CN108586590B - Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge - Google Patents

Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge Download PDF

Info

Publication number
CN108586590B
CN108586590B CN201810411610.9A CN201810411610A CN108586590B CN 108586590 B CN108586590 B CN 108586590B CN 201810411610 A CN201810411610 A CN 201810411610A CN 108586590 B CN108586590 B CN 108586590B
Authority
CN
China
Prior art keywords
grifola frondosa
mercury
polysaccharide peptide
polysaccharide
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810411610.9A
Other languages
Chinese (zh)
Other versions
CN108586590A (en
Inventor
张薇薇
王贺祥
兰进
陈向东
蒋雪珍
郑小杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Medicinal Plant Development of CAMS and PUMC
Original Assignee
Institute of Medicinal Plant Development of CAMS and PUMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Medicinal Plant Development of CAMS and PUMC filed Critical Institute of Medicinal Plant Development of CAMS and PUMC
Publication of CN108586590A publication Critical patent/CN108586590A/en
Application granted granted Critical
Publication of CN108586590B publication Critical patent/CN108586590B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nutrition Science (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to grifola frondosa and application of grifola frondosa polysaccharide peptide in promoting in-vivo mercury discharge. The research of the invention finds that the grifola frondosa has the function of promoting the discharge of mercury in vivo, and has wide application value in the development and application of medicines, foods and health-care products for promoting the discharge of mercury in vivo. Compared with the Grifola frondosa fruiting body and the fermentation mycelium, the Grifola frondosa polysaccharide peptide has the function of better promoting the discharge of mercury in rats.

Description

Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to grifola frondosa and application of grifola frondosa polysaccharide peptide in promoting in-vivo mercury discharge.
Background
Mercury is widely present in human living environment, seriously pollutes the living environment of people and harms the health of people. Mercury in different valence states can exist in three different forms, namely solid phase, liquid phase and gas phase, mercury in different phases can enter a human body through different routes, wherein the most important is respiration and dietary intake, and once mercury enters the human body, mercury can flow through the whole body through a circulatory system and is accumulated in the brain, the kidney and the liver. The mercury can be evaporated into a gas state at normal temperature, the settling velocity is low, the mercury can be stored in the atmosphere for a long time, and the mercury can be circularly dispersed to all places along with the atmosphere.
Grifola frondosa (Grifola frondosa), known under the English name Henofwoods (forest chickens) or Sitting-henmushroom (hens in the nest), belongs to the kingdom Fungi (Fungi), Basidiomycota (Basidiomycota), Hymenomycetes (Hymenomycetes), Aphyllophorales (Aphylophorales), Polyporaceae (Polyporaceae), Grifola (Grifola). Grifola frondosa polysaccharide is an effective immunomodulator, and the structure of the polysaccharide contains a plurality of beta-1, 6-branched chain beta-1, 3-glucan and beta-1, 3-branched chain beta-1, 6-glucan which are highly branched, so that the structure can greatly stimulate the immunoregulatory activity of the grifola frondosa polysaccharide and improve the immunity of the organism.
Active substances capable of adsorbing mercury are not found in grifola frondosa at present.
Disclosure of Invention
The research of the invention finds that the Grifola frondosa (Grifola frondosa) has the function of promoting the discharge of mercury in vivo, and has wide application value in the development and application of medicines, foods and health care products for promoting the discharge of mercury in vivo. Based on the research, the invention provides the application of the grifola frondosa in preparing the medicines, foods and health-care products for promoting the discharge of mercury in the body
The grifola frondosa provided by the invention comprises grifola frondosa sporocarp, fermented mycelia and grifola frondosa polysaccharide peptide.
The research of the invention also finds that the grifola frondosa polysaccharide peptide has better effect of promoting the discharge of mercury in rats compared with grifola frondosa sporocarp and fermentation mycelium.
Furthermore, the molecular weight of the grifola frondosa polysaccharide peptide is 80-110kDa, and preferably about 96 kDa.
Furthermore, in the grifola frondosa polysaccharide peptide, the polysaccharide content is 85-95%, and the protein (or polypeptide) content is 5-15%; preferably, the polysaccharide content is 89.6% and the protein (or polypeptide) content is 10.4%.
Furthermore, the N-terminal sequence of the protein (or polypeptide) in the grifola frondosa polysaccharide peptide is as follows: APPGMHQKQQ, respectively; after mass spectrometry analysis, the sequence also contains 7 sequences with higher reliability, which are respectively:
LVSLSCDPNHTFSIDGHSLTVIEADSVNLKPHTVDSIQIFAAQR；
SLYDVDDDSTVITLADWYHLAAR；
QAILVNDVFPSPLITGNKGDR；
VGPAIPTADATLINGLGR；
YSFVLNADQDVDNYWIR；
SINTLNADLAVITVTK；
NFDGGVNSAILR。
the grifolan polysaccharide peptide can be prepared by water extraction and alcohol precipitation of grifola frondosa sporocarp or fermentation mycelium.
Further, the invention also provides a preparation method of the grifola frondosa polysaccharide peptide, which comprises the following steps:
1) weighing dried Grifola frondosa fruiting body or dried Grifola frondosa mycelium, adding appropriate amount of deionized water, crushing, mixing, and extracting at 80-100 deg.C; centrifuging to obtain supernatant;
2) adding a proper amount of deionized water into the residue obtained by centrifugation in the step 1), uniformly mixing, and extracting at 80-100 ℃; centrifuging to obtain supernatant;
3) mixing the supernatants obtained in the step 1) and the step 2), and performing vacuum rotary evaporation and concentration to obtain a water extraction active substance concentrated solution; then adding a proper amount of ethanol, and standing overnight; centrifuging; and (5) taking the precipitate, and drying to obtain the grifola frondosa polysaccharide peptide.
Preferably, the ratio of the extraction materials to the liquid is 1:5-20, and more preferably 1: 10;
the material-liquid ratio refers to the mass-volume ratio (m/v) of the dried ash tree fruit bodies or dried ash tree mycelium to deionized water in g/mL, or refers to the mass-volume ratio (m/v) of the dry weight of the extraction residue in the step 1) to the deionized water.
Preferably, the extraction temperature is 95 ℃; and/or the extraction time is 3-6 h.
Preferably, the centrifugation rotation speed is 6000-10000rpm, and is further preferably 9,000rpm for 10 min.
Further, the concentration temperature of the vacuum rotary evaporation is 50 to 80 ℃, and more preferably 60 ℃.
Further, the obtained water-extract active substance concentrate is added with ethanol in an amount of 3 to 5 times its volume, and left to stand overnight, more preferably with ethanol in an amount of 4 times its volume.
Further, the drying temperature is 40 to 60 ℃, more preferably 45 ℃.
In a preferred embodiment of the present invention, the method for preparing grifolan polysaccharide peptide comprises the following steps:
1) weighing dried Grifola frondosa fruiting body or dried Grifola frondosa mycelium, adding deionized water according to the material-liquid ratio of 1:10, mixing, and extracting at 95 deg.C (preferably 6 hr); centrifuging at 9,000rpm for 10min to obtain supernatant;
2) adding deionized water into the residue obtained by centrifugation in the step 1) according to the material-liquid ratio of 1:10, uniformly mixing, and extracting at 95 ℃ (preferably for 3 h); centrifuging at 9,000rpm for 10min to obtain supernatant;
3) mixing the supernatants obtained in the step 1) and the step 2), and performing vacuum rotary evaporation and concentration at 60 ℃ to obtain a water extraction active substance concentrated solution; then adding ethanol with the volume of 4 times of that of the mixture, and standing overnight; centrifuging at 9,000rpm for 10 min; and (4) drying the precipitate at 45 ℃ to obtain the grifola frondosa polysaccharide peptide.
On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.
The method can improve the extraction rate of the grifola frondosa polysaccharide peptide and the purity of the grifola frondosa polysaccharide peptide.
Drawings
FIG. 1 shows the process for extracting polysaccharide peptides from Grifola frondosa of example 1.
FIG. 2 shows the polyacrylamide gel electrophoresis of Grifola frondosa polysaccharide peptide prepared in example 1.
FIG. 3 shows the method for measuring the mercury content in example 2.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
In the following grifola frondosa polysaccharide peptides, the measurement of polysaccharide content was performed by the phenol-sulfuric acid method, and the measurement of protein content was performed by the Bradford method.
EXAMPLE 1 separation and extraction of Polypeptides from Grifola frondosa
Taking appropriate amount of Grifola frondosa fruiting body, and extracting Grifola frondosa polysaccharide peptide according to the method shown in figure 1 (in figure 1, "m/v" refers to mass-to-volume ratio, in g/mL).
Through detection, the polysaccharide content in the grifola frondosa polysaccharide peptide is 89.6 percent, and the protein content is 10.4 percent; the polysaccharide content is high, the protein content is low, and the ratio of the polysaccharide content to the protein content is 8.57. The molecular weight of the grifolan peptide was concentrated at 96kDa as demonstrated by Superdex75 gel filtration.
The extraction rate of Grifola frondosa polysaccharide peptide in this example was 0.9%.
Polypeptidyl prepared in this example was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) using 16% tricine gel, as shown in FIG. 2 (wherein GFPP represents grifola frondosa polyglycopeptide), giving a protein molecular weight of 6kDa, and its N-terminal sequence was detected as APPGMHQKQQ, and mass spectrometry gave 7 sequences with higher reliability, LVSLSCDPNHTFSIDGHSLTVIEADSVNLKPHTVDSIQIFAAQR, SLYDVDDDSTVITLADWYHLAAR, QAILVNDVFPSPLITGNKGDR, VGPAIPTADATLINGLGR, YSFVLNADQDVDNYWIR, SINTLNADLAVITVTK, NFDGGVNSAILR respectively.
EXAMPLE 2 determination of in vitro Mercury adsorption Rate
The Grifola frondosa polysaccharide peptide sample prepared in example 1 was accurately weighed, deionized water was added, and vortex oscillation was performed to dissolve the polysaccharide peptide sufficiently, to prepare a 4mg/mL sample solution.
HgCl was treated with 10% nitric acid solution2The mother liquor is diluted to 2 mug/mL and prepared as it is.
Experimental groups: the same volume of sample solution was aspirated along with 2. mu.g/mL of HgCl2Oscillating the solution at the room temperature at 160rpm for 2h, fully reacting, adding 4 times of volume of absolute ethyl alcohol, uniformly mixing, standing at the room temperature for 1h, centrifuging at 9,000rpm for 10min, taking the supernatant, and measuring the mercury content in the supernatant by using an AFS-930 double-channel atomic fluorescence spectrometer.
Control group: the sample solution in the above method was changed to the same volume of deionized water, and the rest was the same.
Figure BDA0001648234300000041
Using different concentrations of grifola frondosa polyglycopeptide and 2 mug/mLHgCl2The adsorption activity test of the equal-volume mixing shows that the adsorption rate of the mercury is increased along with the increase of the concentration of the polysaccharide peptide within a certain range, and the result is shown in table 1.
TABLE 1 in vitro adsorption rate (%) for mercury of Polypeptides of Grifola frondosa
Figure BDA0001648234300000042
Example 3 Mercury removal from acute Mercury-stained SD rats
Reagent related to animal experiment
1)HgCl2Working solution (1,100. mu.g/kg): weighing HgCl as required2Dissolving the powder in deionized water, mixing, and preparing.
2)HgCl2Working solution (600. mu.g/kg): weighing HgCl as required2Dissolving the powder in deionized water, mixing, and preparing.
3) Physiological saline (0.9% NaCl solution): accurately weighing 0.9g of NaCl solid, dissolving in a small amount of deionized water, and metering to 100mL of deionized water.
4) 3% sodium pentobarbital: 3g of pentobarbital sodium powder is weighed, dissolved in 100mL of physiological saline, uniformly mixed and stored at 4 ℃ in a dark place.
5) DMPS (5.26 mg/mL): 263 mgs DMPS powder is accurately weighed, dissolved in 50mL deionized water, mixed evenly and stored at 4 ℃ in the dark.
6) Low dose polysaccharide peptide (12 mg/mL): the grifola frondosa polysaccharide peptide is weighed according to the requirement and dissolved by a certain volume of deionized water.
7) High dose polysaccharide peptide (12 mg/mL): the grifola frondosa polysaccharide peptide is weighed according to the requirement and dissolved by a certain volume of deionized water (note: because the grifola frondosa polysaccharide peptide cannot be fully dissolved to obtain a uniform solution according to the concentration and is in a colloid state, a method of 12mg/mL polysaccharide peptide is adopted for replacing 4 times of intragastric gavage).
8) Low dose Grifola frondosa fruiting body powder feed (1.152g/100g total weight of powder): crushing the rat feed into powder, weighing 1.152g of Grifola frondosa sporophore dry powder, adding the feed powder to 100g, mixing uniformly, adding a proper amount of deionized water to form a dough, processing into a cylindrical feed, and drying at 45 ℃.
9) High dose grifola frondosa sporocarp powder feed (4.608g/100g total weight of powder): crushing the rat feed into powder, weighing 4.608g of Grifola frondosa sporophore dry powder, adding the feed powder to 100g, mixing uniformly, adding a proper amount of deionized water to form a dough, processing into a cylindrical feed, and drying at 45 ℃.
Second, establishment of acute mercuric infection poisoning model of rat
According to the data, no mature and unified method for establishing a mercury poisoning model exists at present, so that the mercury staining dosage is obtained by analysis after repeated experiments. Finally, the SD rat acute mercuric poisoning model is established by injecting HgCl into the abdominal cavity of the SD rat according to the dosage of 1,100 mug/kg body weight2The solution was injected in a volume of 1mL/300g body weight for 3 consecutive days, and administered orally 2 days after recovery.
Three, grouping and administration mode of rats
Experimental rats were randomly divided into 7 groups of 6 rats each, and a negative control group was provided in addition to the 6 groups listed in table 2: SD rats were injected with deionized water intraperitoneally at a dose of 1,100. mu.g/kg body weight, at an injection volume of 1mL/300g body weight, for 3 consecutive days, and after 2 days of recovery, rats were gavaged with deionized water at a volume of 0.8mL/100g body weight.
TABLE 2 grouping and administration of acute mercuric-staining rats
Figure BDA0001648234300000051
Fourthly, collecting and preserving samples
Before the first administration, and after the first administration on days 3, 6 and 15, SD rats are anesthetized with ether, 0.5mL of blood is collected from orbit, and the blood is filled in a trace blood collection tube containing heparin sodium, and the blood collection tube is repeatedly shaken after the blood collection to prevent blood coagulation and is stored at-20 ℃. On day 15, the blood was collected from the orbit, the rat was anesthetized with 3% (m/v) sodium pentobarbital, the liver and kidney were harvested, the superficial blood on the surface was washed with physiological saline, dried with filter paper, frozen at-80 ℃, and the viscera was lyophilized with a lyophilizer and ground into powder.
Fifthly, treating the sample and measuring the mercury content
The detection of the mercury content is completed by adopting microwave digestion and atomic fluorescence spectrometry in a feed titer and safety supervision inspection test center of Ministry of agriculture, and an AFS-930 double-channel atomic fluorescence spectrometer is used as an instrument. The specific method is shown in figure 2. Sixthly, the grifola frondosa promotes the discharge of mercury in the body of the rat infected with acute mercury
Using HgCl at a dose of 1,100. mu.g/kg2The solution is injected into the abdominal cavity of SD rats for 3 days continuously to cause acute mercury poisoning of the SD rats, and the oral administration is carried out after 2 days of recovery. In the experimental process, the change trend of the blood mercury content in the rat body is continuously monitored, and the liver mercury and kidney mercury content after 15 days is measured, so that the removing effect of the grifola frondosa on the mercury in the rat body is verified. The experimental results are shown in table 3, the mercury in the blood of the negative control group rat is always at a low level, which indicates that the experimental environment is basically free from mercury pollution and cannot influence the experimental results. And each mercury-stained group of rats was injected with HgCl intraperitoneally for 3 consecutive days2Obvious 'blood mercury peak' appears after the solution, and the blood mercury content of rats of each mercury staining group has very significant difference (p) compared with that of a control group<0.001) without significant difference in mercury content between rats in each mercury-stained group. After the start of dosing, rats were individually stained with mercuric oxideThe mercuric blood is continuously reduced until the normal mercuric blood level is reached in 15 days without differential reduction (table 3), which shows that the rats can discharge the mercuric blood through self-metabolism and reduce the toxic action of the mercury on the organism, but the table 3 also shows that the mercuric blood reduction speed of the rats of each administration group is higher compared with that of the rats of the model group, which shows that the grifola frondosa sporophore powder and the polyglycopeptide have the similar effect as DMPS, namely the mercuric blood discharge is promoted. It can also be seen from table 4 that the grifola frondosa sporocarp powder and the polyglycopeptide can promote the discharge of liver mercury and kidney mercury.
TABLE 3 Mercury content in blood administered to rats infected with acute mercuric infection
Figure BDA0001648234300000061
Note: compared with the negative control group, the test results show that,***p<0.001，**p<0.01，*p<0.05; in comparison to the set of models,###p<0.001，##p<0.01，#p<0.05。
TABLE 4 Effect of administration of Grifola frondosa on the content of liver and kidney mercury in rats with acute mercurial infections
Figure BDA0001648234300000071
Note: compared with the negative control group, the test results show that,***p<0.001，**p<0.01，*p<0.05; in comparison to the set of models,###p<0.001，##p<0.01，#p<0.05。
example 4 Grifola frondosa effect on promoting Mercury excretion from rats with persistent mercury
Establishment of model of continuous mercuric poisoning of SD rat
The SD rat continuous mercuric poisoning model is established on the basis of the acute mercuric poisoning model, and the SD rat is injected with HgCl in the abdominal cavity according to the dose of 1,100 mug/kg body weight2The solution is injected into a volume of 1mL/300g body weight for 3 days, and is administered by daily administration of 600 μ g/kg body weight from day 6 after 2 daysDose SD rats were injected intraperitoneally with HgCl2The solution is injected into a volume of 1mL/300g body weight, and is orally administered after 2-3 h. In the determination of the mercury removal effect of the grifola frondosa on the body of the acute mercury-stained SD rat, the activities of the grifola frondosa sporophore powder and the polysaccharide peptide high-dose group are higher than those of the low-dose group, so that only the high-dose group is arranged.
II, SD rat grouping and administration mode
The experimental rats were randomly divided into 5 groups of 6 rats each, the administration method was as shown in table 2, and the SD rats were subjected to intraperitoneal injection of deionized water at a dose of 1,100. mu.g/kg body weight in a volume of 1mL/300g body weight for 3 days in a negative control group, and after 2 days of recovery, deionized water was continuously injected every day from day 6, and after 2-3 hours, gastric administration of deionized water was performed at a rate of 0.8mL/100g body weight.
Thirdly, collecting and preserving samples
Before the first administration and every 6 days after the first administration, SD rats are anesthetized with ether, 0.5mL of blood is collected from eye sockets and filled into a trace blood collection tube containing heparin sodium, and the blood collection tube is repeatedly shaken after the blood collection to prevent blood coagulation and is stored at-20 ℃.
Fourthly, processing the sample and measuring the mercury content
Sample handling and assay methods were the same as those for the acute mercuric toxicity model in rats of example 3.
Fifthly, the grifola frondosa promotes the discharge of mercury in rats continuously infected with mercury
Using HgCl at a dose of 1,100. mu.g/kg2The solution is administered to SD rat via intraperitoneal injection for 3 days to cause acute mercuric poisoning of SD rat, and HgCl is administered via intraperitoneal injection at a dose of 600 μ g/kg per day after recovery for 2 days2The solution is orally taken after 2-3h, and the change trend of the blood mercury content in the rat body is continuously monitored, so that the mercury removing effect of the grifola frondosa on the rat body is verified. The change trend of blood mercury in rats is shown in table 5, and the blood mercury level of the negative control group rats is always very low, which indicates that no mercury pollution exists in the environment and no influence of exogenous mercury on the experimental results exists. And each mercury-stained group of rats was injected with HgCl intraperitoneally for 3 consecutive days2Obvious 'blood mercury peak' appears later, and the blood mercury content of rats of each mercury staining group is extremely obvious in average difference compared with that of a control group (p)<0.001), and eachThere was no significant difference in the mercury content in the blood among the rats in the mercury-stained group. After continuous mercury staining and administration, the mercuric blood of the rats in the model group always shows a growing situation, while the mercuric blood of the rats in each administration group is maintained to fluctuate within a relatively stable range. The difference between the high-dose group and the model group is significant (p) compared with the high-dose group of the ash tree flower fruit body powder in 12 days<0.05), and thereafter the difference between each administration group and the model group was more significant (p)<0.01 or p<0.001), which shows that the grifola frondosa sporocarp powder and the polysaccharide peptide can promote the discharge of the mercury from the blood of the rats continuously stained with mercury, like DMPS.
TABLE 5 Mercury content in blood of rats continuously stained with Mercury
Figure BDA0001648234300000081
Note: compared with the negative control group, the test results show that,***p<0.001，**p<0.01，*p<0.05; in comparison to the set of models,###p<0.001，##p<0.01，#p<0.05。
the experimental rat intakes 5g/300g of Grifola frondosa fruiting body powder feed every day, which is equivalent to eating 0.192-0.768g/kg of Grifola frondosa dry fruiting body every day; according to the equivalent dose conversion relation between animals and human bodies in pharmacological tests, 1.86-7.44g of dried grifola frondosa eaten by a 60kg adult per day can effectively promote the discharge of mercury in the bodies; if the fresh Grifola frondosa fruiting body is eaten, 18.6-74.4g of the fruiting body is eaten every day, and the same effect is achieved.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of medicinal plants of academy of Chinese medical science
<120> application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 44
<212> PRT
<213> Grifola frondosa
<400> 1
Leu Val Ser Leu Ser Cys Asp Pro Asn His Thr Phe Ser Ile Asp Gly
1 5 10 15
His Ser Leu Thr Val Ile Glu Ala Asp Ser Val Asn Leu Lys Pro His
20 25 30
Thr Val Asp Ser Ile Gln Ile Phe Ala Ala Gln Arg
35 40
<210> 2
<211> 23
<212> PRT
<213> Grifola frondosa
<400> 2
Ser Leu Tyr Asp Val Asp Asp Asp Ser Thr Val Ile Thr Leu Ala Asp
1 5 10 15
Trp Tyr His Leu Ala Ala Arg
20
<210> 3
<211> 21
<212> PRT
<213> Grifola frondosa
<400> 3
Gln Ala Ile Leu Val Asn Asp Val Phe Pro Ser Pro Leu Ile Thr Gly
1 5 10 15
Asn Lys Gly Asp Arg
20
<210> 4
<211> 18
<212> PRT
<213> Grifola frondosa
<400> 4
Val Gly Pro Ala Ile Pro Thr Ala Asp Ala Thr Leu Ile Asn Gly Leu
1 5 10 15
Gly Arg
<210> 5
<211> 17
<212> PRT
<213> Grifola frondosa
<400> 5
Tyr Ser Phe Val Leu Asn Ala Asp Gln Asp Val Asp Asn Tyr Trp Ile
1 5 10 15
Arg
<210> 6
<211> 16
<212> PRT
<213> Grifola frondosa
<400> 6
Ser Ile Asn Thr Leu Asn Ala Asp Leu Ala Val Ile Thr Val Thr Lys
1 5 10 15
<210> 7
<211> 12
<212> PRT
<213> Grifola frondosa
<400> 7
Asn Phe Asp Gly Gly Val Asn Ser Ala Ile Leu Arg
1 5 10

Claims (11)

1. The application of grifola frondosa polysaccharide peptide in preparing medicines for promoting in vivo mercury discharge;
wherein, the N-terminal sequence of the protein or polypeptide in the grifola frondosa polysaccharide peptide is as follows: APPGMHQKQQ, respectively;
the preparation method of the grifola frondosa polysaccharide peptide comprises the following steps:
1) weighing dried Grifola frondosa fruiting body or dried Grifola frondosa mycelium, adding appropriate amount of deionized water, crushing, mixing, and extracting at 80-100 deg.C; centrifuging at 6000-10000rpm to obtain supernatant;
2) adding a proper amount of deionized water into the residue obtained by centrifugation in the step 1), uniformly mixing, and extracting at 80-100 ℃; centrifuging at 6000-10000rpm to obtain supernatant;
3) mixing the supernatants obtained in the step 1) and the step 2), and performing vacuum rotary evaporation and concentration to obtain a water extraction active substance concentrated solution; then adding a proper amount of ethanol, and standing overnight; centrifuging; and (5) taking the precipitate, and drying to obtain the grifola frondosa polysaccharide peptide.
2. The use of claim 1, wherein the molecular weight of the grifolan peptide is 80-110 kDa.
3. The use of claim 1, wherein the molecular weight of the grifolan peptide is 96 kDa.
4. The use of claim 1, wherein the polysaccharide peptide of Grifola frondosa has a polysaccharide content of 85-95% and a protein content of 5-15%.
5. The use of claim 1, wherein the polysaccharide peptide of Grifola frondosa has a polysaccharide content of 89.6% and a protein content of 10.4%.
6. The use of claim 1, wherein the protein or polypeptide of the Grifola frondosa polysaccharide peptide further comprises the following 7 sequences:
LVSLSCDPNHTFSIDGHSLTVIEADSVNLKPHTVDSIQIFAAQR；
SLYDVDDDSTVITLADWYHLAAR；
QAILVNDVFPSPLITGNKGDR；
VGPAIPTADATLINGLGR；
YSFVLNADQDVDNYWIR；
SINTLNADLAVITVTK；
NFDGGVNSAILR。
7. the use according to claim 1, wherein the preparation method of the grifolan polysaccharide peptide comprises centrifuging at 9,000rpm for 10 min; and/or the presence of a gas in the gas,
the extraction temperature is 95 ℃; and/or the presence of a gas in the gas,
the ratio of the extract to the liquid is 1: 5-20.
8. The use according to claim 7, wherein the extraction time in the preparation method of the grifolan polysaccharide peptide is 3-6 h; and/or the ratio of the extraction materials to the liquid is 1: 10.
9. The use according to claim 1, wherein the preparation method of the grifolan polysaccharide peptide comprises the steps of:
1) weighing dried Grifola frondosa fruiting body or dried Grifola frondosa mycelium, adding deionized water according to the material-liquid ratio of 1:10, mixing, and extracting at 95 deg.C; centrifuging at 9,000rpm for 10min to obtain supernatant;
2) adding deionized water into the residue obtained by centrifugation in the step 1) according to the material-liquid ratio of 1:10, uniformly mixing, and extracting at 95 ℃; centrifuging at 9,000rpm for 10min to obtain supernatant;
3) mixing the supernatants obtained in the step 1) and the step 2), and performing vacuum rotary evaporation and concentration at 60 ℃ to obtain a water extraction active substance concentrated solution; then adding ethanol with the volume of 4 times of that of the mixture, and standing overnight; centrifuging at 9,000rpm for 10 min; and (4) drying the precipitate at 45 ℃ to obtain the grifola frondosa polysaccharide peptide.
10. Application of Grifola frondosa in preparing medicine for promoting mercury excretion in vivo is provided.
11. The use of claim 10, wherein the Grifola frondosa comprises Grifola frondosa fruiting body, fermented mycelium.
CN201810411610.9A 2018-04-26 2018-05-02 Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge Active CN108586590B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2018103815731 2018-04-26
CN201810381573 2018-04-26

Publications (2)

Publication Number Publication Date
CN108586590A CN108586590A (en) 2018-09-28
CN108586590B true CN108586590B (en) 2020-11-10

Family

ID=63619671

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810411610.9A Active CN108586590B (en) 2018-04-26 2018-05-02 Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge

Country Status (1)

Country Link
CN (1) CN108586590B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1213378A (en) * 1996-03-08 1999-04-07 株式会社雪国梅泰克 Antitumor substance extracted from Grifola
CN1398898A (en) * 2002-08-28 2003-02-26 维京仲华（上海）生物医药科技有限公司 Extraction and separation process of ash tree flower sporophore polyglycopeptide
CN1663959A (en) * 2004-03-27 2005-09-07 中国海洋大学 Beta-glucan peptide and its preparing process and application
JP2007031665A (en) * 2005-07-29 2007-02-08 Yukiguni Maitake Co Ltd Glycoprotein extracted from grifola frondosa
KR100740893B1 (en) * 2001-04-19 2007-07-19 충남대학교산학협력단 Anti-Cancer Compositions Containing Glycoprotein KGF-1
WO2008050910A1 (en) * 2006-10-27 2008-05-02 Yukiguni Maitake Co., Ltd. Grifola frondosa-origin substance having antiinfluenza virus activity and method of producing the same
CN103509091A (en) * 2013-10-10 2014-01-15 江苏大学 Grifola frondosa mycelium anti-tumor glycoprotein and preparation method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1213378A (en) * 1996-03-08 1999-04-07 株式会社雪国梅泰克 Antitumor substance extracted from Grifola
KR100740893B1 (en) * 2001-04-19 2007-07-19 충남대학교산학협력단 Anti-Cancer Compositions Containing Glycoprotein KGF-1
CN1398898A (en) * 2002-08-28 2003-02-26 维京仲华（上海）生物医药科技有限公司 Extraction and separation process of ash tree flower sporophore polyglycopeptide
CN1663959A (en) * 2004-03-27 2005-09-07 中国海洋大学 Beta-glucan peptide and its preparing process and application
JP2007031665A (en) * 2005-07-29 2007-02-08 Yukiguni Maitake Co Ltd Glycoprotein extracted from grifola frondosa
WO2008050910A1 (en) * 2006-10-27 2008-05-02 Yukiguni Maitake Co., Ltd. Grifola frondosa-origin substance having antiinfluenza virus activity and method of producing the same
CN103509091A (en) * 2013-10-10 2014-01-15 江苏大学 Grifola frondosa mycelium anti-tumor glycoprotein and preparation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A polysaccharide-peptide with mercury clearance activity from dried fruiting bodies of maitake mushroom Grifola frondosa;Weiwei Zhang等;《Scientific Reports》;20181204;1-9 *

Also Published As

Publication number Publication date
CN108586590A (en) 2018-09-28

Similar Documents

Publication Publication Date Title
CN107822948B (en) Composition with after-sun repair, oxidation resistance and whitening effects and preparation method thereof
WO2001051070A1 (en) Physiologically active substance eem-s originating in mushrooms, process for producing the same and drugs
Elkhateeb et al. To heal or not to heal? Medicinal mushrooms wound healing capacities
CN115724911B (en) Donkey-hide gelatin peptide and application thereof in preparation of health care products related to tonifying qi, nourishing blood or preventing miscarriage
CN111704650B (en) Polypeptide with uric acid reducing effect, composition, compound preparation, preparation method and application
CN112898383A (en) Oligopeptide, active peptide composition and application of oligopeptide and active peptide composition in preparation of product with anti-inflammatory effect
WO2020093510A1 (en) Separation and purification method for polysaccharide in ganoderma lucidum spores
CN102408477A (en) Antler plate protein peptide, as well as preparation method and application thereof
CN112940145A (en) Method for extracting and preparing animal ganoderma lucidum polysaccharide
RU2322091C1 (en) Composition of biologically active substances and method for preparing its nanodispersion
CN106749729B (en) A kind of Smilacina japonica polysaccharide and its preparation method and application
CN104998022B (en) A kind of dragon&#39;s blood leaf extract and preparation method thereof, its pharmaceutical composition, preparation and application
CN107011419B (en) Isatis root active glycopeptide with antiviral activity and preparation method and application thereof
CN108586590B (en) Application of grifola frondosa and grifola frondosa polysaccharide peptide in promoting in vivo mercury discharge
CN103864891A (en) Chinese chive seed antioxidative tripeptide, and preparation and application thereof
CN115844920A (en) Application of caulerpa lentillifera polysaccharide in preparation of medicine for treating osteoporosis
JP2005068112A (en) Extract from grifola frondosa (a kind of mushroom) and glycoprotein and method for producing the same
CN105998091B (en) A kind of extracting method and extract of Antrodia camphorata triterpene substance
CN109294984B (en) Lentinan capsule for efficiently amplifying NK cells in vivo and preparation method thereof
CN1250240C (en) Isatis root extract, and preparing method and use thereof
CN108186693B (en) Preparation method of paecilomyces hepiali active substance for reducing uric acid or treating hyperuricemia, active substance prepared by method and application thereof
TWI618540B (en) Composition for preventing renal toxicity caused by drug toxicity, preparation method thereof and Its use
JP4480204B2 (en) Anti-tumor fraction of Kawariharatake
CN115245558B (en) Preparation method of thick-shell mussel immunocompetent hexapeptide liposome
CN109771454A (en) Purposes of the birch young pilose antler extract in the preparation that preparation treatment and/or prevention high fat diet cause hepatic injury related disease

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant