CN115211491A - Application of broadleaf holly leaf in preparation of jack bean urease inhibitor - Google Patents
Application of broadleaf holly leaf in preparation of jack bean urease inhibitor Download PDFInfo
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- CN115211491A CN115211491A CN202210632865.4A CN202210632865A CN115211491A CN 115211491 A CN115211491 A CN 115211491A CN 202210632865 A CN202210632865 A CN 202210632865A CN 115211491 A CN115211491 A CN 115211491A
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- broadleaf holly
- holly leaf
- jack bean
- bean urease
- urease
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/90—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting the nitrification of ammonium compounds or urea in the soil
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- Life Sciences & Earth Sciences (AREA)
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- Food Science & Technology (AREA)
- Birds (AREA)
- Soil Sciences (AREA)
- Pest Control & Pesticides (AREA)
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- Health & Medical Sciences (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of natural medicines, and provides application of broadleaf holly leaf in preparation of a jack bean urease inhibitor 50 ) The concentration of the broadleaf holly leaf can reach 5.68 +/-0.26 mg/mL, the broadleaf holly leaf has obvious effect of inhibiting the urease activity of the jack beans, and the broadleaf holly leaf is a natural traditional Chinese medicinal material, has easily obtained raw materials, is safe, non-toxic and environment-friendly, and enriches the selectable varieties of the existing jack bean urease inhibitor; the jack bean urease inhibitor is further applied to preparation of plant fertilizers and animal feed additives, is beneficial to reduction of drug resistance, and has good application value and development prospect.
Description
Technical Field
The invention relates to the technical field of natural medicines, in particular to application of broadleaf holly leaf in preparation of a jack bean urease inhibitor.
Background
Urease (EC 3.5.1.5) is also called Urease or urea amidohydrolase. Among the urease enzymes, jack bean urease is a metalloenzyme which is found to contain nickel ions for the first time and has milestone significance in biochemical development history. Urease is widely present in the ecosystem and has an indispensable regulatory role in the metabolic cycle of natural nitrogen. Urease catalyzes the hydrolysis of urea to 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, urease catalyzes and hydrolyzes urea too fast to form ammonia, which can cause volatilization of ammonia and excessive accumulation of nitrate ions and ammonium ions in soil, thereby reducing the utilization rate of ammonia and polluting the environment such as atmosphere, water body, soil and the like; in animal husbandry, nonprotein nitrogen is decomposed too quickly by ruminant rumen urease, and the produced excess ammonia accumulates in ruminant blood, increasing the possibility of poultry blood ammonia poisoning. In addition, high ammonia levels in poultry houses can induce ruminants to develop various diseases and also can contaminate air quality. In the field of medicine, urease in the human body can decompose residual urea metabolites again and be absorbed by the body again, so that the acid-base balance of the human body is damaged, the local pH value is increased, and various diseases of the urinary system and other organs are caused.
The use of urease inhibitors is currently recognized as the most effective way to solve the dual economic and environmental problems caused by urease. Currently, research on urease inhibitors mainly includes three major categories, namely heavy metal salts, organic small molecules and plant extracts. Among them, the use of heavy metals is limited due to their high toxicity. In recent years, urease inhibitors are applied more and more widely at home and abroad, so that the development of safe, efficient and environment-friendly urease inhibitors becomes a hotspot of current research.
Disclosure of Invention
The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides the application of broadleaf holly leaves in preparing the jack bean urease inhibitor, the broadleaf holly leaves can obviously inhibit the activity of jack bean urease, and the broadleaf holly leaves can inhibit the median Inhibitory Concentration (IC) of the jack bean urease activity 50 ) Can reach 5.68 +/-0.26 mg/mL, and the broadleaf holly leaf is a natural medicine and can be used for preparing safe, efficient and environment-friendly urease inhibitor.
In a first aspect of the invention there is provided the use of broadleaf holly leaf in the preparation of a jack bean urease inhibitor.
Specifically, the application of broadleaf holly leaf in preparing a canavalise inhibitor is that the broadleaf holly leaf is derived from ligustrum robustum of ligustrum of Oleaceae.
The broadleaf holly leaf is derived from ligustrum robustum of ligustrum of Oleaceae, is a medicinal and edible medicinal material in Guizhou, sichuan and other areas, and is bitter in taste, slightly sweet and slightly cold in nature; entering liver, gallbladder and stomach meridians; has the effects of clearing away heat and toxic materials; it is traditionally used for conjunctival congestion, pharyngolaryngitis, stomatitis, acute mastitis, and pyogenic infections; modern pharmacological research shows that the broadleaf holly leaf has various pharmacological effects of reducing blood pressure and blood fat, resisting oxidation, inhibiting bacteria, relieving pain and the like. The invention discovers for the first time that the broadleaf holly leaf has the effect of inhibiting the urease activity of jack beans, has obvious effect, has the advantages of easily available raw materials, safety, no toxicity, environmental protection and the like, and can be used for preparing efficient jack bean urease inhibitors.
Preferably, the broadleaf holly leaf is a water extract or an alcohol extract of broadleaf holly leaf.
Preferably, the volume fraction of the alcohol is 60% to 80%.
More preferably, the volume fraction of the alcohol is 70%.
Preferably, the preparation method of the broadleaf holly leaf comprises the steps of taking dried broadleaf holly leaf, pulverizing, adding water or alcohol solvent for soaking, extracting, filtering, collecting filtrate, extracting the decoction dregs once again according to the same operation, combining the two filtrates, and concentrating to obtain extract.
Preferably, the alcoholic solvent is ethanol.
Preferably, the amount of the alcohol solvent is 5-15 times of the mass of the broadleaf holly leaf.
Preferably, the soaking time is 0.5 to 1 hour.
Preferably, the extraction time is 1-3 hours.
In a second aspect of the invention, there is provided a jack bean urease inhibitor.
The invention protects a jack bean urease inhibitor which comprises the broadleaf holly leaf.
Preferably, a pharmaceutically acceptable carrier and/or an auxiliary material is also included.
In a third aspect of the invention, there is provided the use of a jack bean urease inhibitor.
The invention protects the application of the jack bean urease inhibitor in preparing animal feed additives.
Preferably, the animal is a ruminant or a monogastric animal.
The canavalia cylindrical urease inhibitor is added into 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 broilers can be reduced, the utilization rate of the urea is improved, and the ammonia content in air in poultry houses and livestock houses is reduced.
The invention protects the application of the jack bean urease inhibitor in the preparation of plant fertilizers.
Preferably, the plant fertilizer is a nitrogen fertilizer.
The canavalia ensiformis urease inhibitor is added into a nitrogen fertilizer, so that the utilization rate of urea is improved, the yield of crops is increased, and the production performance is improved.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention uses broadleaf holly leaf to prepare the canavalise inhibitor, and the broadleaf holly leaf inhibits the median Inhibitory Concentration (IC) of the canavalise activity 50 ) Can reach 5.68 +/-0.26 mg/mL, the broadleaf holly leaf has obvious activity of inhibiting the jack bean urease, the raw materials are easy to obtain, safe and nontoxic, no harm is caused to the environment, the environment is protected, and the selectable types of the existing jack bean urease inhibitor are enriched;
(2) The invention utilizes broadleaf holly leaves to prepare the jack bean urease inhibitor, and then further applies the jack bean urease inhibitor to the preparation of plant fertilizers and animal feed additives, which is beneficial to reducing the drug resistance of the drugs.
Drawings
FIG. 1 is a graph showing the inhibitory activity of aqueous and alcoholic extracts of Folum Ilicis on jack bean urease;
FIG. 2 is a graph showing the type of inhibition of jack bean urease by Folum Ilicis;
FIG. 3 is a graph showing the progress of the reaction between Kuding tea and jack bean urease.
Detailed Description
In order to make the technical solutions of the present invention more clearly apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
1. Experimental materials
Test drug
Sedoula ureabase (Type III, 40.3U/mg), available from Sigma Aldrich, USA; urea, dithiothreitol and cysteine were all purchased from solibao; glutathione was purchased from melphalan organisms; sodium fluoride and boric acid were purchased from mcelin. Hydroxyethyl piperazine ethanethiosulfonic acid (HEPES), sodium salicylate, sodium nitroprusside, sodium hydroxide, sodium hypochlorite and glycerol were purchased from Guangzhou chemical reagent factories and were all analytically pure.
2. Preparation of jack bean urease
A proper amount of jack bean urease is weighed and dissolved in 20mM HEPES buffer solution (pH value is 7.5) to prepare a jack bean urease solution with the concentration of 10U/mL, and the jack bean urease solution is placed in a refrigerator at the temperature of 4 ℃ for storage for later use.
3. Preparation of urea
An appropriate amount of urea was weighed and dissolved in 20mM HEPES buffer (pH 7.5) to prepare a 150mM urea solution, which was stored in a refrigerator at 4 ℃ for further use.
4. Preparation of Bertholot color developing solution
Solution A: respectively weighing appropriate amount of sodium nitroprusside and sodium salicylate powder, dissolving in 20mM HEPES buffer solution (pH 7.5) to obtain color developing solution A containing 9.73mM sodium nitroferricyanide and 700mM sodium salicylate, and storing at 4 deg.C in dark place;
and B, liquid B: weighing 9g of sodium hydroxide, dissolving in 20mM HEPES buffer solution (pH value is 7.5), cooling, adding 12mL of sodium hypochlorite, mixing uniformly, diluting to 50mL, and storing at 4 ℃ in a dark place for later use.
Example 1 preparation and formulation of aqueous extract of Folum Ilicis
The application of broadleaf holly leaf in preparing the garden sword bean urease inhibitor is characterized in that the broadleaf holly leaf is derived from ligustrum robustum in ligustrum of Oleaceae, the broadleaf holly leaf is a water extract of the broadleaf holly leaf, and the preparation method of the water extract of the broadleaf holly leaf comprises the following steps:
preparing the water extract of the broadleaf holly leaf: taking dried broadleaf holly leaves, pulverizing into powder, weighing 50g, adding pure water with the weight being 10 times of that of the broadleaf holly leaves, soaking for 0.5 hour, heating and refluxing for 2 hours, filtering while the solution is hot, collecting filtrate, extracting the dregs once again according to the same operation, combining the two filtrates, and placing the filtrate in a rotary evaporator for reduced pressure concentration to obtain broadleaf holly leaf extract.
Example 2 preparation and formulation of an alcohol extract of Folum Ilicis
The application of broadleaf holly leaf in preparing the garden canary urease inhibitor is characterized in that the broadleaf holly leaf is derived from ligustrum robustum in ligustrum of oleaceae, the broadleaf holly leaf is an alcohol extract of the broadleaf holly leaf, and the preparation method of the alcohol extract of the broadleaf holly leaf comprises the following steps:
preparing an alcohol extract of broadleaf holly leaf: taking dried broadleaf holly leaf, pulverizing into powder, weighing 50g, adding 70% ethanol which is 10 times of the mass of the broadleaf holly leaf, soaking for 0.5 hour, heating and refluxing for extraction for 2 hours, filtering while hot, collecting filtrate, extracting herb residues once again according to the same operation, combining the filtrates, and concentrating the filtrate in a rotary evaporator under reduced pressure to obtain the broadleaf holly leaf extract without ethanol smell.
Example 3 study of inhibitory Effect of Folum Ilicis on Canavalia urensis urease
Mixing jack bean urease solution with a series of concentrations of the water extract solution of Folum Ilicis prepared in example 1 and the alcohol extract solution of Folum Ilicis prepared in example 2, respectively, and incubating at 37 deg.C for 20 min; adding a urea solution, and reacting for 20 minutes at room temperature in a dark place; adding Berthlot color development solution, developing for 10 min in dark, sucking 200 μ L of incubation solution to 96-well plate, and measuring OD at 595nm on enzyme-linked immunosorbent assay Absolute The value is obtained. Each concentration was done 3 times in parallel. The blank was replaced with the solvent of each dilution, and the OD was measured in the same manner as above Blank space . Determining the corresponding OD according to equation 1 Relative to each other The value is obtained. The residual enzyme activity was determined according to equation 2 and the corresponding median inhibitory concentration was determined by GraphPad(IC 50 ) Expressed as mean ± sem.
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)
The results are shown in FIG. 1, in which FIG. 1A shows that the aqueous extract of Folum Ilicis inhibits jack bean urease activity, and FIG. 1B shows that the alcohol extract of Folum Ilicis inhibits jack bean urease activity, wherein the half Inhibitory Concentration (IC) of the aqueous extract and alcohol extract of Folum Ilicis inhibits jack bean urease activity 50 ) 11.81 plus or minus 0.65mg/mL and 5.68 plus or minus 0.26mg/mL respectively show that the broadleaf holly leaf has obvious effect of inhibiting the activity of jack bean urease, and the alcohol extract of the broadleaf holly leaf has stronger effect of inhibiting the jack bean urease than the water extract of the broadleaf holly leaf.
Example 4 Studies of urease inhibiting type of Folum Ilicis
A certain concentration of jack bean urease and a series of concentrations of the alcohol extract solution of broadleaf holly leaf (0, 1.25, 2.5, 5, 10 mg/mL) prepared in example 2 were mixed well and incubated at 37 ℃ for 20 minutes. Then, a series of concentrations of urea solution (0.469-15 mM) was added at room temperature and the mixture was left to react for 20 minutes in the dark, and then the color was developed and OD was measured according to the method of example 3 Absolute Value 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)]) Drawing a Lineweaver-Burk diagram to obtain an L-B curve, and finally obtaining a kinetic parameter K through the L-B curve in combination with a formula 3 M 、v max And performing secondary mapping by using L-B curve to obtain inhibition constant K i 。
As shown in FIG. 2, FIG. 2A shows the Lineweaver-Burk reciprocal number chart of Kuding tea for inhibiting Canavalia urease, and FIG. 2B shows the slope of the curve in the Lineweaver-Burk reciprocal number chart of Kuding tea for inhibiting Canavalia urease and the inhibitionA dose concentration profile; from the Lineweaver-Burk reciprocal number chart, the kinetic parameter K of broadleaf holly leaf for inhibiting the urease of jack beans along with the change of the concentration of the broadleaf holly leaf is known M A small variation, v max The values gradually decreased with increasing concentration of Folum Ilicis, and it was preliminarily concluded that the type of action of Folum Ilicis for inhibiting the urease activity of Canavalia gladiata is a non-competitive inhibition type. In addition, the inhibition constant K of broadleaf holly leaf 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 broadleaf holly leaf for inhibiting the jack bean urease i It was 2.21. + -. 0.67mg/mL.
Example 5 kinetic study of broadleaf holly leaf for inhibiting jack bean urease
Taking a series of concentration ethanol extract solutions of broadleaf holly leaf (0, 1.25, 2.5, 5, 10 mg/mL) of the same volume and a certain concentration of jack bean urease, mixing the solutions uniformly, incubating the mixture in an incubator at 37 ℃ for 20 minutes, and adding an equal volume of urea solution at room temperature for 20 minutes in a dark place (incubation system) or directly adding an equal volume of urea solution without incubation in the incubator for 20 minutes in a dark place (no incubation system). Then, 200. Mu.L of the reaction solution was transferred to a 1.5mL centrifuge tube at different time intervals (0-30 min), and then color development and OD measurement were performed according to the method of example 3 Absolute value Value 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 control and 3 replicates were measured.
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. Curve fitting was performed on each test point for each concentration of broadleaf holly leaf extract according to equation 4 to determine the rate of binding of broadleaf holly leaf to jack bean urease.
P t ＝V s *t+(V 0 -V s )(l-e -kapp*t )*k app -1 (formula 4)
Wherein P is the amount of the reaction product accumulated t minutes after the start of the reaction, V 0 And Vs is the reaction initiation rate and the reaction equilibrium rate, respectively, and kapp is V 0 And the apparent first order rate constant that is followed when interconverting between Vs.
Fig. 3A and 3B are curves of progress of the reaction between broadleaf holly leaves and jack bean urease without and with incubation, respectively, and it can be seen from the curves of progress of the reaction between broadleaf holly leaves and jack bean urease in fig. 3 that broadleaf holly leaves and jack bean urease first form urease-broadleaf holly leaf complex EI rapidly and then isomerize slowly into urease-broadleaf holly leaf complex EI. From the above results, it was found that the binding mode of broadleaf holly leaf and jack bean urease is a slow binding type.
The experiments show that the broadleaf holly leaf can obviously inhibit the activity of jack bean urease. In addition, kinetic studies have shown that broadleaf holly leaf is a non-competitive, slow-binding urease inhibitor. In conclusion, the broadleaf holly leaf has the effect of resisting the jack bean urease, has an obvious effect, is a potential urease inhibitor, is cheap and easy to obtain, is safe and nontoxic by combining with the broadleaf holly leaf serving as a traditional Chinese medicinal material, has good application value and development prospect in the aspect of inhibiting the jack bean urease activity, and can be applied to preparation of animal feed additives and plant fertilizers.
Claims (9)
1. The application of broadleaf holly leaf in preparing a canavalise inhibitor is characterized in that the broadleaf holly leaf is derived from ligustrum robustum (Ligustrum robustum) in Oleaceae.
2. The use as claimed in claim 1, wherein the broadleaf holly leaf is an aqueous or alcoholic extract of broadleaf holly leaf.
3. Use according to claim 2, wherein the alcohol has a volume fraction of 60% to 80%.
4. A jack bean urease inhibitor comprising the broadleaf holly leaf of any one of claims 1-3.
5. The jack bean urease inhibitor according to claim 4, further comprising a pharmaceutically acceptable carrier and/or adjuvant.
6. Use of the jack bean urease inhibitor according to claim 4 or 5 for the preparation of an animal feed additive.
7. Use according to claim 6, wherein the animal is a ruminant or a monogastric animal.
8. Use of the jack bean urease inhibitor according to claim 4 or 5 for the preparation of plant fertilizers.
9. Use according to claim 8, wherein the plant fertilizer is a nitrogen fertilizer.
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| JP2004091338A (en) * | 2002-08-29 | 2004-03-25 | Maruzen Pharmaceut Co Ltd | Urease inhibitor, ammonia smell suppressant and diaper rash preventing agent |
| CN103315169A (en) * | 2013-05-21 | 2013-09-25 | 程庆宝 | Angora rabbit feed comprising immature chinese holly leaf |
| CN103601602A (en) * | 2013-11-21 | 2014-02-26 | 苏州科技学院 | Method for extracting urease inhibiting ingredient from glossy privet leaf and application of urease inhibiting ingredient |
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