CN100509856C - Cryptoporus volvatus polysaccharide, preparation and application thereof - Google Patents
Cryptoporus volvatus polysaccharide, preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a closed bacterium polysaccharide and a preparing method; application of preparing cure allergic rhinitis medicine through closed bacterium polysaccharide and allergic asthma medicine and application of preparing cure adult respiratory distress syndrome medicine; which is characterized by the following parts: (1) the component of closed bacterium polysaccharide are more definite and the action of inhibit sensitized big rat antigen attack back lung acidophilia inflammation and can be developed to new medicine or functional food or health products be used to treat allergic rhinitis and allergic asthma; (2) the polysaccharides through PEG deposition polysaccharides to produce the closed bacterium polysaccharides are much more higher than polysaccharides preparing through alcohol deposition; and quantity of PEG is about one twentieth of alcohol.
Description
(1) technical field
The present invention relates to a kind of cryptoporus volvatus polysaccharide and preparation method thereof, and the application of this cryptoporus volvatus polysaccharide in preparation treatment of allergic rhinitis, allergic asthma and adult respiratory distress syndrome's medicine.
(2) background technology
Latent pore fungi popular name Cryptoporus volvatus (peck) Scnear, tree lotus bud, tree stump, lotus look bacterium, wooden fish bacterium, incense wood orchid, black son (Yunnan), a bite young pilose antler (Japan) of stepping.Belong to mycota, Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymenomycetes), non-pleat order [Aphyllophorales, old system make Aphyllophorales (Polyporales)], polyporaceae (Polyporaceae), latent pore fungi genus (Cryptoporus).Be distributed in provinces such as Chinese Sichuan, Jiangsu, Zhejiang, Hubei, Jilin, Yunnan, Hainan, Fujian, Korea, Japan, Europe, North America also have distribution.
Pharmaceutical use about latent pore fungi early has report, Lan Mao (1397-1476) " the southern regions of the Yunnan Province book on Chinese herbal medicine " record: bitter, sweet, cold nature.Control blood under the large intestine, the poison of long-pending heat.Treat (inside and outside) nine kinds of hemorrhoid, it is painful that (attached side) controls tooth, and Cryptoporus volvatus (peck) Scnear is bitten on painful tooth, aches and promptly end.Polysaccharide protein among the hot water extract that people (1981) such as Korea S Li Shi obtain has antitumour activity.(1990) " Xinhua's Compendium of Materia Medica " record is write by Plant Research Inst., Jiangsu Prov. etc.: mildly bitter flavor, property is flat.Cough-relieving arranged, relieving asthma, function such as detoxifcation.Be used to control trachitis, asthma, also can be used for children's's wean (the Yunnan Lijing is among the people commonly used), volumes such as Xu Jintang " Chinese medicinal fungi " (1997) the 32 chapters conceal pore fungi and have put down in writing the report both domestic and external of latent pore fungi, pharmaceutical use, morphological structure, life habit, the life history, living condition, culture technique etc. comprehensively.
Artificial culture about latent pore fungi does not appear in the newspapers, but Hua Qihong etc. study the liquid fermentation and culture of latent pore fungi, and applied for Chinese patent (patent No. 94117227.9), then Tai Shi Bioisystech Co., Ltd in Hangzhou makes further research liquid fermentation technology and the pharmaceutical use of latent pore fungi, the liquid fermentate of finding latent pore fungi has better curative effect to allergic asthma and allergic rhinitis, and has applied for Chinese patent (disclosing 01126498.5).
Chemical ingredients about latent pore fungi also has " Chinese Pharmaceutical Journals " 1993.28 (2) such as report: Hua Qihong, Jin nation's beam: 19 kinds of volatile oil component researchs of latent pore fungi have been reported in P75～76; Hua Qihong, Jin nation's beam etc. " plant resources and environment " 1993.2 (1): chemical constitution studies such as latent pore fungi sugar, amino acid, trace element have been reported in P60～61; Ma Weiguang etc. " Yunnan plant research " 1994.16 (2): isolating four ergosterol compounds from wild latent pore fungi have been reported in P196～200; Wu Jinzhong, Huang have reported that the tlc analysis result between amino acid, carbohydrate and water extract, ethanol extraction, the ligroin extraction relatively in latent pore fungi fermenting culture and the wild latent pore fungi in " Colleges Of Traditional Chinese Medicine Of Fujian's journal " 1997.7 (2) P21～26 over year.
But seldom for the effective constituent research that conceals pore fungi.
(3) summary of the invention
The present invention promptly is for the application in preparation treatment of allergic rhinitis, allergic asthma and adult respiratory distress syndrome's medicine of a kind of cryptoporus volvatus polysaccharide and preparation method and this cryptoporus volvatus polysaccharide is provided.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of cryptoporus volvatus polysaccharide (CFSP), described cryptoporus volvatus polysaccharide is extracted by the raw material that contains cryptoporus volvatus polysaccharide and makes, and molecular weight ranges is 2000～200000, and main chain is by 1 → 6 seminose that connects, 1 → 6 semi-lactosi that connects, the wood sugar that 1 → 3 Fucose and 1 → 4,1 → 3 that connects is connected constitutes.
Described cryptoporus volvatus polysaccharide can be prepared by following method: the raw material extracting in water that will contain cryptoporus volvatus polysaccharide, the water extract concentrates the back and adds polyoxyethylene glycol (PEG) mixing, staticly settle, the taking precipitate washing with alcohol, throw out is water-soluble, protein is removed in centrifugal back, and centrifugal again, membrane sepn is held back 2000～200000 molecular weight ranges, and concentrated after drying gets cryptoporus volvatus polysaccharide.
The described raw material that contains cryptoporus volvatus polysaccharide is one of following or the combination of following two or more arbitrary form: 1. natural latent pore fungi; 2. latent pore fungi liquid medium; 3. cryptoporus volvatus filament; 4. latent pore fungi solid culture; 5. cryptoporus volvatus powder.
A kind of method for preparing above-mentioned cryptoporus volvatus polysaccharide, described method is as follows: the raw material extracting in water that will contain cryptoporus volvatus polysaccharide, the water extract concentrates the back and adds polyoxyethylene glycol (PEG) mixing, staticly settle, the taking precipitate washing with alcohol, throw out is water-soluble, and protein is removed in centrifugal back, centrifugal again, membrane sepn is held back 2000～200000 molecular weight ranges, and concentrated after drying gets cryptoporus volvatus polysaccharide.
Further, described method is as follows: the raw material that will contain cryptoporus volvatus polysaccharide adds 5～10 quality water doubly, 70～90 ℃ were extracted 1～4 hour down, the water extract is centrifugal that supernatant concentration to proportion is 1.0～1.3, get concentrated solution, every 100mL concentrated solution adds polyoxyethylene glycol 5～50g, fully stir back standing over night precipitation, centrifugal throw out 95% washing with alcohol that gets, protein is removed in water-soluble, the centrifugal back of throw out, centrifugal again, membrane sepn is held back 2000～200000 molecular weight ranges, and concentrated after drying promptly gets described cryptoporus volvatus polysaccharide.
In the aforesaid method, the described raw material that contains cryptoporus volvatus polysaccharide is one of following or the combination of following two or more arbitrary form: 1. natural latent pore fungi; 2. latent pore fungi liquid medium; 3. cryptoporus volvatus filament; 4. latent pore fungi solid culture; 5. cryptoporus volvatus powder.
The method of extracting purified polysaccharide at present mainly contains 1) ethanol precipitation: this method is used the most general, also have most versatility, but the operation energy consumption height needs amount of alcohol big, wastes time and energy, and yield is low.2) ultrafiltration process: hold back the polysaccharide that extracts certain molecular weight by ultra-filtration membrane.This method also has very wide range of application, and energy consumption is low, and extraction rate is fast, does not need organic solvent.But the polysaccharide content that extracts is low, the residual height of various molecular weights material.3) complexometry: this method mainly utilizes the specific combination adding complexing agent of some acidic polysaccharose to make it to separate out, and complexing agent commonly used has calcium chloride, copper sulfate, cetyl trimethylammonium bromide etc.This method energy consumption is low, and the polysaccharide content of extraction is higher, but range of application is narrow, only limits to the mixed polysaccharide of acidic polysaccharose or some characteristic.4) post partition method: this method utilizes the difference of various column packing character to come purified polysaccharide.Such as present popular macroporous resin purification polysaccharide, ion-exchange purification glycoprotein or the like.The purity of polysaccharide that this method is extracted is higher, but complicated operation carries out pre-treatment to pillar, stepwise elution, and the regeneration of pillar, a large amount of liquid waste disposal etc. in the production process, yield is low, the cost height.And extract often contains post material residue, will detect the residual quantity of toluene, dimethylbenzene as the polysaccharide behind the macroporous resin extraction.
Extracting method of the present invention is to precipitate polysaccharide with PEG, is exactly specifically: the raw water that will contain cryptoporus volvatus polysaccharide is carried, and adds polyoxyethylene glycol (PEG), stir, dissolving is left standstill, precipitation, centrifugal throw out, throw out 95% washing with alcohol of getting, throw out is water-soluble, and is centrifugal, protease hydrolysis, boil, centrifugal, membrane sepn, concentrated after drying get mass content at the cryptoporus volvatus polysaccharide more than 50%.Technological difficulties solved by the invention are how to isolate Crude polysaccharides from aqueous extract.Discovery will be far above with ethanol sedimentation gained polysaccharide content with PEG precipitation polysaccharide products obtained therefrom polysaccharide content, and only is about 1/20th of ethanol consumption with the amount of PEG, thus yield height of the present invention, the content height, cost is low, and easy and simple to handle.Used 95% ethanol refers to that the ethanol volumn concentration is 95% the aqueous solution among the present invention.
Described polyoxyethylene glycol is preferably PEG1000～20000.
Described removal protein method can be proteolysis such as restriction endonuclease, excision enzyme or prozyme, reduce proteinic molecular weight and make to dissociate out, also can use organic solvent such as trichoroacetic acid(TCA), macroporous resin adsorption with sugared bonded albumen, add heavy metallic salt, the method precipitating proteins such as boil.Be preferably among the present invention and be protease hydrolysis.
Preferably, described method is as follows: cryptoporus volvatus powder adds the water of 5 times of quality, and 80 ℃ were extracted 2 hours, and extracting solution is got supernatant liquor; Precipitation adds suitable quantity of water and fully stirs extraction, the centrifugal supernatant liquor that gets of extracting solution, twice supernatant liquor merges, be evaporated to volume-diminished and get a concentrated solution for, constantly stir down, every 100mL concentrated solution adds 15 gram PEG6000, stirring makes abundant dissolving, and room temperature is placed and spent the night, and makes precipitation fully, centrifugal must precipitate with 95% alcohol reflux colourless substantially to extracting solution, resolution of precipitate in suitable quantity of water, the centrifugal insolubles of removing, on reset and add protein incision enzyme, after the hydrolysis hydrolyzed solution is boiled, put and be chilled to room temperature, the centrifugal insolubles of removing, the supernatant ultrafiltration membrance filter of the molecular weight 500,000 that dams, filtrate concentrates with the ultra-filtration membrane of the molecular weight 20,000 that dams, and the concentrated solution freeze-drying promptly gets described cryptoporus volvatus polysaccharide.
Perhaps, described method is as follows: natural latent pore fungi is pulverized the water that the back adds 2.5 times of quality, and 80 ℃ were extracted 2 hours, and extracting solution gets supernatant liquor; Precipitation adds suitable quantity of water and fully stirs extraction, extracting solution centrifuging and taking supernatant liquor, twice supernatant liquor merges, be evaporated to volume-diminished and get a concentrated solution for, constantly stir down, every 100mL concentrated solution adds 25 gram PEG20000, stirring makes abundant dissolving, room temperature is placed and to be spent the night, and makes precipitation fully, centrifugal must precipitate with 95% alcohol reflux colourless substantially to extracting solution, get resolution of precipitate in suitable quantity of water, the centrifugal insolubles of removing, on reset and add protein incision enzyme, after the hydrolysis hydrolyzed solution is boiled, put and be chilled to room temperature, the centrifugal insolubles of removing, supernatant be with the dialysis tubing flowing water dialysis of the molecular weight 5000 that dams 48 hours, dialyzed solution with 0.45 micron membrane filtration after 60 ℃ of evaporates to dryness promptly get described must cryptoporus volvatus polysaccharide.
Described cryptoporus volvatus polysaccharide can be applicable to prepare treatment of allergic rhinitis medicine, allergic asthma medicine and adult respiratory distress syndrome's medicine, certainly also this cryptoporus volvatus polysaccharide food prepared therefrom, protective foods or food additives be can be used for, allergic rhinitis, allergic asthma, and adult respiratory distress syndrome's patient are applied to.Described medicine can be made into the receivable formulations of multiple human body such as capsule, tablet, granule, oral liquid, injection liquid.
Allergic rhinitis and asthma are very close on the immunology pathogenesis, sensitinogen can excite similar histopathological response at last lower respiratory tract respectively, nasal cavity and tunica mucosa bronchiorum all have helper T cell, eosinophilic granulocyte and the mast cells infiltration of great expression Th2 cytokine, inflammatory mediators such as produced simultaneously histamine, leukotriene, prostaglandin(PG), platelet activation factor, chemokine and bradykinin cause similar Inflammatory response process at the upper respiratory tract with lower respiratory tract.After the patient carried out the rechallenge of nose allergen, eosinophilic granulocyte soaks in the lower respiratory tract mucous membrane increased, and the cell quantity of expression of adhesion molecules increases simultaneously, showed that the alterative inflammation reaction of local mucous membrane may produce similar reaction at other position.It is one of mechanism of its treatment of allergic rhinitis and asthma that cryptoporus volvatus polysaccharide can significantly suppress eosinophilic granulocyte.
(adult respiratory distress syndrome ARDS) is the respiratory insufficiency that is caused by acute lung injury to the adult respiratory distress syndrome.Its pathogenesis is very complicated, think that at present a large amount of neutrophil leucocytes are gathered in lung, adhere to the alveolar capillary endothelium under chemokine and adhesion molecule effect, discharge oxyradical, proteolytic enzyme and inflammatory mediator etc., damage alveolar epithelial cells and capillary endothelial cell.The damage of alveolar-capillary membrane and the effect of inflammatory mediator are increased alveolar epithelium and capillary endothelium permeability, cause permeability, cause the pulmonary diffusion function obstacle.Adult respiratory distress syndrome's pathogeneticing characteristic is the gathering of neutrophil leucocyte.Cryptoporus volvatus polysaccharide can significantly suppress the gathering of gathering, multinuclear granulocyte and the plymphomonocyte of polymorphonuclear leukocyte, is one of mechanism of treatment adult respiratory distress syndrome.
The present invention extracts a kind of mixed polysaccharide (CFSP) from latent pore fungi fermented product, by adopting the pharmacodynamic study to the influence of inflammatory cell (eosinophilic granulocyte EOS) in the broncho-pulmonary perfusate of sensitization rat antigen attack back is index, has confirmed that this mixed polysaccharide is the main pharmacodynamics composition in the latent pore fungi fermented product; Anti-inflammatory action experiment by Sephadex G 200 inducing mouse abdominal cavity inflammation has confirmed that further this cryptoporus volvatus polysaccharide is the main active ingredient in the latent pore fungi fermented product.
And then this mixed polysaccharide carried out structural research, find that this polysaccharide mainly is made up of seminose, semi-lactosi, wood sugar, four kinds of monose of Fucose, main chain is by 1 → 6 seminose that connects in its molecule, 1 → 6 semi-lactosi, the wood sugar composition that 1 → 3 Fucose and 1 → 4,1 → 3 that connects is connected.
CFSP1, CFSP2, CFSP3 irritate stomach (10mg/kg) and intravenous injection (10mg/kg) has all significantly suppressed the mouse peritoneal inflammatory cell gathering that Sephadex G 200 (2mg/kg) abdominal injection causes, compare with model group, Fig. 1 is seen in P＜0.05～0.001.CFSP1, CFSP2, CFSP3 filling stomach (10mg/kg) and intravenous injection (10mg/kg) have all significantly suppressed the gathering of intraperitoneal polymorphonuclear leukocyte, and P＜0.05～0.01 the results are shown in Figure 2.CFSP1, CFSP2, CFSP3 filling stomach (10mg/kg) and intravenous injection (10mg/kg) have all significantly suppressed the gathering of multinuclear granulocyte and plymphomonocyte, compare with model group, and Fig. 3 is seen in P＜0.05～0.001.
Cryptoporus volvatus polysaccharide of the present invention and preparation thereof are mainly reflected in the beneficial effect of using: (1) cryptoporus volvatus polysaccharide refinement of the present invention concrete effective component, can suppress sensitization rat antigen very significantly and attack the effect of lung's acidophilia inflammation afterwards; Significantly suppressed the mouse peritoneal inflammatory cell gathering that Sephadex G 200 (2mg/kg) abdominal injection causes, the gathering of polymorphonuclear leukocyte, multinuclear granulocyte and lymph monokaryon born of the same parents' gathering.More spell out the concrete effective component of cryptoporus volvatus polysaccharide, can be developed to new drug or functional foodstuff or healthcare products and be used for allergic rhinitis and allergic asthma and adult respiratory distress syndrome, had broad prospect of application; (2) described cryptoporus volvatus polysaccharide preparation method will be far above with ethanol sedimentation gained polysaccharide content with PEG precipitation polysaccharide products obtained therefrom polysaccharide content, and only is about 1/20th of ethanol consumption with the amount of PEG, the yield height, and the content height, cost is low, and easy and simple to handle.
(4) Figure of description
The restraining effect of Fig. 1 mouse peritoneal total white blood cells that to be CFSP1, CFSP2, CFSP3 and dexamethasone cause Sephadex G 200 (2mg/kg) abdominal injection; (T check, mean ± SD; Compare with model group
*P＜0.05,
*P＜0.01,
* *P＜0.001, (10mg/kg iv) compares with the CFSP1 group
##P＜0.01,
###P＜0.001)
Fig. 2 is the influence to Sephadex G 200 inducing mouse abdominal cavity inflammation cell divides of CFSP1, CFSP2, CFSP3 and dexamethasone; (T check, mean ± SD; Compare with model group
*P＜0.05,
*P＜0.01) Fig. 3 is CFSP1, CFSP2, the CFSP3 influence to multinuclear granulocyte and plymphomonocyte, with model group relatively
*P＜0.05,
*P＜0.01,
* *P＜0.001, (10mg/kg, iv) group relatively with CFSP1
##P＜0.01,
###P＜0.001.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the preparation of cryptoporus volvatus polysaccharide
Get cryptoporus volvatus powder 2000 grams, add 10 premium on currency, 80 ℃ were extracted 2 hours.Centrifugal 10 minutes of 3500 rev/mins of extracting solutions, supernatant liquor 1.Precipitation adds 2 premium on currency, fully stir and extract, 3500 rev/mins centrifugal 10 minutes, supernatant liquor 2.Supernatant liquor 1 and supernatant liquor 2 are merged, be evaporated to about 6 liters, under continuous the stirring, add 1000 gram PEG6000, continue stirring and PEG was fully dissolved in 1 hour.Room temperature is placed and to be spent the night, and makes precipitation fully, with centrifugal 10 minutes of 3500 rev/mins of solution, must precipitate then, abandons supernatant.Precipitation is colourless substantially to extracting solution with 95% ethanol refluxing extraction 6 hours in apparatus,Soxhlet's, volatilizes the ethanol in the precipitation, weighs, and must precipitate 82 and restrain.To precipitate with 2 premium on currency and dissolve, 3500 rev/mins of centrifugal insolubless of removing, on reset and add the 500mg protein incision enzyme, regulate suitable pH value and temperature, hydrolysis was boiled hydrolyzed solution 10 minutes after 5 hours, put and was chilled to room temperature, 3500 rev/mins of centrifugal insolubless of removing, supernatant concentrates with the ultra-filtration membrane of the molecular weight 20,000 that dams with the ultrafiltration membrance filter of the molecular weight 500,000 that dams, filtrate, and the concentrated solution freeze-drying gets cryptoporus volvatus polysaccharide 46 grams.With the sulfuric acid-phynol method measuring result is total polysaccharides content 87%, and color is greyish white, soluble in water, molecular weight 20,000～200,000.
Embodiment 2: the preparation of cryptoporus volvatus polysaccharide
Get natural latent pore fungi 200 grams, pulverize, add 500 ml waters, 80 ℃ were extracted 2 hours.Centrifugal 10 minutes of 3500 rev/mins of extracting solutions, supernatant liquor 1.Precipitation adds 200 ml waters, fully stir and extract, 3500 rev/mins centrifugal 10 minutes, supernatant liquor 2.Supernatant liquor 1 and supernatant liquor 2 are merged, be evaporated to about 200 milliliters, under continuous the stirring, add 50 gram PEG20000, continue stirring and PEG was fully dissolved in 1 hour.Room temperature is placed and to be spent the night, and makes precipitation fully, with centrifugal 10 minutes of 3500 rev/mins of solution, must precipitate then, abandons supernatant.Precipitation is colourless substantially to extracting solution with 95% ethanol refluxing extraction 6 hours in apparatus,Soxhlet's, volatilizes the ethanol in the precipitation, weighs, and must precipitate 4.2 and restrain.To precipitate with 200 ml waters and dissolve, 3500 rev/mins of centrifugal insolubless of removing, on reset and add the 50mg protein incision enzyme, regulate suitable pH value and temperature, hydrolysis was boiled hydrolyzed solution 10 minutes after 5 hours, put and was chilled to room temperature, 3500 rev/mins of centrifugal insolubless of removing, supernatant is with the dialysis tubing flowing water dialysis of the molecular weight 5000 that dams 48 hours, dialyzed solution with 0.45 micron membrane filtration after 60 degree evaporates to dryness promptly get cryptoporus volvatus polysaccharide 2 and restrain.With the sulfuric acid-phynol method measuring result is total polysaccharides content 65%.
Embodiment 3: the preparation of cryptoporus volvatus polysaccharide
Get cryptoporus volvatus filament 2000 grams, add 10 premium on currency, 80 ℃ were extracted 2 hours.Centrifugal 10 minutes of 3500 rev/mins of extracting solutions, supernatant liquor 1.Precipitation adds 2 premium on currency, fully stir and extract, 3500 rev/mins centrifugal 10 minutes, supernatant liquor 2.Supernatant liquor 1 and supernatant liquor 2 are merged, be evaporated to about 1 liter, under continuous the stirring, add 200 gram PEG6000, continue stirring and PEG was fully dissolved in 1 hour.Room temperature is placed and to be spent the night, and makes precipitation fully, with centrifugal 10 minutes of 3500 rev/mins of solution, must precipitate then, abandons supernatant.Precipitation was with 95% alcohol reflux 6 hours, and the precipitation oven dry after the extraction is weighed, and must precipitate 8.2 and restrain.To precipitate with 500 ml waters and dissolve, 3500 rev/mins of centrifugal insolubless of removing, on reset and add the 100mg protein incision enzyme, regulate suitable pH value and temperature, hydrolysis was boiled hydrolyzed solution 10 minutes after 5 hours, put and was chilled to room temperature, 3500 rev/mins of centrifugal insolubless of removing, supernatant concentrates with the ultra-filtration membrane of the molecular weight 20,000 that dams with the ultrafiltration membrance filter of the molecular weight 500,000 that dams, filtrate, and the concentrated solution freeze-drying gets cryptoporus volvatus polysaccharide 4 grams.With the sulfuric acid-phynol method measuring result is total polysaccharides content 88%, and color is greyish white, soluble in water, molecular weight 20,000～200,000.
Embodiment 4:
Get cryptoporus volvatus polysaccharide 50 grams among the embodiment 1, add starch 45.5 grams, medicinal silicon-dioxide 0.5 gram, mixing, promptly gets cryptoporus volvatus polysaccharide capsule (every contains cryptoporus volvatus polysaccharide 50mg) by encapsulated 1000.
Embodiment 5:
400 gram sucrose are made simple syrup with thermosoling, standby.Get embodiment 1 gained cryptoporus volvatus polysaccharide 5 grams, be dissolved in water, be diluted to 2000 milliliters, add simple syrup and Sorbic Acid 8 grams, mixing adds water to 5000 milliliters, packing, and sterilization promptly gets cryptoporus volvatus polysaccharide oral liquid (1mg/ml).
Embodiment 6:
Get cryptoporus volvatus polysaccharide 0.5 gram among the embodiment 1,5 kilograms of milk powder, mixing, packing promptly gets functional milk powder (10mg/100g).
Embodiment 7: the cryptoporus volvatus polysaccharide compositional analysis
Adopt hydrolysis, reduction, acetylation method that embodiment 1 is made cryptoporus volvatus polysaccharide (CFSP1), embodiment 2 makes cryptoporus volvatus polysaccharide (CFSP2), and embodiment 3 makes cryptoporus volvatus polysaccharide (CFSP3) and forms glycan analysis, the results are shown in Table 1:
The composition sugar of table 1 cryptoporus volvatus polysaccharide
| Fucose | Pectinose | Wood sugar | Seminose | Semi-lactosi | Glucose | |
| CFSP1 | 12.59％ | 1.67％ | 19.94％ | 41.19％ | 19.61％ | 5.00％ |
| CFSP2 | 6.59％ | 0.72％ | 19.95％ | 49.14％ | 19.62％ | 3.98％ |
| CFSP3 | 7.25％ | ——? | 27.34％ | 49.49％ | 15.92％ | —— |
Table 1 shows that the composition sugar of the cryptoporus volvatus polysaccharide that the present invention makes has 6 kinds, be Fucose, pectinose, wood sugar, seminose, semi-lactosi, glucose, but content has only 4 kinds to be seminose, semi-lactosi, wood sugar, Fucose greater than 10% main composition sugar, and wherein mannose content is the highest.
Cryptoporus volvatus polysaccharide part methylization to gained of the present invention, adopt GC-MS analysis part methylate, the result shows that the main chain of CFSP1, CFSP2, CFSP3 constitutes by seminose, semi-lactosi, wood sugar, the main mode of connection of seminose and semi-lactosi is 1 → 6, on 2, there is tapping point, Fucose is 1 → 3 to connect, and wood sugar is 1 → 4 to be connected with 1 → 3 and not have tapping point.The end of polysaccharide is mainly seminose and semi-lactosi, and mode of connection is 1 →.
Need to prove that this test result is that the method that we adopt is just measured one of method of polysaccharide structures, can adopt other method that cryptoporus volvatus polysaccharide is carried out structural analysis fully for the people that this respect technology is arranged.Because personnel's difference of analyzing, the analytical procedure difference that adopts, the sensitivity difference of instrument, the possibility of result that draws can be different, lower such as pectinose content among the CFSP2, can not survey pectinose among the CFSP3, form sugar and methylate that quantitative results is not equal as a result, influence is bigger relatively for the lower component of content for this.
Embodiment 8: cryptoporus volvatus polysaccharide is to the restraining effect of inflammatory cell in the broncho-pulmonary perfusate
1, experiment title: cryptoporus volvatus polysaccharide is attacked the effect of the inflammatory cell in the broncho-pulmonary perfusate of back to sensitization rat antigen.
2, experiment purpose: estimate cryptoporus volvatus polysaccharide is attacked the inflammatory cell in the broncho-pulmonary perfusate of back to sensitization rat antigen effect.
3, material:
3.1 tried thing: embodiment 1,2,3 gained cryptoporus volvatus polysaccharides (CFSP1, CFSP2, CFSP3) and cryptoporus volvatus powder (CFS)
3.2 albumin, egg (V grade), U.S. Sigma chemical Co.
3.3 dexamethasone sodium phosphate, Gaoyou pharmaceutical factory
3.4 urethane (Urethane), Shanghai chemical reagents corporation of China Drug Co.
3.5P ARI MASTER compresses inhalation machine (Type:084G6305), Made in Germany
3.6 the SD rat, ♀, cleaning level is purchased the Experimental Animal Center in Medical College of Zhejiang Univ., conformity certification number: the accurate word 22-9601018 of Zhejiang laboratory animal implementation condition
4, method:
1) grouping: totally 60 of SD rats, divide 6 groups, see Table 2.
2) sensitization: albumin is dissolved in 10% aluminum hydroxide gel, is made into 2% Protalbinic acid gelating soln.The subcutaneous injection of every rat four-footed palm, every some 0.1ml; Repeat sensitization more once after 10 days.
3) administration: from sensitization beginning in the 10th day administration.Tried thing CFS normal saline solution 0.5g/kg and irritated stomach (ig), CFSP1, CFSP2, CFSP3 normal saline solution 10mg/kg irritate stomach (ig) (dosage is according to cryptoporus volvatus polysaccharide Mass Calculation in the normal saline solution), dexamethasone (DXM) 0.5mg/kg abdominal injection (ip) is more than tried the equal administration of thing 10 days.
4) antigen is attacked: sensitization the 18th day, the sensitization rat placed in the 4L bell glass in 1 hour after the administration, with PARI MASTER compression inhalation machine aerosol 1.0% ovum protein 30min, next day repeat attack once.Rat put back to use for experiment after mouse cage is supported 24h again.
5) bronchovesicular perfusion: the bronchovesicular perfusion is pressed literature method, urethane 1g/kgip rat, and skin of neck is cut in the anesthesia back, isolate tracheae, tracheostomize inserts trachea cannula, with the Hanks solution 10ml that contains heparin, divide 2 times, each 5ml injects air flue from trachea cannula, washes back and forth 3 times at every turn, washing fluid is collected in vitro, the about 70-80% of the rate of recovery (7-8ml).
6) white blood count and differential counting: 1% Glacial acetic acid 1:1 dilutes perfusate, uses tally at microscopically meter sum.Perfusate is applied on the slide, does the back and dye differential count under oily mirror then with the Wright-Giemsa staining fluid.
5, result: model group is after 1.0% ovum protein is attacked for 2 times, and eosinophilic granulocyte showed increased, plymphomonocyte percentage obviously reduce.Show that the attack of sensitization rat antigen has very significantly allergic inflammation reaction process.CFS and therefrom isolating CFSP1, CFSP2, CFSP3 can obviously suppress sensitization rat antigen and attack eosinophilic granulocyte rising in the bronchovesicular perfusate of back, and its effect is suitable, proves that CFSP1, CFSP2, CFSP3 are effective components main among the CFS.Concrete outcome sees Table 2.
Table 2: after cryptoporus volvatus powder and cryptoporus volvatus polysaccharide are attacked sensitization rat antigen
The restraining effect of inflammatory cell in the broncho-pulmonary perfusate (x ± SD)
Statistical method: Mann-Whitney Rank Sum Test or T-test, compare with model group
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Embodiment 9: cryptoporus volvatus polysaccharide is to the effect of Sephadex G 200 inducing mouse abdominal cavity inflammation
1, experiment title: cryptoporus volvatus polysaccharide is to the effect of Sephadex G 200 inducing mouse abdominal cavity inflammation.
2, experiment purpose: estimate the restraining effect of cryptoporus volvatus polysaccharide to Sephadex G 200 inducing mouse abdominal cavity inflammation.
3, material
3.1 animal
The ICR mouse, male and female half and half, body weight 20 ± 2 grams.Purchase Shanghai medicine institute, conformity certification number: the moving word the 220010014th of doctor in the Chinese Academy of Sciences.
3.2 medicine and reagent
Given the test agent: embodiment 1,2,3 gained cryptoporus volvatus polysaccharides (CFSP1, CFSP2, CFSP3) are with after the stroke-physiological saline solution dissolving, and it is standby to be stored in 4 ℃ of refrigerators;
Dexamethasone sodium phosphate injection (DXM): 5mg/mL, sky, Hubei medicine medicine company limited-liability company, lot number: 20030605;
Sephadex G 200:Pharmacia import lot number of the repackaged products 79-09-19;
Heparin: Huamei Bio-Engrg Co.,, lot number: 0203;
Vltra tears: Shangyu Hai Shen is so kind as to give;
Calf serum: folium ilicis chinensis company in Hangzhou produces, lot number: 20041008.
3.3 key instrument
Opticmicroscope: the Japanese Olympus C011 of company type 928388
Eppendorf high speed freezing centrifuge: German Eppendorf company.
Electronic balance: the Shanghai balance equipment FA1104 of factory type
3.4 reagent preparation
Sephadex G 200: Sephadex G 200 is formulated as the suspension of 0.1mg/ml and autoclaving with 1% Gonak.
Irrigating solution: 1% calf serum D-Hanks damping fluid (containing the 5IU/ml heparin), face and use preceding preparation.
4, experimental technique
4.1Sephadex G 200 inducing mouse abdominal cavity inflammation cell aggregation tests
4.1.1 grouping
This experiment is divided into 5 groups, i.e. model group; The CFSP1 group (10mg/kg, ig); The CFSP2 group (10mg/kg, ig); The CFSP3 group (10mg/kg, ig); CFSP1 intravenous injection group (10mg/kg, iv); The DXM positive controls (0.5mg/kg, ig), press the amount of cryptoporus volvatus polysaccharide in the normal saline solution and calculate by described consumption.
4.1.2 medication
Cause scorching preceding to each group mouse administration every day 1 time, for three days on end.Cause scorching back administration every day 1 time, continuous 2 days.Model group physiological saline 0.3mL/10g irritates stomach, every day 1 time, continuous 5 days; Each administration group is carried out administration respectively according to grouping.
4.1.3 animal causes inflammation
Cause inflammation before the 4th administration.Model group, administration group and positive control drug group are all used Sephadex G200 (0.1mg/mL) abdominal injection, every mouse 2mg/kg.
4.1.4 peritoneal lavage
Adopt the cervical vertebra dislocation method to put to death mouse after causing scorching 48h in the abdominal cavity.Every mouse peritoneal injection irrigating solution 5mL gently rubs moments later sucking-off peritoneal lavage fluid.
4.1.5 white blood cell count(WBC)
Peritoneal lavage fluid is shaken up the back take out 50 μ L, with 4 times of white corpuscle diluted, take a morsel and drip on cell counting count board, at microscopically counting total white blood cells.
4.1.6 Arneth's count
Behind the centrifugal 10min of peritoneal lavage fluid low temperature 2000rpm, abandoning supernatant is played even back smear with pipettor.After treating that smear is at room temperature done, the Wright 6～8min that dyes, Ji's nurse Sa was redyed 1 minute.Carefully dyestuff is rinsed well with tap water, under 40 times of mirrors of opticmicroscope, carried out the cell divide counting.
5, result
CFSP1, CFSP2, CFSP3 irritate stomach (10mg/kg) and intravenous injection (10mg/kg) has all significantly suppressed the mouse peritoneal inflammatory cell gathering that Sephadex G 200 (2mg/kg) abdominal injection causes, compare with model group, Fig. 1 is seen in P＜0.05～0.001.CFSP1, CFSP2, CFSP3 filling stomach (10mg/kg) and intravenous injection (10mg/kg) have all significantly suppressed the gathering of intraperitoneal polymorphonuclear leukocyte, and P＜0.05～0.01 the results are shown in Figure 2.CFSP1, CFSP2, CFSP3 filling stomach (10mg/kg) and intravenous injection (10mg/kg) have all significantly suppressed the gathering of multinuclear granulocyte and plymphomonocyte, compare with model group, and Fig. 3 is seen in P＜0.05～0.001.
Claims (6)
1. cryptoporus volvatus polysaccharide, it is characterized in that described cryptoporus volvatus polysaccharide is made by the raw material extraction that contains cryptoporus volvatus polysaccharide, molecular weight ranges is 2000～200000, and main chain is by 1 → 6 seminose that connects, 1 → 6 semi-lactosi that connects, the wood sugar that 1 → 3 Fucose and 1 → 4,1 → 3 that connects is connected constitutes; The described raw material that contains cryptoporus volvatus polysaccharide is one of following or the combination of following two or more arbitrary form: 1. natural latent pore fungi; 2. latent pore fungi liquid medium; 3. cryptoporus volvatus filament; 4. latent pore fungi solid culture; 5. cryptoporus volvatus powder.
2. cryptoporus volvatus polysaccharide as claimed in claim 1, it is characterized in that described cryptoporus volvatus polysaccharide is prepared by following method: the raw material extracting in water that will contain cryptoporus volvatus polysaccharide, the water extract concentrates the back and adds the polyoxyethylene glycol mixing, staticly settle, the taking precipitate washing with alcohol, throw out is water-soluble, centrifugal, isolating protein, centrifugal again, membrane sepn is held back 2000～200000 molecular weight ranges, and concentrated after drying gets cryptoporus volvatus polysaccharide.
3. the preparation method of cryptoporus volvatus polysaccharide as claimed in claim 1 or 2, it is characterized in that described method is as follows: the raw material that will contain cryptoporus volvatus polysaccharide adds 5～10 quality water doubly, 70～90 ℃ were extracted 1～4 hour down, the water extract is centrifugal that supernatant concentration to proportion is 1.0～1.3, get concentrated solution, every 100mL concentrated solution adds PEG1000～200005～50g, fully stir back standing over night precipitation, centrifugal throw out 95% washing with alcohol that gets, throw out is water-soluble, protein is removed in centrifugal back, centrifugal again, membrane sepn is held back 2000～200000 molecular weight ranges, and concentrated after drying promptly gets described cryptoporus volvatus polysaccharide; The described raw material that contains cryptoporus volvatus polysaccharide is one of following or the combination of following two or more arbitrary form: 1. natural latent pore fungi; 2. latent pore fungi liquid medium; 3. cryptoporus volvatus filament; 4. latent pore fungi solid culture; 5. cryptoporus volvatus powder.
4. the application of cryptoporus volvatus polysaccharide as claimed in claim 1 or 2 in preparation treatment of allergic rhinitis medicine.
5. the application of cryptoporus volvatus polysaccharide as claimed in claim 1 or 2 in preparation treatment allergic asthma medicine.
6. the application of cryptoporus volvatus polysaccharide as claimed in claim 1 or 2 in preparation treatment adult type respiratory distress syndrome medicine.
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| CN102920747B (en) * | 2012-11-15 | 2014-04-16 | 中国农业大学 | Application of cryptoporus volvatus in preparation of products for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN102920748B (en) * | 2012-11-15 | 2014-04-16 | 中国农业大学 | Application of cryptoporus volvatus in preparation of products for curing porcine reproductive and respiratory syndrome (PRRS) |
| CN103908477A (en) * | 2014-01-21 | 2014-07-09 | 中国医学科学院药用植物研究所 | Use of cryptoporus volvatus(Peck)Shear extract in in-vivo and in-vitro inhibition of influenza viruses |
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Non-Patent Citations (4)
| Title |
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| 隐孔菌多糖成分对致敏大鼠气道高反应性和炎症细胞的影响. 汤慧芳，陈季强，谢强敏等.浙江大学学报（医学版），第32卷第4期. 2003 |
| 隐孔菌多糖成分对致敏大鼠气道高反应性和炎症细胞的影响. 汤慧芳，陈季强，谢强敏等.浙江大学学报（医学版），第32卷第4期. 2003 * |
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| 隐孔菌胶囊的制备工艺和质量标准研究. 赵航.浙江大学硕士学位论文. 2004 * |
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