阅读说明：本技术 氯化两面针碱在制备脲酶抑制剂中的应用 (Application of nitidine chloride in preparation of urease inhibitor ) 是由 鲁强 李彩兰 刘玫瑰 于 2021-08-12 设计创作，主要内容包括：本发明属于天然化合物应用技术领域,公开了氯化两面针碱在制备脲酶抑制剂中的应用。本发明指出氯化两面针碱抑制脲酶的效果显著,且具有结构简单,安全性好等特点,具备良好的应用价值与开发前景,可作为脲酶抑制剂在植物肥料、饲料添加剂和医药领域等领域进行应用。(The invention belongs to the technical field of natural compound application, and discloses application of nitidine chloride in preparation of a urease inhibitor. The invention indicates that the nitidine chloride has obvious effect of inhibiting urease, has the characteristics of simple structure, good safety and the like, has good application value and development prospect, and can be used as a urease inhibitor to be applied in the fields of plant fertilizers, feed additives, medicines and the like.)

1. The application of nitidine chloride in preparing urease inhibitor is characterized in that the structural formula of nitidine chloride is shown as formula (I):

2. a urease inhibitor, which is characterized by comprising nitidine chloride or a pharmaceutically acceptable salt thereof.

3. The urease inhibitor of claim 2 further comprising a pharmaceutically acceptable excipient.

4. Use of a urease inhibitor according to claim 2 or 3 for the preparation of an animal feed additive.

5. Use according to claim 4, wherein the animal is a ruminant or a monogastric animal.

6. Use of a urease inhibitor according to claim 2 or 3 for the preparation of plant fertilisers.

7. Use of the urease inhibitor according to claim 2 or 3 in the medical field.

Technical Field

The invention belongs to the technical field of natural compound application, and particularly relates to application of nitidine chloride in preparation of a urease inhibitor.

Background

Urease, also known as urease or urea amide hydrolase, is a Ni-containing substance widely existing in various organisms and ecological environments²⁺The metalloenzyme of (1). Urease can catalyze the hydrolysis of urea into ammonia, and thus plays an important role in the metabolic process of nitrogen. However, the catalytic hydrolysis speed of urea is too high due to the overhigh activity of urease, and a large amount of ammonia gas is released, so that various hazards are brought to agriculture, animal husbandry, environment and even human health. In agriculture, too high urease activity can cause the catalytic hydrolysis speed of urea to be too fast, release a large amount of ammonia gas, seriously reduce the nitrogen utilization rate of various organisms, destroy the pH environment of soil, and cause the increase of agricultural production cost and the pollution of agricultural products. In the field of animal husbandry, nonprotein nitrogen is decomposed too fast under the action of ruminant rumen urease, excessive volatile ammonia is generated and accumulated in blood, and the animal is easy to suffer from blood ammonia poisoning and even death. In addition, the high ammonia content in the poultry house can induce the occurrence of various diseases of ruminants and pollute the air quality. In the field of medicine, urease in a human body can decompose and reabsorb metabolic product urea in the human body again, so that the pH value balance of the human body is changed, the pH values of body fluid and urine are increased, urinary system and gastrointestinal tract infection is induced, and various diseases such as gastritis, gastric ulcer, kidney and lithangiuria are caused.

At present, researchers at home and abroad research and use urease inhibitors for prevention and treatment aiming at the serious harm of urease to human production and life. Heretofore, urease inhibitors have been widely used in the fields of medicine, agriculture and animal husbandry, and the classes thereof mainly include heavy metal ions, hydroxamic acids, phosphoramide-type inhibitors, phosphate urease inhibitors, metal complexes and the like. However, the effectiveness of the existing urease inhibitor is mostly influenced by factors such as temperature, time, pH environment and the like, so that the existing more mature urease inhibitor in China is acetohydroxamic acid, and has the characteristics of simple synthesis, low cost and low toxicity. However, the major problem is that the long-term use of the acetohydroxamic acid can cause the microorganisms to generate adaptability and drug resistance, thereby causing the acetohydroxamic acid to lose the effect.

Therefore, it is desirable to provide a novel urease inhibitor product with good effect to reduce the dependence on acetohydroxamic acid and thus reduce the occurrence of drug resistance.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of nitidine chloride in the preparation of the urease inhibitor, and indicates that nitidine chloride has an obvious effect of inhibiting urease, has the characteristics of simple structure, good safety and the like, has good application value and development prospect, and can be applied to the fields of plant fertilizers, feed additives, medicines and the like as the urease inhibitor.

The invention provides an application of nitidine chloride in preparation of a urease inhibitor, wherein the structural formula of the nitidine chloride is shown as a formula (I):

the nitidine chloride is extracted and separated from Zanthoxylum nitidum of Rutaceae, is a natural benhydrophenanthridine alkaloid with molecular formula of C₂₁H₁₈NO₄Cl, molecular weight 383.82, chemical structural formula as shown in formula (I). In recent years, researches show that nitidine chloride has the characteristics of high efficiency and low toxicity, and has various pharmacological actions such as anti-inflammation, antibiosis, analgesia, malaria resistance, tumor resistance and the like. The invention firstly discovers that nitidine chloride has the effect of inhibiting the activity of urease, so that nitidine chloride can be applied to preparing the urease inhibitor.

Preferably, the urease is jack bean urease. Experiments show that the nitidine chloride has good inhibition effect on the activity of jack bean urease and can be equivalent to a first-line urease inhibitor acetoxyhydroxamic acid.

The invention also provides a urease inhibitor which comprises nitidine chloride or pharmaceutically acceptable salts thereof.

Preferably, the urease inhibitor further comprises pharmaceutically acceptable auxiliary materials.

The invention also provides application of the urease inhibitor in preparation of an animal feed additive.

Preferably, the animal is a ruminant or a monogastric animal.

The nitidine chloride or the pharmaceutically acceptable salt thereof is added into the feed as an animal feed additive, so that the decomposition speed of urea in ruminants or monogastric animals such as dairy cows, beef cattle, mutton sheep and broiler chickens can be reduced, the utilization rate of the urea is improved, and the ammonia content in air in poultry houses and animal houses is reduced.

The invention also provides application of the urease inhibitor in preparation of plant fertilizers. The urease inhibitor is added into nitrogen fertilizer, and can raise urea utilization rate, increase crop yield and raise production performance.

The invention also provides application of the urease inhibitor in the field of medicines. The urease inhibitor can avoid excessive increase of pH values of body fluid and urine of a human body, reduce the incidence rate of diseases of the urinary system and the digestive system, and can be applied as a medicament for resisting gastritis, gastric ulcer or lithangiuria.

Compared with the prior art, the invention has the following beneficial effects:

the invention develops the new application of nitidine chloride as a urease inhibitor in the fields of plant fertilizers, feed additives and medicines, enriches the selectable varieties of the existing urease inhibitors and is beneficial to reducing the drug resistance of medicines. Meanwhile, the nitidine chloride is a natural medicinal component and has the characteristics of high efficiency and low toxicity, so that the nitidine chloride has the advantages of better safety and longer action time compared with the existing urease inhibitor.

Drawings

FIG. 1 shows the urease inhibiting activity of nitidine chloride and acetoxyhydroxamic acid; wherein A represents the inhibitory activity of nitidine chloride, and B represents the inhibitory activity of acetohydroxamic acid;

FIG. 2 is a study of the type of inhibition of nitidine chloride on urease; wherein A represents a Lineweaver-Burk reciprocal diagram of the nitidine chloride for inhibiting urease, B represents a relation diagram of the slope of a curve in the Lineweaver-Burk reciprocal diagram of the nitidine chloride for inhibiting urease and the concentration of an inhibitor, and C represents a relation diagram of the intercept of the curve in the Lineweaver-Burk reciprocal diagram of the nitidine chloride for inhibiting urease and the concentration of the inhibitor;

FIG. 3 is a graph showing the progress of the reaction between nitidine chloride and urease; wherein A and B are reaction progress curves of nitidine chloride and urease under the conditions of no incubation and incubation respectively;

figure 4 is a graph of the effect of thiol-containing compounds on the urease inhibiting activity of nitidine chloride, where P <0.01 compared to the urease group and # is P <0.01 compared to nitidine chloride + urease group;

FIG. 5 is a graph of the effect of inorganic compounds on nitidine chloride inhibition of urease activity; in the figure, # is P <0.01 compared to the urease group, # is P <0.05 compared to the nitidine chloride + urease group, and # is P <0.01 compared to the nitidine chloride + urease group.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

Experimental Material

Test drug

Nitidine chloride, purchased from Doctorifen Biotech limited, was greater than 98% pure. Canavalia ureabase (enzyme from Canavalia ensiformis, Type III, 40.3U/mg), acetohydroxamic acid, available from Aladdin, Inc.; urea, dithiothreitol and cysteine are all purchased from Solibao; glutathione was purchased from melphalan organisms; sodium fluoride and boric acid were purchased from mcelin. HEPES, sodium salicylate, sodium nitroprusside, sodium hydroxide, sodium hypochlorite and glycerol are purchased from Guangzhou chemical reagent factories and are all analytically pure.

Preparation of test drugs

Weighing appropriate amount of nitidine chloride and acetohydroxamic acid, respectively, preparing into corresponding concentration with 20mM HEPES buffer solution (pH 7.5), and storing at 4 deg.C in dark place.

Preparation of jack bean urease

A proper amount of jack bean urease is weighed, dissolved in 20mM HEPES buffer solution (pH is 7.5) to prepare 10U/mL jack bean urease solution, and the jack bean urease solution is placed in a refrigerator for freezing storage at 4 ℃ for later use.

Preparation of urea

An appropriate amount of urea was weighed, dissolved in 20mM HEPES buffer (pH 7.5) to prepare a 150mM urea solution, and stored in a refrigerator at 4 ℃ for further use.

Preparation of Bertholot color developing solution

Solution A: an appropriate amount of sodium nitroprusside and sodium salicylate powder are respectively weighed, dissolved in 20mM HEPES buffer solution (pH 7.5) to prepare a developing solution A containing 9.73mM sodium nitroferricyanide and 700mM sodium salicylate, and the developing solution A is placed at 4 ℃ and kept away from light for later use.

And B, liquid B: weighing 9g of sodium hydroxide, dissolving in 20mM HEPES buffer solution (pH 7.5), cooling, adding 12mL of sodium hypochlorite, mixing, diluting to 50mL, and storing at 4 ℃ in a dark place for later use.

Example 1: research on inhibition effect of nitidine chloride on urease

Uniformly mixing a jack bean urease solution and a test drug solution with a series of concentrations, and incubating for 20 minutes at 37 ℃; adding a urea solution, and reacting for 20 minutes at room temperature in a dark place; adding Berthlot color development solution, developing for 10 minutes in a dark place, sucking 200 mu L of incubation solution to a 96-well plate, and measuring the OD absolute value at 595nm on an enzyme-linked immunosorbent assay. Each concentration was done 3 times in parallel. The blank was replaced with the solvent of each dilution and the OD blank was determined as above for the rest of the runs. The corresponding relative value of OD is obtained according to equation 1. The Residual enzyme activity (RA) was determined according to equation 2, and the corresponding median inhibitory concentration IC was determined by GraphPad₅₀。

OD_{Relative to each other}＝OD_Absolute–OD_{Blank space}(formula 1)

Residual enzyme activity (%) ═ OD_{Relative value} ^{Test article}/OD_{Relative to each other} ^{Blank space}X 100% (formula 2)

As shown in FIG. 1, nitidine chloride inhibited the median Inhibitory Concentration (IC) of jack bean urease activity₅₀) 33.2 + -4.8 μ M, comparable to the putative first-line urease inhibitor acetoxyhydroxamic acid (IC)₅₀31.7 ± 5.8 μ M), indicating that nitidine chloride significantly inhibits urease activity.

Example 2: studies on inhibition types of nitidine chloride on urease

Mixing a certain concentration of jack bean urease with a series of concentrations of nitidine chloride solution (0, 25, 50, 100 μ M), and incubating at 37 deg.C for 20 min. A series of concentrations of urea solution (0.9375-30mM) were then added at room temperature and the reaction was protected from light for 20 minutes, followed by development and OD measurement according to the method of example 1_AbsoluteValue and finding the corresponding OD_{Relative to each other}The value is obtained. The solvent to which the substance was added was used as a blank and 3 replicates were run. The experiment was conducted by counting the inverse of the reaction rate (1/V, i.e., 1/OD)_{Relative to each other}) Reciprocal of substrate concentration (1/[ urea)]) Making a Lineweaver-Burk diagram, and finally obtaining a kinetic parameter K through an L-B curve combined with a formula 3_M、v_maxAnd obtaining the inhibition constant K by secondary plotting through an L-B curve_i、K_is。

As shown in FIG. 2, it can be seen from the Lineweaver-Burk Biobloid chart that the kinetic parameter K of the nitidine chloride for inhibiting jack bean urease is changed with the change of the concentration of the nitidine chloride_MA small variation, v_maxThe value gradually decreases with the increase of the concentration of nitidine chloride, so that the action type of the nitidine chloride for inhibiting the jack bean urease can be preliminarily inferred to be a non-competitive inhibition type. In addition, the inhibition constant K of the nitidine chloride for inhibiting the jack bean urease can be obtained by carrying out secondary mapping on a Lineweaver-Burk double reciprocal diagram by combining the type of the nitidine chloride for inhibiting the jack bean urease_iIs 18 +/-5.1 mu M, K_is10.3. + -. 2.2. mu.M.

Example 3: dynamics research of nitidine chloride for inhibiting urease

Uniformly mixing a series of volumes of nitidine chloride solutions (0, 25, 50 and 100 mu M) with the same concentration and a certain concentration of jack bean urease, incubating for 20min at 37 ℃, adding an equal volume of urea solution at room temperature for reaction for 20min in a dark place (incubation system) or directly adding an equal volume of urea solution for reaction for 20min in a dark place without incubation in the incubator (non-incubation system). The reaction solution was then transferred to a 1.5mL centrifuge tube at various time intervals (0-30min), developed according to the method of example 1 and OD measured_AbsoluteValue and finding the corresponding OD_{Relative to each other}The value is obtained. The solvent to which the substance was added was used as a blank and 3 replicates were run.

OD obtained from the above two reaction systems_{Relative to each other}Substituting the value into the standard curve of the ammonia content to calculate the corresponding ammonia concentration. And then performing curve fitting on each test point of each concentration of nitidine chloride according to a formula 4 to judge the rate of combination of nitidine chloride and jack bean urease.

P_t＝V_s*t+(V₀-V_s)*(l-e^-kapp*t)*k_app ^-1(formula 4)

Wherein P is the amount of reaction product accumulated t minutes after the start of the reaction, V₀And V_sRespectively, the reaction initiation rate and the reaction equilibrium rate, k_appIs V₀And V_sThe apparent first order rate constant that follows when interconverting.

As can be seen from the reaction progress curves of nitidine chloride and jack bean urease in fig. 3, nitidine chloride and jack bean urease first rapidly form urease-nitidine chloride complex EI, and then slowly isomerize into urease-nitidine chloride complex EI. Therefore, the combination mode of the nitidine chloride and the jack bean urease is judged to be slow combination type.

Example 4: effect of mercapto Compounds on urease Activity

Respectively collecting equal amount of jack bean urease solution and sulfhydryl compound-containing solution (dithiothreitol, cysteine or glutathione solution, concentration is 1.25mM)Mixing, and pre-incubating in 37 deg.C incubator for 20 min. Adding 0.1mM nitidine chloride solution, mixing, incubating at 37 deg.C for 20min, adding urea, reacting at room temperature for 20min, developing according to the method of example 1, and measuring OD_AbsoluteValue and finding the corresponding OD_{Relative to each other}And the corresponding residual enzyme activity RA is determined. The solvent to which the substance was added was used as a blank and 3 replicates were run.

As shown in FIG. 4, when the system contains a sulfhydryl compound (dithiothreitol, cysteine or glutathione solution), the enzyme activity of jack bean urease can be maintained even in the presence of nitidine chloride. The result shows that one action site of the nitidine chloride for inhibiting the activity of the jack bean urease is a sulfhydryl group in a urease amino acid series.

Example 5: effect of inorganic Compounds on urease Activity

Equal amounts of jack bean urease solution and inorganic compound solution (1.25mM boric acid or sodium fluoride solution) were mixed well and pre-incubated in a 37 ℃ incubator for 20 minutes. Adding 0.1mM nitidine chloride solution, mixing, incubating at 37 deg.C for 20min, adding urea at room temperature for reaction for 20min, developing according to the method of example 1, and measuring OD_AbsoluteValue and finding the corresponding OD_{Relative to each other}And the corresponding residual enzyme activity RA is determined. The solvent to which the substance was added was used as a blank and 3 replicates were run.

As shown in fig. 5, when both jack bean urease and nitidine chloride and boric acid or sodium fluoride were present in the system, the residual enzyme activity was reduced, even lower than that when the nitidine chloride alone was used with jack bean urease, indicating that boric acid or sodium fluoride did not protect the jack bean urease activity, and that the nitidine chloride may be synergistic with the boric acid or sodium fluoride to inhibit the jack bean urease activity. The result further indicates that the action site of the nitidine chloride for inhibiting the activity of the jack bean urease can be a sulfhydryl group in a urease amino acid series.

The experiments show that the nitidine chloride can obviously inhibit the activity of jack bean urease, and the inhibition effect is equivalent to that of acetohydroxamic acid. In addition, inhibition type studies indicate that nitidine chloride is a non-competitive urease inhibitor. Further mechanism research shows that the nitidine chloride mainly interacts with a sulfhydryl active site in a jack bean urease structure, so that the activity of jack bean urease is inhibited. In conclusion, the nitidine chloride has the effect of resisting the jack bean urease, and the effect is obvious. And the nitidine chloride has the advantages of simple structure, safety and the like, has good application value and development prospect in the aspect of inhibiting the activity of urease, and is a potential urease inhibitor.