Urease inhibitor for preventing / treating ammonia poisoning in ruminants

Abstract

The present invention relates to use of trimethylglycine and a salt thereof as a urease inhibitor in preventing / treating ammonia poisoning in ruminants. Trimethylglycine and the salt thereof have a high inhibitory effect on urease in the rumen of ruminants, and can be used as a novel urease inhibitor to improve the safety of urea as a non-protein nitrogen feed, thus effectively preventing / treating ammonia poisoning caused by excessively fast decomposition of urea and the accumulation of ammonia in blood.

Description

A urease inhibitor for the prevention / treatment of ammonia poisoning in ruminants Technical Field

[0001] This invention relates to trimethylglycine and its salts as novel rumen urease inhibitors for the prevention / treatment of ammonia poisoning in ruminants. Background Technology

[0002] Ruminants (such as cattle and sheep) have rumen microorganisms that can use non-protein nitrogen (NPN) as a nitrogen source to synthesize microbial proteins that are then utilized by the animals. Urea is the most commonly used NPN in ruminant feeding. Urea has a high nitrogen content, approximately 46%. The nitrogen content of 1 kg of urea is roughly equivalent to the nitrogen content of 2.8 kg of crude protein, or the amount of crude protein contained in 6.5 kg of soybean meal. Therefore, compared to protein feeds such as soybeans and soybean meal, which are more expensive, urea is a cheap nitrogen source for ruminants (Sun Meijie. Effects of different urea addition levels on rumen fermentation and microbial community structure in fattening Hu sheep. Journal of Nanjing Agricultural University, 2022, 45(2): 323-332).

[0003] Ruminants are able to utilize urea because bacteria in their rumen produce urease (also known as urease). Urease is a hydrolytic enzyme that catalyzes the breakdown of urea into ammonia and carbon dioxide. Rumen microorganisms utilize the ammonia released from urea decomposition to combine with the breakdown products of carbohydrates in their feed to form amino acids, which are then used to synthesize microbial proteins. These microbial proteins are then digested and absorbed in the abomasum and small intestine to synthesize proteins in the animal's body and its products.

[0004] Urease, produced by rumen microorganisms, catalyzes the rapid hydrolysis of urea into ammonia. Studies have shown that 1 kg of rumen contents can decompose 1 g of urea into ammonia within 1 hour, meaning the rate of ammonia production from urea decomposition is four times the rate at which rumen microorganisms utilize ammonia. This rapid decomposition of urea results in a large amount of ammonia that cannot be utilized by rumen microorganisms, reducing urea utilization efficiency. Furthermore, excessive ammonia accumulation, absorbed into the bloodstream by the rumen wall and posterior gastrointestinal tract, cannot be metabolized by the liver quickly enough, easily leading to ammonia poisoning in animals. Symptoms include convulsions, difficulty breathing, and frothing at the mouth, and in severe cases, death (Niu Ying. A New Exploration to Improve Urea Utilization in Cattle and Sheep. Modern Agriculture, 2020, 7: 62). Therefore, the main limiting factor for urea utilization in ruminants is the excessively rapid decomposition of urea into ammonia by urease in the rumen, leading to ammonia poisoning. Thus, research on the prevention and treatment of ammonia poisoning in ruminants is an important topic in current ruminant husbandry research, and urease inhibitors are a promising technology.

[0005] Currently, there are over 100 patented urease inhibitors, mainly classified into three categories: metal salts, small organic molecules, and plant extracts. Only a few urease inhibitors, such as acetyloxyxamic acid, butyl thiophosphate triamine (NBPT), propyl thiophosphate triamine (NPPT), and hydroquinone (HQ), have been put into practical use. However, existing urease inhibitors still suffer from numerous problems in use, including low biological activity, low cost-effectiveness, and poor biosafety, and have not yet been widely applied in the prevention / treatment of ammonia poisoning in ruminants.

[0006] Trimethylglycine, also known as betaine or glycine betaine, is a quaternary ammonium alkaloid. In its pure form, it is a white, prismatic or leaf-shaped crystal, soluble in water, methanol, and ethanol. As a natural plant-based ingredient, trimethylglycine is safe, green, non-toxic, and pollution-free, and it is already widely synthesized and produced at a low price.

[0007] This study is the first to discover that trimethylglycine and its salts have significant rumen urease inhibitory activity, and have advantages such as high cost-effectiveness and good biocompatibility. They can be used as novel rumen urease inhibitors in ruminants for the prevention / treatment of ammonia poisoning in ruminants, thereby improving the safety of using urea as a non-protein nitrogen feed. Summary of the Invention

[0008] The purpose of this invention is to provide a novel rumen urease inhibitor, trimethylglycine, and its salts, for the prevention / treatment of ammonia poisoning in ruminants. The structural formula of trimethylglycine is:

[0009] The urease inhibitor also includes acceptable salts, including hydrochloride, citrate, malate, phosphate, maleate, fumarate, sulfonate, or methanesulfonate.

[0010] A urease inhibitor for the prevention / treatment of ammonia poisoning in ruminants, wherein the ruminants include various types of cattle, sheep, deer, camels, etc., and the ammonia poisoning in ruminants refers to the poisoning symptoms caused by excessively high blood ammonia concentration due to improper consumption of urea by ruminants. The urease inhibitor contains trimethylglycine or an acceptable salt thereof, and the proportion of the urease inhibitor used is 0.1 wt% to 30 wt% of the urea mass of the feed; more preferably 1 wt% to 10 wt%.

[0011] This invention includes the application of the urease inhibitor described above in the preparation of various types of products for the prevention / treatment of ammonia poisoning in ruminants.

[0012] 1. Preparation of rumen urease solution

[0013] Rumen contents were collected from Yiling cattle via a rumen fistula. After thorough mixing, the contents were centrifuged at 26,000g for 15 minutes at 4°C. The rumen bacterial pellet was collected and stored at -80°C. Before the experiment, 20g of the rumen bacterial pellet was resuspended in HEPES buffer (pH 7.5, 50mmol / L) and then subjected to low-temperature high-pressure disruption (22,000 p.si (cell pressure)) using a high-pressure cell disruptor. After centrifugation at 12,000g for 10 minutes at 4°C, the supernatant was used as the rumen urease solution.

[0014] 2. Assay for rumen urease inhibitory activity

[0015] The basic principle of urease inhibitor activity testing is to determine the level of urease inhibitor activity by detecting the change in the ability of urease to catalyze the decomposition of urea and release ammonia after the urease inhibitor reacts with urease. The most commonly used method is the indophenol method. First, the inhibitor is allowed to react with urease for a period of time. Then, it is allowed to catalyze the decomposition of urea. The resulting ammonia gas dissolves in a buffer solution, and indophenol is used for color development. The OD value is then measured using a spectrophotometer. Finally, the inhibition rate of the inhibitor on urease is calculated using the following formula:

[0016] Blank: No inhibitors or urea were added to the above test solutions;

[0017] Control: No inhibitors were added to the above test solutions.

[0018] The activity assay for rumen urease was performed according to the method reported by Weatherburn. The specific steps are as follows: 25 μL of urease solution and 25 μL of the test compound (concentration prepared with DMSO-phosphate buffer according to the experimental setup) were added to a 96-well plate. The plate was co-incubated at 37°C for 30 min. Then, 50 μL of phosphate buffer containing 25 mmol of urea was added, and the plate was co-incubated at 37°C for 30 min. Next, 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added, and the plate was co-incubated at 37°C for 30 min. The absorbance was measured at 620 nm. A blank control group and a normal control group were also included, with three replicates for each group. The inhibition rate and half-maximal inhibitory concentration (IC50) of the inhibitor were calculated. 50 .

[0019] 3. Ruminant animal feeding experiments

[0020] Several ruminants of similar weight were selected, and three treatment groups were set up: (1) normal diet; (2) diet + urea (1% to 3% of the diet); (3) diet + urea (1% to 3% of the diet) + rumen urease inhibitor (trimethylglycine or its salt). The urease inhibitor can be used alone or mixed with urea in the feed, and the proportion used is 1% to 20% of the amount of urea used. The experimental period was 4 weeks. The animals were observed daily to check for ammonia poisoning symptoms, and their weight was weighed to calculate the average daily weight gain.

[0021] This study found that trimethylglycine and its salts have significant inhibitory activity against rumen urease, with an IC50 value of [missing value]. 50 With a concentration of 28.5 μM to 36.3 μM, it can be used as a novel rumen urease inhibitor for the prevention and treatment of ammonia poisoning in ruminants, thereby improving the safety and economic benefits of ruminant farming.

[0022] The symptoms of ammonia poisoning described in this invention (i.e., when peripheral blood ammonia exceeds about 1 mg / 100 ml of blood) are accompanied by symptoms including muscle contraction, ataxia, excessive salivation, swelling, and respiratory failure. Detailed Implementation

[0023] Example 1

[0024] Add 25 μL of rumen urease solution and 25 μL of trimethylglycine solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer) to a 96-well plate and co-incubate at 37 °C for 30 min. Then add 50 μL of phosphate buffer containing 25 mmol urea and co-incubate at 37 °C for 30 min. Next, add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) and co-incubate at 37 °C for 30 min. Measure the absorbance at 620 nm. A blank control group and a normal control group were also included. The half-maximal inhibitory concentration (IC50) of trimethylglycine against rumen urease was calculated. 50 The value is 31.7 μM.

[0025] Example 2

[0026] Add 25 μL of rumen urease solution and 25 μL of trimethylglycine hydrochloride solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer) to a 96-well plate and co-incubate at 37 °C for 30 min. Then add 50 μL of phosphate buffer containing 25 mmol urea and co-incubate at 37 °C for 30 min. Next, add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) and co-incubate at 37 °C for 30 min. Measure the absorbance at 620 nm. A blank control group and a normal control group were also included. The half-maximal inhibitory concentration (IC50) of trimethylglycine hydrochloride against rumen urease was calculated. 50 It is 28.5 μM.

[0027] Example 3

[0028] Add 25 μL of rumen urease solution and 25 μL of trimethylglycine citrate solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer) to a 96-well plate and co-incubate at 37 °C for 30 min. Then add 50 μL of phosphate buffer containing 25 mmol urea and co-incubate at 37 °C for 30 min. Next, add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) and co-incubate at 37 °C for 30 min. Measure the absorbance at 620 nm. A blank control group and a normal control group were also included. The half-maximal inhibitory concentration (IC50) of trimethylglycine citrate against rumen urease was calculated. 50 The value is 33.8 μM.

[0029] Example 4

[0030] Add 25 μL of rumen urease solution and 25 μL of trimethylglycine malate solution (1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 80 μM, 100 μM, prepared with DMSO-phosphate buffer) to a 96-well plate and co-incubate at 37 °C for 30 min. Then add 50 μL of phosphate buffer containing 25 mmol urea and co-incubate at 37 °C for 30 min. Next, add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) and co-incubate at 37 °C for 30 min. Measure the absorbance at 620 nm. A blank control group and a normal control group were also included. The half-maximal inhibitory concentration (IC50) of trimethylglycine malate against rumen urease was calculated. 50The value is 36.3 μM.

[0031] Example 5

[0032] Yiling cattle of similar weight were selected, and three treatment groups (7 animals per group) were set up: (1) normal diet (corn starch, the same below); (2) diet + urea (1% of the diet); (3) diet + urea (1% of the diet) + trimethylglycine, with the trimethylglycine used at 1% of the urea quality. The experiment lasted for 4 weeks. The animals were observed for ammonia poisoning symptoms and weighed daily. No ammonia poisoning symptoms were observed in any group during the entire experimental period. The average daily weight gain of group 1 was 820±73g, group 2 was 1015±106g, and group 3 was 1272±68g.

[0033] Example 6

[0034] Yiling cattle of similar weight were selected, and three treatment groups (7 cattle per group) were set up for the experiment: (1) normal diet (the same group as Group 1 in Example 5); (2) diet + urea (1.5% of the diet); (3) diet + urea (1.5% of the diet) + trimethylglycine hydrochloride, with the proportion of trimethylglycine hydrochloride being 2% of the urea quality. The experiment lasted for 4 weeks, and the animals were observed for ammonia poisoning symptoms and weighed daily. During the entire experimental period, one cow in Group 2 showed ammonia poisoning symptoms in the third week. After timely administration of trimethylglycine hydrochloride, the symptoms were relieved and the cow was removed from the experiment. No ammonia poisoning symptoms were observed in the other groups. The average daily weight gain of Group 1 was 820±73g, the average daily weight gain of Group 2 was 1076±96g, and the average daily weight gain of Group 3 was 1429±72g.

[0035] Example 7

[0036] Yiling cattle of similar weight were selected, and three treatment groups (7 cattle per group) were set up: (1) normal diet (the same group as Group 1 in Example 5); (2) diet + urea (2% of the diet); (3) diet + urea (2% of the diet) + trimethylglycine, with the trimethylglycine added at 3% of the urea content. The experiment lasted for 4 weeks, and the animals were observed for ammonia poisoning symptoms and weighed daily. During the entire experimental period, 3 cattle in Group 2 showed ammonia poisoning symptoms in the second week. After timely administration of trimethylglycine, the symptoms were relieved and the experiment in Group 2 was stopped. No ammonia poisoning symptoms were observed in the other groups. The average daily weight gain of Group 1 was 820±73g, and the average daily weight gain of Group 3 was 1482±95g.

[0037] Example 8

[0038] Yiling cattle of similar weight were selected, and three treatment groups (7 cattle per group) were set up: (1) normal diet (the same group as Group 1 in Example 5); (2) diet + urea (2.5% of the diet); (3) diet + urea (2.5% of the diet) + trimethylglycine citrate, with the trimethylglycine citrate added at 5% of the urea content. The experiment lasted for 4 weeks, and the animals were observed for ammonia poisoning symptoms and weighed daily. During the entire experimental period, 5 cattle in Group 2 showed ammonia poisoning symptoms in the first week. After timely administration of trimethylglycine citrate, the symptoms were relieved, and the experiment in Group 2 was stopped. No ammonia poisoning symptoms were observed in the other groups. The average daily weight gain of Group 1 was 820±73g, and the average daily weight gain of Group 3 was 1506±83g.

[0039] Example 9

[0040] White goats of similar weight were selected, and three treatment groups (nine goats in each group) were set up: (1) normal diet; (2) diet + urea (1.5% of the diet); (3) diet + urea (1.5% of the diet) + trimethylglycine, with the trimethylglycine used at 2% of the urea quality. The experiment lasted for 4 weeks, and the animals were observed for ammonia poisoning symptoms and weighed daily. During the entire experimental period, one goat in group 2 showed ammonia poisoning symptoms in week 4. After timely administration of trimethylglycine, the symptoms were relieved and the goat was removed from the experiment. No ammonia poisoning symptoms were observed in the other groups. The average daily weight gain of group 1 was 197±45g, group 2 was 245±67g, and group 3 was 288±59g.

[0041] Example 10

[0042] White goats of similar weight were selected, and three treatment groups (nine goats in each group) were set up for the experiment: (1) normal diet (the same group as group 1 in Example 9); (2) diet + urea (2% of the diet); (3) diet + urea (2% of the diet) + trimethylglycine hydrochloride, with the trimethylglycine hydrochloride added at 3% of the urea quality. The experiment lasted for 4 weeks, and the animals were observed for ammonia poisoning symptoms and weighed daily. During the entire experimental period, three goats in group 2 showed ammonia poisoning symptoms in week 3. After timely administration of trimethylglycine hydrochloride, the symptoms were relieved and the goats were removed from the experiment. No ammonia poisoning symptoms were observed in the other groups. The average daily weight gain of group 1 was 197±45g, group 2 was 268±53g, and group 3 was 321±46g.

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