mg / ml): weigh 0.5349 g of NH4Cl, dissolve and make up to 1 L.

[0059] Nessler's reagent is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., analytical pure.

[0060] Arecoline: norarecolin: arecolin: norarecoline: arecoline = 2:3:2:6 (mass ratio).

[0061] 2. Test method

[0062] Kinetic evaluation of rumen urease inhibitory compounds: For each of the two rumen urease inhibitory compounds, 19 test tubes were prepared, with tube 0 serving as a blank. 70 μL of urea buffer (according to Table 1) at various concentrations was added to each tube, shaken, and then 15 μL of the candidate compound at various concentrations was added, ensuring that the liquid volumes in each tube were equal. For the treatment without inhibitor, an equal amount of solvent was added as for the treatment with inhibitor. The tubes were preheated in a 37°C water bath for 2 minutes. 15 μL of the diluted enzyme solution was added to each tube, shaken thoroughly, and allowed to react at 37°C for 30 minutes. The ammonia nitrogen content of the solution was determined using Nessler's reagent, and the solution was incubated for 10 minutes to develop color. (The 15 μL enzyme solution in tube 0 was added last, and all other procedures were the same as for tube 6 in the no-inhibitor group. After cooling, tube 0 was used as a blank. The absorbance (A420) of each tube was read in a spectrophotometer at a wavelength of 420 nm.) The specific experimental concentration gradient is shown in Table 1.

[0063] Table 1 Table of small molecule compounds that inhibit rumen urease and the concentration of substrate urea

[0064]

[0065]

[0066] 3. Test results

[0067] The Km and Vmax values ​​corresponding to the enzyme kinetic curves of arecoline, arecoline, norarecoline, norarecoline and total areca alkaloids were obtained by linear fitting of the reciprocal of the reaction rate with respect to the reciprocal of the urea concentration in Graphpad Prism. Figure 2 .

[0068] Depend on Figure 2 It can be seen that when no arecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.5461 mg / ml and Vmax = 1137.5270 μg / min*L; when 0.0325 mg / ml of arecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.2383 mg / ml and Vmax = 837.5209 μg / min*L; and when 0.065 mg / ml of arecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.1689 mg / ml and Vmax = 732.6007 μg / min*L. As the arecoline concentration increased, Km and Vmax also decreased, indicating that arecoline has an uncompetitive inhibitory effect on ruminal urease.

[0069] Depend on Figure 2It can be seen that when arecoline was not added, the parameters of the kinetic curve for ruminal urease were Km = 0.5461 mg / ml and Vmax = 1137.5270 μg / min*L; when 0.0325 mg / ml of arecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.2311 mg / ml and Vmax = 860.5852 μg / min*L; and when 0.065 mg / ml of arecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.2792 mg / ml and Vmax = 798.722 μg / min*L. Therefore, arecoline exhibits a mixed inhibitory effect on ruminal urease.

[0070] Depend on Figure 2 It can be seen that when no demethylarecoline was added, the parameters of the kinetic curve of rumen urease were Km = 0.5461 mg / ml and Vmax = 1137.5270 μg / min*L; when 0.0325 mg / ml of demethylarecoline was added, the parameters of the kinetic curve of rumen urease were Km = 0.2870 mg / ml and Vmax = 807.7544 μg / min*L; when 0.065 mg / ml of demethylarecoline was added, the parameters of the kinetic curve of rumen urease were Km = 0.1875 mg / ml and Vmax = 608.2725 μg / min*L. As the concentration of demethylarecoline increased, Km and Vmax also decreased, indicating that demethylarecoline has an uncompetitive inhibitory effect on rumen urease.

[0071] Depend on Figure 2 It can be seen that when no demethylarecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.5461 mg / ml and Vmax = 1137.5270 μg / min*L; when 0.0325 mg / ml of demethylarecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.2319 mg / ml and Vmax = 930.2326 μg / min*L; and when 0.065 mg / ml of demethylarecoline was added, the parameters of the kinetic curve for ruminal urease were Km = 0.2511 mg / ml and Vmax = 888.0994 μg / min*L. Therefore, demethylarecoline has a mixed inhibitory effect on ruminal urease.

[0072] Depend on Figure 2It can be seen that when no total alkaloids of areca catechu were added, the parameters of the kinetic curve of rumen urease were Km = 0.5461 mg / ml and Vmax = 1137.5270 μg / min*L; when 0.0325 mg / ml of total alkaloids of areca catechu were added, the parameters of the kinetic curve of rumen urease were Km = 0.3267 mg / ml and Vmax = 679.8096 μg / min*L; when 0.065 mg / ml of total alkaloids of areca catechu were added, the parameters of the kinetic curve of rumen urease were Km = 0.4097 mg / ml and Vmax = 503.2112 μg / min*L. Therefore, total alkaloids of areca catechu have a mixed inhibitory effect on rumen urease.

[0073] 4. Test conclusion:

[0074] 1. Arecoline has an anti-competitive inhibitory effect on rumen urease.

[0075] 2. Arecaline has a mixed inhibitory effect on rumen urease.

[0076] 3. Demethylarecoline has an anti-competitive inhibitory effect on rumen urease.

[0077] 4. Demethylarecoline has mixed inhibitory effects on rumen urease.

[0078] 5. Areca alkaloids have mixed inhibitory effects on rumen urease.

[0079] Example 3 Analysis of the Binding Sites of Areca Alkaloids and Rumen Urease

[0080] 1. Test materials

[0081] PyMoOL software (2.3.0, SDGR), AutoDock Vina software (1.1.2, Scripps), and AutoDockTools software.

[0082] 2. Test methods

[0083] We used AutoDock Vina software to perform molecular docking of betel nut alkaloids and rumen urease. We then used PyMoOL software to visualize the docking results and analyze the amino acid sites where betel nut alkaloids bind to rumen urease. The specific steps are as follows:

[0084] (1) The three-dimensional structure of rumen microbial urease was constructed as the receptor using the SWISS-MODEL server, and the structures of the required natural compounds were downloaded from the PubChenm website (https: / / pubchem.ncbi.nlm.nih.gov / ) as the ligand.

[0085] (2) The receptor and ligand were opened in AutoDockVina software respectively. The receptor was processed by deleting water molecules, adding hydrogen, and calculating point charges. The ligand was processed by adding hydrogen, adding charges, and adding atom types.

[0086] (3) Use the Grid menu in the software to set the docking area of ​​the receptor. The center coordinates of the docking area are (60.604, 95.952, 94.265), and the size (X, Y, Z) is set to 72, 74, 92. Then save the file.

[0087] (4) Start running AutoGrid, open the processed receptor and ligand, select the genetic algorithm for docking, and select the longest calculation time. After the run is completed, output and save the file.

[0088] (5) AutoDock was used to analyze the docking results, and the optimal result set by the software by default was selected from the 10 docking results as the final docking result of the experiment.

[0089] (6) Use PyMOL software to plot the molecular docking results and mark the positions of hydrogen bonds and major interacting amino acids.

[0090] 3. Test results

[0091] The molecular docking technology was used to analyze the binding pattern of areca alkaloids and rumen urease, showing the action sites of areca alkaloids and rumen urease, such as Figure 3 、 4 , 5, and 6, which show that arecoline forms a hydrogen bond with the ARG-336 amino acid near the nickel ion in the active center of rumen urease; arecoline forms a hydrogen bond with the ALA-363 amino acid near the nickel ion in the active center of rumen urease; norarecoline forms a hydrogen bond with the ARG-336 and HIS-219 amino acids near the nickel ion in the active center of rumen urease; and norarecoline forms a hydrogen bond with the KCX-217 and HIS-219 amino acids near the nickel ion in the active center of rumen urease.

[0092] The study aimed to investigate the stability of areca alkaloids in the rumen environment and their effects on urea decomposition by rumen microorganisms (ratio of total areca alkaloids to AHA)

[0093] 1. Test materials

[0094] Reagents: All experimental water used was ultrapure water, and all reagents used in the experiment were analytically pure and purchased from Sinopharm Group. Carbon dioxide (purity > 99.999%) and nitrogen (purity > 99.999%) were used. Total arecoline: demethylarecoline: arecoline: norarecoline: arecoline = 2:3:2:6 (mass ratio).

[0095] Instrument: incubator (DRP-9082, Shanghai Sensing Instrument Co., Ltd.); anaerobic operation box (PLAS-LAB 855-AC & 855-ACB, LANSING, MI, USA); compound detection instrument and parameters as follows: 100th electron analysis balance (Sartorius Practum, SQP type, Sartorius Science Instrument (Beijing) Co., Ltd.); Milli-Q Plus purified water instrument (Millipore, Brussels, Belgium); vortex mixer (Vortex-Genie2, Scientific Industries, USA); centrifugal concentrator (Genevac miVac, Tegent Scientific Ltd., UK). The chromatographic column was selected as ACQUITY UPLC HSS T3 1.8 μm, 3.0×100 mm columns (Waters, Dublin, Ireland); the instrument used liquid chromatography tandem quadrupole time-of-flight mass spectrometry LC-QTOF MS (Agilent, UHPLC (1290)-QTOF (6530)). The ionization mode of mass spectrometry was electrospray ionization (ESI), the acquisition mode was information-dependent acquisition (IDA), and the positive and negative ion modes were used. The spray voltage was 3.5KV; the collision energy was 35V; the scanning range was 50-750m / z; the sheath gas (nitrogen) flow rate was 40arb; the auxiliary / sweep gas (nitrogen) flow rate was 15arb; the auxiliary gas heater temperature was 300℃, and the capillary temperature was 320℃.

[0096] 2. Test method

[0097] 2.1. Preparation of rumen anaerobic culture medium

[0098] First, four kinds of salt solutions were prepared: salt solution A (3g K2HPO4); salt solution B (0.6g CaCl2, 3g K2HPO4, 6g NaCl, 0.6g MgSO4·7H2O); salt solution C (6g K2HPO4); salt solution D (1.6g CaCl2·2H2O, 6g K2HPO4, 12g NaCl, 2.5g MgSO4·7H2O). All the salt solutions were dissolved with distilled water to a constant volume of 1000mL.

[0099] The composition of the anaerobic medium was as follows (per liter): 60 mL clarified rumen fluid; 0.5 g each of glucose, soluble starch, cellobiose, maltose, protein hydrolysate, tryptone, and yeast extract; 8 g NaHCO3; 1 mL trace element solution (containing per liter 300 mg H3BO3, 100 mg ZnSO4·7H2O, 30 mg MnCl2·4H2O, 20 mg CoCl2·6H2O, 30 mg Na2MoO4·2H2O, 10 mg Na2SeO3, 20 mg NiCl2, 10 mg CuCl2·2H2O, and 150 mg FeCl2·4H2O); 15 mL salt solution A; 15 mL salt solution B; 0.5 g cysteine ​​hydrochloride; 10 mL hemin solution (50 mg hemin dissolved in Na Prepare a culture medium with the above ingredients, aerate with CO2 until colorless, adjust the pH to 6.8, and dispense 10 mL of the culture medium into anaerobic tubes in an anaerobic incubator. Sterilize the medium at 121°C for 15 minutes.

[0100] Separately, urea anaerobic dilution solution was prepared by dissolving 3 g of urea in 50 ml of anaerobic diluent (3.8 mL of C salt solution, 3.8 mL of D salt solution, 5 mL of 8% Na2CO3 solution, 1 mL of 0.1% resazurin, and 0.5 g of cysteine ​​hydrochloride in 1 L of distilled water).

[0101] 2.2 Evaluation of rumen stability and urease inhibition effect of total alkaloids of areca in vitro anaerobic culture of rumen microorganisms

[0102] In an anaerobic incubator, add 100 μL of urea anaerobic dilution to the anaerobic culture medium. Mix 200 μL of rumen fluid and 100 μL of the tested rumen urease inhibitor small molecule compound solution and inoculate it into the anaerobic culture medium. The final concentration of the inhibitor in the culture medium is 100 μmol / L. Methanol is also set as a negative control group. Three replicates are performed for each group. The anaerobic culture tube is placed in an incubator at 39°C and cultured for 48 hours as the first generation. After 48 hours of each generation, 200 μL of culture and 100 μL of the tested urease inhibitor are mixed and inoculated into new anaerobic culture medium (the negative control is aspirated with 100 μL of methanol solution (solvent)), and cultured for 48 hours to the fourth generation.

[0103] Each generation, 200 μL samples were taken at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, and after centrifugation at 12000 g for 5 min, 180 μL supernatant was taken, 1.8 μL HCL (8 mol / L) was added, and then it was frozen for determination of NH4-N. The determination method was Nash reagent method.

[0104] At 0 h and 48 h, 200 μL samples were taken from the first and fourth generations, and after centrifugation at 12000 g for 5 min, the supernatant was taken for determination of compound concentration and calculation of 48 h degradation rate of the compound. The specific steps were as follows: 1. The sample was added with 200 μL methanol under the condition of 4°C, and incubated for 30 min with shaking at 1500 rpm. 2. Centrifugation (4°C, 12000 g, 10 min). 3. The supernatant was transferred to a 1.5 ml centrifuge tube. 4. Concentration was performed by centrifugal concentrator. 5. Resolubilization was performed with 100 μL of 1% acetonitrile, and the supernatant was taken for determination (LC-MS).

[0105] 3. Test results

[0106] 3.1 Degradation rate of arecoline in the in vitro anaerobic culture system of rumen microorganisms

[0107] At 0 h and 48 h of the first and last generations (fourth generation) of in vitro anaerobic culture of rumen microorganisms, samples were taken, and the contents of arecoline and acetylhydroxamic acid were determined by mass spectrometry-chromatography method, and the 48 h rumen degradation rate of the compounds in the culture system was calculated, and the results are shown in Table 1. Figure 7 As shown in Table 1, the 48 h degradation rates of the standard inhibitor acetylhydroxamic acid and arecoline in the fourth generation of rumen microorganisms in the in vitro anaerobic culture system were significantly higher than those in the first generation (P<0.05), and the degradation rate of arecoline in the in vitro anaerobic culture system of the first and fourth generations of rumen microorganisms was lower than that of acetylhydroxamic acid (P<0.05).

[0108] 3.2 Effect of arecoline on urea decomposition in the in vitro anaerobic culture system of rumen microorganisms

[0109] The effects of arecoline and acetylhydroxamic acid on urea decomposition in the in vitro anaerobic culture system of rumen microorganisms are shown in Table 2. Figure 8 As shown in Table 2, the decomposition rate of urea in the first two generations of in vitro anaerobic culture was faster than that in the last two generations, and with the increase of culture generations, the overall decomposition rate of urea gradually slowed down. In each generation of in vitro anaerobic culture, the decomposition amount of urea in the culture solution of the arecoline treatment group and the acetylhydroxamic acid treatment group was less than that in the control group, indicating that arecoline and acetylhydroxamic acid could inhibit the utilization of urea in the culture medium by rumen microorganisms in the in vitro anaerobic culture system, and the inhibition effect was stable among each generation of subculture. In the fourth generation of in vitro culture system, the urea decomposition rate of the acetylhydroxamic acid treatment group was slightly higher than that of the arecoline group, and the inhibition effect began to decline.

[0110] 3.3 Effect of total alkaloids of areca on ammonia nitrogen production in anaerobic culture system of rumen microorganisms in vitro

[0111] Effects of total alkaloids of areca and acetylhydroxamic acid on ammonia nitrogen production in anaerobic culture system of rumen microorganisms Figure 9 As shown. The generation rate (slope) and total generation amount of ammonia nitrogen in the first two generations of in vitro anaerobic culture are greater than those in the next two generations. As the culture generations increase, the overall generation rate of ammonia nitrogen gradually slows down. In each generation of in vitro anaerobic culture, the amount of ammonia nitrogen generated in the culture fluid of the areca alkaloids treatment group and the acetohydroxamic acid treatment group was less than that of the control group, indicating that areca alkaloids and acetohydroxamic acid can inhibit the nitrogen metabolism of rumen microorganisms in the in vitro anaerobic culture system, and the inhibitory effect is stable between generations of subculture. Starting from the third generation, the ammonia nitrogen generation rate and total amount of the acetohydroxamic acid treatment group were slightly greater than those of the areca alkaloids group, indicating that the inhibitory effect began to decline.

[0112] 4. Experimental Conclusion

[0113] The effects of total alkaloids of areca and acetohydroxamic acid on the urea and ammonia nitrogen production in the anaerobic culture system of rumen microorganisms were evaluated in vitro. The results are as follows:

[0114] 1. Areca alkaloids and acetylhydroxamic acid can inhibit the decomposition of urea and the production of ammonia nitrogen in the anaerobic culture system of rumen microorganisms in four generations of culture.

[0115] 2. The 24h degradation rates of total alkaloids of areca in the first and fourth generations of subculture were lower than that of the standard inhibitor acetylhydroxamic acid, indicating that total alkaloids of areca were more stable in the rumen environment.

Claims

1. The application of betel nut alkaloids in the preparation of rumen bacterial urease inhibitors, characterized in that: The betel nut alkaloid is selected from the following components: at least one of norarecoline, norarecoline, arecoline and arecoline.

2. The use according to claim 1, characterized in that The betel nut alkaloid is selected from norarecaline, and the concentration of the norarecaline is greater than or equal to 28.3ug / ml.

3. The use according to claim 1, characterized in that The areca alkaloid is selected from norarecoline, and the concentration of the norarecoline is greater than or equal to 26.58 ug / ml.

4. The use according to claim 1, characterized in that The areca alkaloid is selected from arecoline, and the concentration of the arecoline is greater than or equal to 77.34ug / ml.

5. The use according to claim 1, characterized in that The betel nut alkaloid is selected from arecoline; the concentration of the arecoline is greater than or equal to 55.75ug / ml.

6. The use according to claim 1, characterized in that The betel nut alkaloid is selected from the group consisting of norarecoline, arecoline, norarecoline and arecoline.

7. The use according to claim 6, characterized in that The mass ratio of norarecoline:arecoline:norarecoline:arecoline in the betel nut alkaloids is (1-6): (1-5): (1-3): (1-8).

8. The use according to claim 7, characterized in that The mass ratio of demethylarecoline:arecoline:norarecoline:arecoline in the betel nut alkaloids is 2:3:2:

6.

9. The use according to claim 8, characterized in that The concentration of the betel nut alkaloids is greater than or equal to 35.00 ug / ml.

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