CN104147038A - Fungal polysaccharide and composition thereof with anti-gastric ulcer effects - Google Patents
Fungal polysaccharide and composition thereof with anti-gastric ulcer effects Download PDFInfo
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- CN104147038A CN104147038A CN201410440557.7A CN201410440557A CN104147038A CN 104147038 A CN104147038 A CN 104147038A CN 201410440557 A CN201410440557 A CN 201410440557A CN 104147038 A CN104147038 A CN 104147038A
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Abstract
The invention relates to a fungal polysaccharide and a composition thereof with anti-gastric ulcer effects. According to the invention, a crude polysaccharide is obtained from fruiting bodies of pleurotus eryngii, white beech mushroom, calocybe gambosa, pleurotus citrinopileatus and hypsizygus mammoreus bigelow and the crude polysaccharide is purified with a purification method adopting a mixed pillar consisting of 717 cation exchange resin and 732 anion exchange resin; the anti-gastric ulcer effect of the purified fungal polysaccharide and the composition thereof is obviously strengthened; meanwhile, substances which possibly stimulate an immune system to generate allergy are removed, so that the fungal polysaccharide and the composition thereof are safer to use and are also beneficial to quality control of a product. According to the invention, the fungal polysaccharide and the composition thereof are proved to have obvious effects of preventing and treating gastric ulcer by experiments for the first time.
Description
Technical field
The invention relates to the preparation method of fungus polysaccharide and compositions thereof, and this fungus polysaccharide and the new purposes of compositions in treatment and preventing gastric ulcer thereof, technical field of Chinese medicines belonged to.
Background technology
In the world, fungus polysaccharide is called as " biological respinse modifier ", be called for short " BRM ", there is pharmacological action widely, as regulated immunologic function, antitumor, antioxidation, antiulcer, antibacterium, antiviral and the poly-effect of anticoagulant, improve liver function and conciliate virulence, improve the utilization rate of animal hypoxia-bearing capability and oxygen, reduce the viscosity of blood, increase myocardial contraction, improve the rhythm of the heart, blood sugar lowering, calmness, ease pain, relieving asthma, cough-relieving, the effect such as reduce phlegm.Although fungus polysaccharide effect is very remarkable, be described as the healthy bodyguard of 21 century, the domestic masses of China are also very strange to this novel substance of fungus polysaccharide, masses also do not know what is fungus polysaccharide, it has any effect, where is present in, and many people have also heard this noun in end.Even if medical personnel, also has the detailed effect of much also not knowing fungus polysaccharide, thereby fail to give full play to the effect of fungus polysaccharide.Meanwhile, polysaccharide compound has good biocompatibility, when particularly oral, substantially has no side effect.A lot of pharmacy workers endeavour to learn the applied research of polysaccharide in recent years, and fungus polysaccharide has become at the important raw material of medicine and health food research and development field.
Gastric ulcer is the common frequently-occurring disease of modern society, high morbidity.Its pathogeny is complicated.Along with the raising of society living standards of the people, life stress also increases increasingly, and the sickness rate of gastric ulcer is high; And this disease course of disease is long, and relapse rate is high; The secondary disease gastrorrhagia being caused by gastric ulcer, gastric perforation can bring great misery to patient; According to clinical statistics source investigation, showing: gastric ulcer is very large to the development tendency of cancer, is very likely a stage of precancerous lesion.Therefore the research of the treatment of the digestive diseases such as gastric ulcer and mechanism has been caused to numerous medical experts and materia medica expert's concern.
The medicine that is applied at present clinical treatment gastric ulcer has a variety of, but great majority be take and controlled its disease as the medicine of main " symptomatic treatment ".Such as there being the medicine such as cimetidine, A Moxi fourth of gastric acid secretion inhibiting; There are the proglumide, omeprazole of the synthetic class of short prostaglandin etc.; Also have for the preparation sucralfate of protecting gastric mucosa, colloidal bismuth etc.; Also there is the antibiotics medicine for helicobacter pylori infections.But the side effect that these medicines have is larger, what have is expensive, because this class disease of gastric ulcer needs long-term prescription, can cause patient's financial burden.Therefore develop a kind of safe, economical and practical medicament for anti-gastric ulcer very meaningful.
Based on above reason, the invention provides the novel fungus polysaccharide composition and method of making the same with the effect for the treatment of gastric ulcer.
About the preparation method of several fungus polysaccharides such as Pleurotus eryngii, white jade mushroom, Tricholoma mongolicum Imai, Pleurotus citrinopileatus, crab flavour mushroom and fungus polysaccharide compositions thereof and be used for the treatment of and preventing gastric ulcer purposes, before the present invention, have no report.
Summary of the invention
In the present invention, the polysaccharide that extracts purification from the single funguses such as Pleurotus eryngii, white jade mushroom, Tricholoma mongolicum Imai, Pleurotus citrinopileatus, crab flavour mushroom is called fungus polysaccharide of the present invention; By extract single fungus polysaccharide after purification again equal proportion mix after resulting polysaccharide mixture be all called fungus polysaccharide compositions.
The present invention recorded extracting method to comprising several fungus polysaccharides such as Pleurotus eryngii, white jade mushroom, Tricholoma mongolicum Imai, Pleurotus citrinopileatus, crab flavour mushroom and compositions thereof, purification process with and the pharmacologically active of anti-gastric-ulcer.According to the feature of several funguses, scientific and rational extraction and purifying process have been designed, particularly in purification process, used the purification process of the mixed column of 717 cation exchange resiies and 732 anion exchange resin composition to carry out purification to crude polysaccharides, the effect of the fungus polysaccharide anti-gastric-ulcer after purification obviously strengthens.Activity research result shows it is no matter the pharmacological action that single fungus polysaccharide or fungus polysaccharide compositions all have certain anti-gastric-ulcer, and the activity of the fungus polysaccharide after purification is obviously better than unpurified fungus polysaccharide.
Main purpose of the present invention is to provide a kind of the have fungus polysaccharide for the treatment of gastric ulcer effect and the preparation method of compositions thereof.Particularly it applies the purification process of the mixed column of 717 cation exchange resiies and 732 anion exchange resin composition in purification.Fungus sporophore through decocting boil, concentrate, ethanol precipitation goes out crude polysaccharides, crude polysaccharides is removed the impurity such as salt, albumen, pigment through ion exchange resin, prepare purified polysaccharide, polysaccharide after purification has improved therapeutical effect, reduce possible side effect, therefore purifying process of the present invention is the tangible outstanding feature of tool and significant technological progress simultaneously.
The preparation method of fungus polysaccharide of the present invention is: 1. with the crude polysaccharides of preparation after water extraction, ethanol precipitation, dry, thoroughly wave after most ethanol, and with water dissolution, make the aqueous solution of 20-30%.2. this aqueous solution is by 717, the 732 mixture iron exchange resin posts that form, with pure water eluting.3. collect the water elution liquid of 3 column volumes, concentrated, spraying is dried or lyophilization, obtains purification fungus polysaccharide.
Another object of the present invention has been to provide fungus polysaccharide and the application of compositions in prevention and treatment gastric ulcer thereof.In the document of having reported, there is no fungus polysaccharide of the present invention and compositions thereof for the report of anti-gastric-ulcer.The present invention experimental results show that by gastric ulcer animal model and in vitro gastric cells fungus polysaccharide of the present invention and compositions truly have the pharmacologically active of anti-gastric-ulcer.And the polysaccharide pharmacologically active after purification is obviously better than crude polysaccharides.
For reaching above-mentioned and other object, the invention provides fungus polysaccharide and the compositions thereof with the effect for the treatment of gastric ulcer.This fungus polysaccharide can be selected from the polysaccharide of all edible funguses and pharmaceutical fungus.This fungus comprises Pleurotus eryngii, Tricholoma mongolicum Imai, white jade mushroom, Pleurotus citrinopileatus, the sporophore of crab flavour mushroom or the combination of above-mentioned fungus.
Fungus polysaccharide of the present invention is with oral way administration, can be applicable to treatment and preventing gastric ulcer.This fungus polysaccharide or its compositions can be used alone or with other drug use in conjunction.
Content of the present invention can be crossed following technical scheme and implement, and the content that those skilled in the art can be explained by this description is understood content of the present invention and feature.
One, the extraction of fungus polysaccharide and purification
The sporophore of the spendable fungus Pleurotus eryngii of the present invention, Tricholoma mongolicum Imai, white jade mushroom, Pleurotus citrinopileatus, crab flavour mushroom, but be not limited to above-mentioned fungus sporophore.
Get 10 kilograms of fresh fungus sporophores, remove impurity, cutting, adds 4 times of amount ordinary waters and decocts 3 hours, collects decoction liquor, and residue adds 3 times of water gagings and decocts 3 hours, discards residue, merges decoction liquor, and being concentrated into relative density is 1.2.Under stirring, slowly add 95% ethanol of 3 times of amounts, standing over night.Collecting precipitation, drying under reduced pressure, obtains crude polysaccharides 1200-1350 gram.Crude polysaccharides is made 20% aqueous solution with water dissolution, the mixed column (L: Ф consisting of 2 gram of 717 cation exchange resin and 732 anion exchange resin (1:1) with every gram of crude polysaccharides in aqueous solution; 1:7), continue with the pure water eluting of 3 column volumes, collect eluent, suitably concentrated, lyophilization or spraying are dry, the purified polysaccharide 750-850 gram of the salt that is removed, albumen, peptide class and pigment etc.
Polysaccharide antiulcer effect after purification obviously strengthens, and has removed simultaneously and may produce irritated material by stimulating immune system, makes its use safer, is also conducive to the quality control of product.
According to the present invention, for the fungus polysaccharide of this preparation method, can be a kind of fungus polysaccharide, the polysaccharide composition of preparation after also can be two kinds and above fungus polysaccharide and mixing.
Two, the analysis of crude polysaccharides and purified polysaccharide physicochemical property
The assay of total sugar adopts phenol-sulfuric acid method to measure
(1); Acid sugar m-hydrobiphenal
(2)method is measured; Protein and peptide class adopt lowry method to measure
(3).Forming sugar measures with PMP Derivatization Method
(4).The results are shown in Table 1.By the variation of the component contents such as neutral sugar, protein in analysis of physical and chemical property crude polysaccharides and purified polysaccharide, illustrate that the content of sugar in the polysaccharide of purification obviously improves, and protide component content obviously lowers, its form glycosyl this without changing (table 2).Result shows that purification process of the present invention can obviously improve the sugared content of listed fungus polysaccharide (but being not limited to listed fungus), reduces the content of impurity.
Table 1 fungus polysaccharide analysis of physical and chemical property
The composition glycan analysis of table 2 fungus polysaccharide
Three. the effect of crude polysaccharides, purified polysaccharide treatment gastric ulcer animal.
With dehydrated alcohol inducing mouse gastric ulcer model, fungus polysaccharide and compositions antiulcer action thereof are tested.
1 material and equipment
1.1 laboratory animal
Kunming mouse, body weight quality (20 ± 2) g (is provided quality certification SCXK (Ji) 2012-0004 by Jilin University's medical board Experimental Animal Center.)
1.2 medicines and reagent
The fruitbody polysaccharide of Pleurotus eryngii, Tricholoma mongolicum Imai, white jade mushroom, Pleurotus citrinopileatus, crab flavour mushroom is all according to described method preparation; Positive drug: pharmaceutical factory of WEILEXIN granule Jilin University, lot number: 20120311
Dehydrated alcohol, Beijing Chemical Plant, lot number 20120402
2 experimental techniques
Mice, male and female half and half, at random by mice group, every group of l0 only, is respectively model group, the high, medium and low dosage of sample (400,200,100mg/kg) group, positive drug (WEILEXIN granule) is group (7.5g/kg).Model group distilled water, other group is by various dose administration, successive administration 6 days.The 6th day mice freely drinks water, fasting is after 24 hours, every group of all normal administration after 30 minutes in the 7th day, every equal gavage 0.2mL dehydrated alcohol of mice, after 1 hour, mice is put to death in cervical vertebra dislocation, cut abdominal cavity open, ligation pars cardiaca, makes stomach free, first with normal saline, carefully rinses gastric foul, be soaked in again 10% neutral formalin solution, fix 10 minutes, put and in filter paper, blot liquid, then in optical microphotograph Microscopic observation gastric mucosa.
Calculate ulcer index and suppression ratio and calculated by formula below, by ulcer or rotten to the corn size, score: the damaged 1mm of being less than of every 3 point-like ulcer 1 minute
The product in the hemorrhage maximum major diameter of strip and maximum wide footpath
The every millimeters long of the wide person of 1mm is 1 minute
The every millimeters long of the wide person of 2mm is 2 minutes
The every millimeters long of the wide person of 3mm is 3 minutes
Ulcer area S=∏ * d1 * d2 * 1/4 (∏ is pi, and d1, d2 are respectively maximum transverse diameter and the vertical footpath of getting by ulcer center)
Ulcer area 1-12 13-25 26-37 38-50 be greater than 50 or perforation
(mm
2)
Ulcer index 12345
The results are shown in Table 3.
The inhibitory action of table 3 fungus polysaccharide to gastric ulcer
Note: with model group comparison, * * P<0.01; * P<0.05; #, purification Polysaccharide in Pleurotus eryngii: purification crab flavour mushroom polysaccharide (1:1).
Compare with blank group, model group ulcer index and ulcer area value obviously raise, and result has extremely significant difference (P<0.01), illustrates that dehydrated alcohol inducing mouse gastric ulcer model is successfully prepared.
With model group comparison, WEILEXIN group has inhibitory action to ulcer index and ulcer area, has utmost point significant difference (P<0.01), and experimental result shows that WEILEXIN is rational as the selection of positive drug.
The high dose group of each sample and model group comparison, all have inhibitory action to ulcer index and ulcer area, still, after purification, the activity of polysaccharide obviously increases, show by ion-exchange resin purification, not only can obviously improve sugared content, also can obviously improve the activity of each sample.
Each group and model group comparison of polysaccharide after purification, high dose group all has utmost point significant difference (P<0.01), and middle dosage group has significant difference, and low dose group effect is not remarkable.Result shows, each sample has obvious dose-effect relationship.Meanwhile, the activity of compositions display and single fungus polysaccharide is basic identical.
Four. crude polysaccharides, the impact of purified polysaccharide on impaired gastric cells
By choosing the MC of immortalization (being the GES-1 cell that MNNG transforms) as the external model of gastric ulcer cell.Cell line has relatively stable genetic state after building up.Generally believe that gastric ulcer is a stage of gastric precancerous lesion, so this cell model can be made the screening model of medicament for anti-gastric ulcer.
1 material and equipment
Cell derived: MC cell is by Beijing tumour hospital of university heredity chamber.Sample and positive drug are the same.
2 experimental techniques
2.1 cell culture
Above-mentioned culture medium for MC cell (containing 10% hyclone), at 37 ℃, is cultivated in the incubator of 5%C02.Within 1-2 days, change liquid once, to covering with the ratio of Tissue Culture Flask 90% use 2% trypsin with 1:2 or 1:3, go down to posterity.
2.2 MTT experiments
2.2.1 cell viability experiment
By cell suspension inoculation to 96 orifice plates, every hole about 1000-10000 cell, every pore volume 100ul, establishes 5, multiple hole.Cultivate 5 days.Get each time point 4h, 12h, 24h, 48h, 72h, 96h, 120h, every hole adds MTT solution 10ul, and lucifuge continues to hatch 4h, discards culture supernatant, and every hole adds 150ul DMSO, vibration 10min.Select 490nm wavelength, measure each hole absorbance value in microplate reader, record result, take the time as abscissa, the meansigma methods of getting light absorption value is that vertical coordinate is drawn cell growth curve.
2.2.2 fungus polysaccharide is tested MC impact cell
The MC cell of logarithmic (log) phase growth is made to 5 * 10
4/ ml cell suspension, is inoculated in the culture plate in 96 holes, each 100 μ l of every hole.After 24h cell attachment, add after pastille culture medium and continue to cultivate, blank groups of cells, background group and administration group high dose and low dose group are established in experiment.After drug effect 48h, the every hole of supernatant discarded adds MTT10 μ l, and lucifuge is cultivated 4h in 37 ℃ of incubators, again centrifugal abandon supernatant after every hole add dimethyl sulfoxine 150mL, micro oscillator vibration 5min, elisa reading instrument colorimetric (wavelength 490nm), reads OD value automatically.Suppression ratio calculates according to following formula:
Suppression ratio=(OD
the matched group of not dosing is average-OD
administration group is average)/OD
the matched group meansigma methods of not dosing* 100%
3 results and discussion
The cell growth curve of the drafting obtaining with said method is as Fig. 1.
By above growth curve, illustrate that this cell is in normal growth conditions, about 72 hours, cell is stable state, and 24-72 hour cell is grown in logarithmic (log) phase.
The impact of table 4 fungus polysaccharide on MC cell
#, purification Polysaccharide in Pleurotus eryngii: purification crab flavour mushroom polysaccharide (1:1).
This experimental result shows, each sample all has certain inhibitory action to MC cell, no matter is crude polysaccharides, purified polysaccharide, or compositions, and inhibitory action increases with concentration.The effect that after purification, each fungus polysaccharide suppresses MC cell obviously increases.
Above pharmacodynamic study result shows that fungus polysaccharide of the present invention and compositions thereof have the effect of prevention and treatment gastric ulcer.
Accompanying drawing explanation
Fig. 1 is MC cell growth curve figure
The specific embodiment
The present invention is described in detail by the following examples, but does not mean that the present invention only limits to this, and specific embodiments is as follows:
embodiment 1
Get 10 kilograms of fresh Pleurotus eryngii sporophore, remove impurity, cutting, adds 4 times of amount ordinary waters and decocts 3 hours, collects decoction liquor, and residue adds 3 times of water gagings and decocts 3 hours, discards residue, merges decoction liquor, and being concentrated into relative density is 1.2 (60 ℃ of surveys).Under stirring, slowly add 95% ethanol of 3 times of amounts, standing over night.Collecting precipitation, drying under reduced pressure, obtains crude polysaccharides.Crude polysaccharides is made 20% aqueous solution with water dissolution, the mixed column (L: Ф consisting of 2 gram of 717 cation exchange resin and 732 anion exchange resin (1:1) with every gram of crude polysaccharides in aqueous solution; 1:7), continue with the pure water eluting of 3 column volumes, collect eluent, suitably concentrated, lyophilization or spraying are dry, 780 grams of the purified polysaccharides of the salt that is removed, albumen, peptide class and pigment etc.
embodiment 2
Get 10 kilograms of fresh crab flavour mushroom sporophore, remove impurity, cutting, adds 3 times of amount ordinary waters and decocts 3 hours, collects decoction liquor, and residue adds 4 times of water gagings and decocts 2 hours, discards residue, merges decoction liquor, and being concentrated into relative density is 1.3 (60 ℃ of surveys).Under stirring, slowly add 95% ethanol of 3 times of amounts, standing over night.Collecting precipitation, drying under reduced pressure, obtains crude polysaccharides.Crude polysaccharides is made 20% aqueous solution with water dissolution, the mixed column (L: Ф consisting of 2 gram of 717 cation exchange resin and 732 anion exchange resin (1:1) with every gram of crude polysaccharides in aqueous solution; 1:7), continue with the pure water eluting of 3 column volumes, collect eluent, suitably concentrated, lyophilization or spraying are dry, 795 grams of the purified polysaccharides of the salt that is removed, albumen, peptide class and pigment etc.
experimental example 3
Get purification Polysaccharide in Pleurotus eryngii and each 400g of crab flavour mushroom polysaccharide, mix homogeneously, obtains 800 grams of fungus polysaccharide compositionss.
Claims (5)
1. have a fungus polysaccharide for treatment and preventing gastric ulcer, this fungus polysaccharide is from the sporophore of Pleurotus eryngii, white jade mushroom, Tricholoma mongolicum Imai, Pleurotus citrinopileatus, crab flavour mushroom.
2. have treatment and the fungus polysaccharide compositions of preventing gastric ulcer, said composition is to obtain after the fungus polysaccharide equal proportion mixing of the two or more independent preparations in the sporophore of Pleurotus eryngii, white jade mushroom, Tricholoma mongolicum Imai, Pleurotus citrinopileatus, crab flavour mushroom.
3. according to the fungus polysaccharide described in claim, its preparation method is:
A. get fresh fungus sporophore, add 3～5 times of water gagings and decoct 2 times, each 2～4 hours, merge decoction liquor, concentrated, add 2～4 times of amount ethanol precipitations, precipitation is dry, obtains crude polysaccharides;
B. crude polysaccharides is made 15～25% aqueous solution with water dissolution, and the mixed column consisting of 717 cation exchange resiies and 732 anion exchange resin, with pure water eluting, collect eluent, concentrated, dry, the purified polysaccharide of the salt that is removed, albumen, peptide class and pigment etc.
4. according to the fungus polysaccharide described in claim 1-3 or its compositions, it is characterized in that making oral drug preparation containing the fungus polysaccharide of dose therapeutically effective or its compositions and one or more pharmaceutically acceptable pharmaceutical excipients.
5. according to the fungus polysaccharide described in claim 1-4 or its compositions, it is characterized in that can be used alone, also can with other drug use in conjunction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940242A (en) * | 2015-06-01 | 2015-09-30 | 吉林农业大学 | Pleurotus citrinopileatus Singer polysaccharide protein compound, preparation method and use thereof |
| CN106265775A (en) * | 2016-10-08 | 2017-01-04 | 齐鲁师范学院 | A kind of compositions of prophylactic treatment stress gastric ulcer and preparation method and application |
| CN111138558A (en) * | 2020-02-18 | 2020-05-12 | 安徽大学 | A kind of natural immunity enhancing polysaccharide PCP and its preparation method and use |
-
2014
- 2014-08-30 CN CN201410440557.7A patent/CN104147038A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 关力等: "杏鲍菇子实体多糖的提取和纯化工艺研究", 《黑龙江医药》 * |
| 郭永月等: "白玉菇多糖提取方法的比较和优化", 《南京师大学报（自然科学版）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940242A (en) * | 2015-06-01 | 2015-09-30 | 吉林农业大学 | Pleurotus citrinopileatus Singer polysaccharide protein compound, preparation method and use thereof |
| CN106265775A (en) * | 2016-10-08 | 2017-01-04 | 齐鲁师范学院 | A kind of compositions of prophylactic treatment stress gastric ulcer and preparation method and application |
| CN106265775B (en) * | 2016-10-08 | 2020-10-09 | 齐鲁师范学院 | Composition for preventing and treating stress gastric ulcer and preparation method and application thereof |
| CN111138558A (en) * | 2020-02-18 | 2020-05-12 | 安徽大学 | A kind of natural immunity enhancing polysaccharide PCP and its preparation method and use |
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Application publication date: 20141119 |