CN112250737B - Auricularia polytricha glycopeptide with lead-removing function and preparation method and application thereof - Google Patents
Auricularia polytricha glycopeptide with lead-removing function and preparation method and application thereof Download PDFInfo
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- CN112250737B CN112250737B CN202011154452.7A CN202011154452A CN112250737B CN 112250737 B CN112250737 B CN 112250737B CN 202011154452 A CN202011154452 A CN 202011154452A CN 112250737 B CN112250737 B CN 112250737B
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- glycopeptide
- lead
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- auricularia polytricha
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- 108010015899 Glycopeptides Proteins 0.000 title claims abstract description 103
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
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Abstract
The invention discloses auricularia polytricha glycopeptide with a lead removing function and a preparation method and application thereof. The technical scheme provided by the invention is the application of glycopeptide in preparing a medicine for treating lead poisoning, wherein the glycopeptide is named APL and is derived from auricularia polytricha; the N-terminal sequence of the APL is shown as a sequence 1 in a sequence table. The APL contains mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid. Experiments prove that compared with a model group, each dose group containing the glycopeptide APL extracted by the invention can effectively remove lead in liver, particularly the APL high dose group has a liver lead removal rate of 17.96 percent and an effect superior to a positive medicament liver lead removal rate of 16.01 percent. The glycopeptide APL of the present invention can be used for treating lead poisoning.
Description
Technical Field
The invention relates to the technical field of biology, in particular to auricularia polytricha glycopeptide with a lead-removing function and a preparation method and application thereof.
Background
Lead is a toxic heavy metal, has the characteristics of high utilization rate, low recovery rate and difficult degradation, and is extremely harmful to human bodies. In addition to lead released from the environment due to natural causes, a great amount of pollution is generated by human activities such as raw ore mining, smelting and production of lead-containing products, and the lead pollution situation is very severe due to weak environmental awareness, imperfect environmental protection mechanism and the like. Lead pollution is mainly present in the atmosphere, soil and food, and lead in the environment can finally enter human bodies through biological enrichment or direct contact.
Lead metabolism in human body is quite slow, half-life period in blood and soft tissue exceeds one month, lead is mostly combined with erythrocytes or distributed in each tissue, the content of lead in liver and kidney is highest, lead has toxic effect on each system and organ, and lead mainly affects nervous system, reproductive system, cardiovascular system, urinary system, blood and the like. Lead has direct toxic effects on central and peripheral nervous systems, and can cause character changes such as depression or hyperactivity, mental retardation, sensory functions such as visual, auditory, olfactory disorders, and muscle damage. The existing medicines for treating lead poisoning mainly comprise chelating agent medicines, metallothionein, antioxidants and traditional Chinese medicines, different treatment methods can cause other problems while discharging lead, the chelating agent medicines can discharge trace elements necessary for a human body while discharging lead, some medicines can cause the phenomena of nausea, dizziness and weakness, even kidney injury, the natural extract has definite lead discharging curative effect, and the natural extract has great potential in research and development with small toxic and side effects.
Disclosure of Invention
The invention aims to solve the technical problem of how to reduce the content of heavy metal lead in human or animal bodies or how to effectively eliminate lead poison in the human or animal bodies or how to reduce the damage of the heavy metal lead to the human or animal bodies.
In order to solve the technical problems, the invention provides the application of glycopeptide in preparing a medicament for treating lead poisoning.
In the application of the glycopeptide in preparing the medicine for treating lead poisoning, the glycopeptide is named APL and is derived from auricularia polytricha; the N-terminal sequence of the APL is shown as a sequence 1 in a sequence table.
In the above application, the glycopeptide may be derived from fruiting body of Auricularia polytricha.
In the above application, the APL contains mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid.
In the application, the molar ratio of mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid is 27.8: 8: 19.3: 22.7: 8.7: 30: 9.
in the above application, the APL has a molecular weight of 34000 daltons.
In the above application, the glycopeptide is extracted from an aqueous extract of a fruit body of Auricularia polytricha, which is a water-soluble substance extracted from an fruiting body of Auricularia polytricha with water.
In the above application, the glycopeptide may be prepared as follows:
a method of preparing a glycopeptide, comprising:
b-1) preparing crude glycopeptide of auricularia polytricha with protein removed, wherein the preparation method of the crude glycopeptide of auricularia polytricha with protein removed comprises the steps of precipitating an aqueous extract of fruiting bodies of auricularia polytricha by using ethanol, collecting precipitates, and removing protein in the precipitates to obtain the crude glycopeptide of auricularia polytricha with protein removed; the water extract of the fruit body of the auricularia polytricha is a water-soluble substance extracted from the fruit body of the auricularia polytricha by water;
b-2) separating and purifying the glycopeptide from the crude glycopeptide of the protein-removed auricularia polytricha to obtain the glycopeptide named APL.
In the step B-2), the separation and purification of glycopeptide from the crude glycopeptide of protein-removed auricularia polytricha comprises:
b-2-1) carrying out anion exchange column chromatography on the crude auricularia polytricha glycopeptide with the protein removed, wherein the anion exchange group adopted in the anion exchange column chromatography is DEAE, the adopted elution procedure is two-step elution, the first step of elution is carried out by using water, and the second step of elution is carried out by using the following solution with the pH value of 7.0: the solute is 0.8M NaCl, the solvent is water (namely 0.8M NaCl aqueous solution), the elution peak obtained by the second step of elution is collected and named as elution peak D2;
b-2-2) carrying out gel filtration chromatography on the elution peak D2 to obtain the glycopeptide with the molecular weight of 34000 daltons, namely the APL glycopeptide.
In the above glycopeptide preparation method, the water may be deionized water.
In the above glycopeptide preparation method, the chromatography medium for gel filtration chromatography may be Superdex 200, and the elution buffer may be 0.2M NH with pH of 8.54HCO3An aqueous solution. 0.2M NH at pH 8.54HCO3The aqueous solution is 0.2M NH in solute4HCO3The solvent is a solution of water. The water may be ultrapure water.
The glycopeptides described above are also within the scope of the present invention.
In order to solve the technical problems, the invention also provides a medicine for treating lead poisoning.
The medicine for treating lead poisoning contains the glycopeptide.
The active ingredient of the medicine for treating lead poisoning can be the glycopeptide, the active ingredient of the medicine for treating lead poisoning can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the medicine for treating lead poisoning can be determined by a person skilled in the art according to the lead removal effect.
The above-mentioned medicament for treating lead poisoning may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; the pharmaceutically acceptable carrier can be humectant and binder, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, Mel, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; the pharmaceutically acceptable carrier can be a disintegrating agent, such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.
Herein, the treatment of lead poisoning may be embodied as at least one of:
A1) inhibiting the elevation of blood lead in animals exposed to lead;
A2) reducing lead deposition in the animal;
A3) reducing the blood lead content of animals suffering from lead poisoning;
A4) promoting the discharge of lead in the animal body;
A5) improving lead clearance of animal liver;
A6) reduces the damage of lead to various systems and/or organs of the animal body.
The animal described herein can be a mammal.
The experimental results show that compared with the model group, each dose group containing the glycopeptide APL extracted by the invention can effectively remove the lead in the liver, and particularly, the liver lead removal rate of the APL high dose group reaches 17.96%, and the effect is superior to the positive medicament liver lead removal rate of 16.01% (Table 4). The glycopeptide APL of the present invention can be used for treating lead poisoning.
Drawings
FIG. 1 is a DEAE-Cellulose elution curve.
FIG. 2 is a FPLC-Superdex 200 elution curve.
FIG. 3 is an infrared spectrum analysis of the glycopeptide APL. The ordinate is absorbance, and the abscissa is wave number (cm)-1)。
FIG. 4 is an HPLC profile of monosaccharide and uronic acid analysis of glycopeptide APL. The ordinate is abundance (mAU) and the abscissa is time (min).
Detailed Description
The glycopeptide APPI in the following examples was prepared according to the method in example 1 of the chinese invention patent application publication No. CN108727474A, published on 11/02/2018.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of Auricularia polytricha glycopeptide and its application in lead removal
Preparation of auricularia polytricha glycopeptide
1 test Material
Auricularia polytricha (Mont.) Sacc.) No. 3 (Zhaoshuang et al. optimization of fermentation conditions for polysaccharide production from Auricularia polytricha mycelia. food science, vol. 37, No. 4 in 2012), hereinafter referred to as Auricularia polytricha.
2 separation and purification of auricularia polytricha glycopeptide
2.1 extraction of crude glycopeptides by Water extraction and alcohol precipitation
Placing the freeze-dried Auricularia polytricha fruiting body into a high-speed universal crusher, repeatedly crushing for 4 times, each time for 20s, and making into dry powder with uniform texture. Crushing the treated dry powder, weighing and quantifying, adding deionized water according to the proportion of 1:25, and standing overnight in a refrigerator at 4 ℃ to obtain the auricularia polytricha mixed solution. Shaking the mixed solution of the auricularia polytricha evenly before water bath, sealing, carrying out high-temperature water bath at the temperature of 4h and 90 ℃ by using a water bath shaking table under the control of the temperature and the rotating speed, centrifuging the mixture at 6000r/min for 30min after water bath, collecting supernatant (water-soluble substances), measuring the volume of the supernatant, adding absolute ethyl alcohol according to the proportion of 1:4 to separate out polysaccharide, covering tin foil paper after even stirring, standing for 12 hours for solid-liquid separation, centrifuging 6000g for 20min, collecting precipitate, drying the precipitate in a 60 ℃ oven until the mass is constant, and grinding the precipitate into powder, wherein the powder is the crude auricularia polytricha glycopeptide.
2.2 isolation and purification of crude glycopeptides of Auricularia polytricha
Removing protein in the crude auricularia polytricha glycopeptide in the step 1.2.1 by using a Seveage method to obtain the crude auricularia polytricha glycopeptide with the protein removed, wherein the specific method comprises the following steps: and (3) dissolving the crude auricularia polytricha glycopeptide obtained in the step 1.2.1 in deionized water to obtain a crude auricularia polytricha glycopeptide solution. Adding Sevag reagent (prepared by mixing chloroform and n-butanol at a volume ratio of 4: 1) into crude glycopeptide solution of Auricularia polytricha in 1/3 volume, vortex and shake for 5min, centrifuging at 4500g for 15min, sucking supernatant, and removing gel precipitate generated by free protein. Sevag reagent, which was a volume of 1/3 supernatant, was added to the supernatant, vortexed for 5min, centrifuged at 4500g for 15min, and the supernatant aspirated, which was repeated several times until a supernatant free of protein layer was obtained. And (3) reserving the supernatant after each deproteinization, merging the supernatants, adding absolute ethyl alcohol to obtain glycopeptide precipitate, drying at 60 ℃, and dissolving with deionized water to obtain the crude glycopeptide solution of the auricularia polytricha with the deproteinization removed.
2.3 anion exchange column chromatography separation and purification of crude glycopeptide solution of Auricularia polytricha from which proteins are removed
Purifying by anion exchange column chromatography using DEAE-Cellulose, wherein the anion exchange group is DEAE, and the DEAE-Cellulose elution conditions are as follows: deionized water is used for balancing a DEAE-Cellulose chromatographic column (the specification of the chromatographic column is 1cm (inner diameter) × 30cm (column length)), the sample is loaded, the sample is the crude sugar peptide solution of the auricularia polytricha from which the protein is removed, two-step elution is carried out, the flow rate is 1.5mL/min, the eluted liquid is uninterruptedly collected from the beginning of the elution procedure, 6mL of the eluted liquid is collected in each tube, and the polysaccharide concentration of the eluate collected in each tube is measured by a sulfuric acid-phenol method. Eluting the first step with deionized water, collecting the elution peak obtained from the first step, and defining the elution peak as elution peak D1 (namely the elution volume is 7-18 mL); the second elution was eluted with the following solution at pH 7.0: the solute was 0.8M NaCl and the solvent was deionized water, and the peak eluted in the second step was collected and defined as peak D2 (i.e., an eluate with an elution volume of 37mL to 48 mL) (FIG. 1).
Respectively dialyzing the elution peak D1 and the elution peak D2 in distilled water for 10-12 hours, adding 4 times of volume of absolute ethyl alcohol, standing for 12 hours, centrifuging to collect precipitate, drying the precipitate at 60 ℃, and grinding into powder to respectively obtain a D1 component (from the elution peak D1) and a D2 component (from the elution peak D2).
The D2 component is determined to have the function of lead removal by in vitro lead adsorption detection.
Adding 150 mul of 10ppm lead single element standard solution into 150 mul of sample, mixing uniformly at room temperature, placing in an oscillator at 160rpm/min, fully shaking for 3h, quickly adding absolute ethyl alcohol with four times of volume after reaction, mixing uniformly, standing for 1h at room temperature, centrifuging for 10min at the rotating speed of 9000rpm/min, sucking supernatant, fixing the volume of the obtained supernatant to 5ml with 5% dilute nitric acid, and mixing uniformly. The sample is sent to a feed titer and safety supervision inspection test center of the department of agriculture to detect the lead content by a Z-2000 atomic absorption spectrophotometer-graphite furnace method, and deionized water is used as a reference instead of the sample.
Adsorption rate of glycopeptide to lead:
2.4 molecular Sieve chromatography purification of the D2 fraction
The fraction D2 was subjected to FPLC-Superdex 200 gel filtration chromatography (FPLC is Rapid protein liquid chromatograph, model ATKAexplorer, from GE Co., Ltd.; Superdex 200 is chromatography medium from GE Co., Ltd.), and elution buffer was 0.2M NH pH 8.54HCO3Solution (solute 0.2M NH)4HCO3The solvent is a solution of ultrapure water), the specification of the chromatography column is 30cm (column length) × 1cm (inner diameter), and the flow rate is 0.4 mL/min. And collecting the eluent by a partial collector, continuously collecting the eluted liquid from the beginning of the elution procedure, collecting 3mL of the eluent by each tube, and measuring the polysaccharide concentration of the eluent collected by each tube by a sulfuric acid-phenol method. The peak of the polysaccharide pool (elution volume of 13-18 mL of eluate) was collected and defined as the APL peak (FIG. 2).
2.5 Ultrafiltration concentration to obtain glycopeptide APL
Dialyzing APL elution peak in distilled water for 10-12 hr, ultrafiltering and concentrating at 4 deg.C with molecular weight cutoff of 5000 Dalton, freezing at-80 deg.C, and lyophilizing frozen sample to obtain powder (APL).
Molecular characterization of 3 glycopeptide APL
3.1 Infrared Spectroscopy (IR) analysis of glycopeptide APL
1-2mg of glycopeptide APL iS respectively taken, tabletting iS carried out by a KBr tabletting method, and detection and analysis are carried out by a Fourier transform infrared spectrometer Nicolet iS 5.
The infrared analysis result shows that the glycopeptide APL has a structure of a characteristic sugar group such as-OH, a stretching vibration absorption peak of a hydroxyl group, C ═ O, C — H absorption peak, and the like, and indicates that the glycopeptide APL has a polysaccharide structure (fig. 3).
3.2 monosaccharide and uronic acid analysis of glycopeptide APL
3.2.1, reagents
Trifluoroacetic acid, acetonitrile (chromatographically pure), phosphate buffer (pH 6.8), monosaccharide and uronic acid standards (mannose, rhamnose, glucose, xylose and galactose, galacturonic acid, glucuronic acid).
3.2.2 sample analysis method
3.2.2.1, complete acid hydrolysis:
weighing appropriate amount of lyophilized glycopeptide APL, adding 0.5mL of 2mol/L trifluoroacetic acid solution, and hydrolyzing at 120 deg.C for 120 min. And (5) drying by a nitrogen blowing instrument.
Treating a standard substance: firstly, preparing a 10mg/ml standard solution, and placing at-20 ℃. Taking out and melting, adding 5 μ L of each standard substance into a sealable glass tube, and mixing. Then, 0.5mL of a 2mol/L TFA solution was added, and the mixture was hydrolyzed at 120 ℃ for 120min simultaneously with the sample. And (5) drying by using an air pump.
3.2.2.2 PMP derivatization:
0.5ml of each of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone (PMP) reagent and 0.3mol/L NaOH solution dissolved in anhydrous methanol was added to the sample obtained after the hydrolysis and drying, and after the mixture was sufficiently mixed, the mixture was reacted in a water bath at 70 ℃ for 30 min. Cooled to room temperature, 0.3mol/L HCl 0.5ml is added and mixed well. 0.5ml of chloroform was added, followed by sufficient shaking extraction and centrifugation (5000rpm, 5min) to remove the chloroform layer, followed by extraction three times in total. The water layer (not less than 0.4ml) is filtered by a 0.22 μm filter membrane and is ready to be loaded.
3.2.2.3, apparatus conditions:
a chromatographic column: a SHISEIDO C18 column (4.6X 250mm,5 μm),
mobile phase 0.1mol/L Phosphate Buffer (PB) pH 6.8 acetonitrile 82:18 (v/v);
the flow rate is 1.0mL/min per minute;
the column temperature was 25 ℃;
sample size of 10 μ L
The wavelength was 245 nm.
The instrument comprises the following steps: agilent 1200 high performance liquid chromatograph
The assay results are shown below (fig. 4), and indicate that the glycopeptide APL contains monosaccharides, including mannose, rhamnose, glucose, galactose and xylose, and uronic acid; examples of uronic acids include glucuronic acid and galacturonic acid (Table 1). Mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid in a molar ratio of 27.8: 8: 19.3: 22.7: 8.7: 30: 9.
TABLE 1 monosaccharide and uronic acid content of glycopeptide APL
1.3.3 determination of N-terminal amino acid sequence of glycopeptide APL
The N-terminal amino acid sequence of the glycopeptide APL was determined by automated EDMAN degradation and the N-terminal sequence was determined using a protein sequencer equipped with an HPLC system from Hewlett Packard 1000A. The result shows that the N-terminal amino acid sequence of the APL is HDDMGMSAMM (sequence 1 in the sequence table), which indicates that the glycopeptide APL has a polypeptide structure.
1.3.4 molecular weight of glycopeptide APL
Gel filtration chromatography is used for measuring the molecular weight of the glycopeptide APL, and the result shows that the molecular weight of the glycopeptide APL is 34000 daltons.
Second, application of auricularia polytricha glycopeptide in lead elimination
And (3) dissolving the glycopeptide APL prepared in the step (2.5) in physiological saline to obtain a glycopeptide APL solution. The glycopeptide APPI prepared in step 6 of the Chinese patent application with the publication number of CN108727474A in example 1 is dissolved in physiological saline to obtain the glycopeptide APPI solution. Wherein, the characterization of the glycopeptide APPI is as described in paragraphs 0081-0093 of the Chinese invention patent application with publication number CN 108727474A. Dissolving disodium ethylene diamine tetraacetate calcium salt (EDTA-2NaCa) in physiological saline to obtain EDTA-2NaCa solution.
The experiment was set up in triplicate, 40 male SD rats (8 weeks old, weight 150-2Solution (solute is Pb (Ac)2Solvent is physiological saline) 0.5mL, Pb (Ac)2The dose of (2) is 20mg/kg body weight. Stopping the injection of Pb (Ac)2The solutions were recovered for 3d and randomly divided into 8 groups of 5, each group consisting of model group (negative control group), positive control group (positive drug EDTA-2NaCa injection), APL treatment group (APL low dose group, APL medium dose group, APL high dose group), and APPI treatment group (APPI low dose group, APPI medium dose group, and APPI high dose group). Administration was as follows:
APL low dose group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of glycopeptide APL solution administered by gavage, Pb (Ac) each time2The administered dose of (3) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administered dose of APL of each glycopeptide is 40mg/kg body weight/d.
Dose groups in APL: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of glycopeptide APL solution administered by gavage, Pb (Ac) each time2The administered dose of (3) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administered dose of APL of each glycopeptide is 80mg/kg body weight/d.
APL high dose group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of glycopeptide APL solution administered by gavage, Pb (Ac) each time2The administered dose of (3) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administered dose of APL of each glycopeptide is 160mg/kg body weight/d.
APPI low dose group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of glycopeptide APPI solution for intragastric administration, each time Pb (Ac)2The administered dose of (2) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administered dose of each glycopeptide APPI is 40mg/kg body weight/d.
Dosage group in APPI: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL solution and intragastric administrationGlycopeptide APPI solution 1.0mL, Pb (Ac) each time2The administration dose of (2) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administration dose of each glycopeptide APPI is 80mg/kg body weight/d.
APPI high dose group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of glycopeptide APPI solution for intragastric administration, each time Pb (Ac)2The administration dose of (2) is 5mg/kg body weight (small dose of plumbous toxin is given) and the administration dose of each glycopeptide APPI is 160mg/kg body weight/d.
Positive control group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of the solution and 1.0mL of EDTA-2NaCa solution administered by gavage, each time Pb (Ac)2The dose of (2) was 5mg/kg body weight (small dose of plumbism) and the dose of EDTA-2NaCa was 300mg/kg body weight/d per administration.
Model group: each mouse was intraperitoneally injected with Pb (Ac) 1 time per day for 30 consecutive days20.5mL of solution and 1.0mL of physiological saline, Pb (Ac) each time2The dose of (2) was 5mg/kg body weight (small dose of lead toxin was given) (Table 2).
From the beginning of administration, blood is taken from the orbit every 6 days, a blood sample is stored in a refrigerator at the temperature of 20 ℃ below zero, a rat is anesthetized at the end point of an experiment, the liver of the rat is picked up, the blood sample and the liver sample are ground into powder after being frozen and dried in vacuum, the blood sample and the liver sample are subjected to acidolysis and digestion at the end period of the storage experiment in the refrigerator at the temperature of 20 ℃ below zero, the sample is sent to a feed titer and safety supervision and inspection test center for detecting the lead content by using a Z-2000 atomic absorption spectrophotometer-graphite furnace method, a lead standard solution (Beijing Nanagang Chuanglian Biotechnology research institute) is used as a standard substance, and the lead content is quantitatively analyzed by using a standard curve method (external standard method).
The formula of the hepatic lead removal rate is as follows:
lead removal (%) - (model group mean-experimental group mean)/model group mean × 100%.
All data were processed statistically using independent sample t-tests of SPSS12.0(SPSS inc., USA) statistical software.
TABLE 2 rat groups and corresponding dosing treatments
TABLE 3 in vivo test rats blood lead content (μ g/L)
Note: "+" shows significant difference (P <0.05) from the model group, "#" shows very significant difference (P <0.01) from the model group, N ═ 5.
TABLE 4 in vivo test for removing hepatic lead of rats in each group
| Liver lead removal (%) | |
| Model set | 0 |
| Positive control group | 16.01 |
| APL Low dose group | 9.92 |
| APL Medium dose group | 14.95 |
| APL high dose group | 17.96 |
| APPI Low dose group | 3.09 |
| APPI Medium dose group | 2.56 |
| APPI high dose group | 2.33 |
After lead enters a human body, the lead firstly enters a blood circulation system in an ion form, and researches show that compared with a model group, each dosage group containing the glycopeptide APL extracted by the invention can effectively inhibit the increase of the blood lead of rats under the lead exposure condition after 12 days of administration treatment, the difference is extremely obvious (table 3), and the lead removing effect is more obvious along with the increase of the dosage, but compared with the model group, the APPI group does not show a remarkable trend of the decrease of the blood lead even under the condition of high-dosage administration. After lead enters the human body, it enters organs along with blood circulation and accumulates in body tissues, thus causing damage to the immune system, urinary system, and the like of the body. The liver is the most important organ of the gland of detoxification metabolism in vivo, and the detection of liver lead can reflect the deposition condition of lead in vivo, and the experimental result shows that each dosage group containing the glycopeptide APL extracted by the invention can effectively remove lead in the liver compared with a model group, particularly the APL high dosage group has the liver lead removal rate of 17.96 percent and the effect is superior to the positive medicament liver lead removal rate of 16.01 percent (Table 4). In conclusion, the glycopeptide APL extracted by the invention can effectively remove lead in the liver of the rat after the lead enters the rat body. The glycopeptide APL of the present invention may be used for the treatment of lead poisoning.
Sequence listing
<110> agriculture and forestry academy of sciences of Beijing City
<120> auricularia polytricha glycopeptide with lead-removing function, preparation method and application thereof
<130> GCSQ202585
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Auricularia polytricha (Auricularia polytricha)
<400> 1
His Asp Asp Met Gly Met Ser Ala Met Met
1 5 10
Claims (3)
1. The application of glycopeptide in preparing a medicine for treating lead poisoning, wherein the glycopeptide is named APL and is derived from auricularia polytricha; the N-terminal sequence of the glycopeptide is shown as a sequence 1 in a sequence table;
the APL is prepared according to a method comprising the following steps:
b-1) preparing crude glycopeptide of auricularia polytricha with protein removed, wherein the preparation method of the crude glycopeptide of auricularia polytricha with protein removed comprises the steps of precipitating an aqueous extract of fruiting bodies of auricularia polytricha by using ethanol, collecting precipitates, and removing protein in the precipitates to obtain the crude glycopeptide of auricularia polytricha with protein removed; the water extract of the fruit body of the auricularia polytricha is a water-soluble substance extracted from the fruit body of the auricularia polytricha by water;
b-2) separating and purifying glycopeptide from the crude glycopeptide of the protein-removed auricularia polytricha to obtain the glycopeptide named APL; the step B-2), wherein the separation and purification of glycopeptide from the protein-removed crude peptide of Auricularia polytricha comprises:
b-2-1) carrying out anion exchange column chromatography on the crude auricularia polytricha glycopeptide with the protein removed, wherein the anion exchange group adopted in the anion exchange column chromatography is DEAE, the adopted elution procedure is two-step elution, the first step of elution is carried out by using water, and the second step of elution is carried out by using the following solution with the pH value of 7.0: collecting the elution peak obtained in the second step of elution with 0.8M NaCl as solute and water as solvent, and naming the elution peak as elution peak D2;
b-2-2) carrying out gel filtration chromatography on the elution peak D2 to obtain the glycopeptide with the molecular weight of 34000 daltons, namely APL.
2. Use according to claim 1, characterized in that: the glycopeptide contains mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid; in the glycopeptide, the molar ratio of mannose, rhamnose, glucose, galactose, xylose, glucuronic acid and galacturonic acid is 27.8: 8: 19.3: 22.7: 8.7: 30: 9.
3. use according to claim 1 or 2, characterized in that: the treatment of lead poisoning is embodied by at least one of:
A1) inhibiting the elevation of blood lead in animals exposed to lead;
A2) reducing lead deposition in the animal;
A3) reducing the blood lead content of animals suffering from lead poisoning;
A4) promoting the discharge of lead in the animal body;
A5) improving lead clearance of animal liver;
A6) reduces the damage of lead to various systems and/or organs of the animal body.
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