CN100387703C - Fatly phospho embrella Pa-I.H bacterial strain and method of culturing fatty phospho embrella and medicine made from its active component - Google Patents
Fatly phospho embrella Pa-I.H bacterial strain and method of culturing fatty phospho embrella and medicine made from its active component Download PDFInfo
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Abstract
The present invention relates to a pholiota adiposa fungus Pa-I. H strain, a method of artificially culturing a pholiota adiposa fungus by using the strain, medicinal applications of the pholiota adiposa fungus and a medical composition of active components of the pholiota adiposa fungus. A separated pholiota adiposa fungus Pa-I. H strain is utilized to artificially culture pholiota adiposa sporophores by adopting a solidity-liquid-solidity technological line; the method comprises the following steps of slant culture, submerged fermentation, strain culture, mycelium culture and sporophore culture; the pholiota adiposa fungus strain of the present invention does not degrade after passage for 18 times, and belongs to a strain with strong ageing resistance, high stability and strong metabolic capability. In addition, the present invention discloses extracts of the pholiota adiposa fungus, such as polysaccharides and nucleic acids of the pholiota adiposa fungus, and applications of the extracts in the prevention and treatment of tumours and other diseases, in improvement in immunity and in medicines with the action of analgesia and anti-inflammation.
Description
Technical field:
The invention provides a kind of new fatly phospho umbrella Pa-IH bacterial strain and with the method for this bacterial classification artificial culture fatly phospho umbrella bacterium, disclose its pharmaceutical usage simultaneously and, belong to and relate to fungi artificial culture and medicinal technical field with the pharmaceutical composition of its activeconstituents
Background technology:
Fatly phospho umbrella Pholiota adiposa (Fr.) Que is commonly called as thorn mushroom, yellow mushroom, yellow umbrella, belongs to Basidiomycotina, Hymenomycetes, Agaricales, Stropharia rugoso-annulata section, phosphorus Agaricus on taxonomy.Its sporophore is born on the trunk of willow and birch etc. in Jilin Province autumn.The Pa-IH bacterial strain) and use this bacterial strain and grow the fatly phospho umbrella sporophore through cultivation experiments new bacterial strain provided by the present invention is (to call in the following text:, confirm to separate this bacterial strain that obtains and belong to really that the phosphorus umbrella belongs to, the fatly phospho umbrella kind through identifying the errorless back of checking.Do not see bibliographical information by retrieval.
Summary of the invention:
The invention provides a kind of new fatly phospho umbrella Pa-IH bacterial strain.
The present invention also provides the method that reaches with this bacterial classification artificial culture fatly phospho umbrella bacterium, is adapted to suitability for industrialized production.
The present invention discloses its pharmaceutical usage.
The present invention further provides the fatly phospho umbrella polysaccharide that extracts with this bacterium and fatly phospho umbrella nucleic acid component and be main medicine and be used for prevention and tumor treatment and analgesia, anti-inflammatory action, enhance immunity power etc. with the other medicines prescription.
Fatly phospho umbrella kind Pa-IH bacterial strain on May 23rd, 2003 in China typical culture collection center (Wuhan University) preservation, and receive preservation registration number CCTCC NO:M203043.
Have following sign: the bacterium colony outward appearance: mycelia velveteen shape, short, aerial hyphae is undeveloped, the initial stage is white, the later stage is faint yellow.
1, microscopy:
(1) mycelia is sturdy, and branch is many.
(2) color depth, even in the methylene blue simple stain.
(3) the visible no cavity of raised growth point.
(4) clamp connexion is arranged, and flourishing.
(5) no living contaminants.
2, physiological and biochemical property:
(1) the growth optimum temps is 24～26 ℃.
(2) the best pH value 6.0～6.5 of growth.
(3) to the demand of oxygen, good gas.
(4) substratum growth optimum moisture 60%.
(5) growth optimum humidity 60～65%.
3, medium component: PDA synthetic medium.
Show that according to document and above-mentioned experimental result of planting again this fatly phospho umbrella bacterium (being the Pa-IH bacterial strain) belongs to phosphorus umbrella genus, fatly phospho umbrella kind.
Fatly phospho umbrella of the present invention is not degenerated more than 18 times for kind going down to posterity, and belongs to resistance of aging, bacterial strain that stable metabolic capacity is strong.How a kind of performance of bacterial classification not only requires its viability strong, growth rate is fast, production cost is low, good product quality, can metabolic capacity that consider it again be stablized, active constituent content height, output and yield number, be to determine the important examination condition of bacterial classification performance, this bacterial strain has guaranteed the above-mentioned requirements condition.
Method with this bacterial classification artificial culture fatly phospho umbrella bacterium is as follows:
Utilize the fatly phospho umbrella bacterium Pa-IH bacterial strain after separating to carry out the solid-state-liquid state-solid-state operational path of artificial culture fatly phospho umbrella sporophore employing, comprise the steps: that slant culture, liquid submerged fermentation, seed culture, mycelium are cultivated and sporophore is cultivated.
Described slant culture, used substratum are the PDA synthetic medium, PH6～6.5,24～26 ℃ of culture temperature, incubation time 10～12 days.
Described shake-flask seed is cultivated, and used medium component comprises peptone 1.5%, yeast extract paste 0.1%, maltose 1%, glucose 1%, wheat bran 5%, KH
2PO
40.15%, MgSO
47H
2O0.075%, vitamins B
10.001%, PH6～6.5, inoculum size is the inclined-plane and the ratio that shakes bottle 1: 6,24～26 ℃ of culture temperature, incubation time 7～10 days, the rotating speed of rotary shaking table are 170～200 rev/mins.
Related mycelium is cultivated, selected substratum is mixtures such as hardwood crumbs, soya bean stalk powder, corn cob meal, Semen Maydis powder, wheat bran, gypsum, lime, and preferred weight percent content is wood chip 30%, soya bean stalk powder 20%, corn cob 20%, Semen Maydis powder 20%, wheat bran 7%, white sugar 1%, gypsum 1%, lime 1%.24～26 ℃ of culture temperature, incubation time 25～35 days, PH6～6.5.
Described management of producing mushroom comprises that take off bag handles in good time, 13～34 ℃ of temperature, and humidity 85～90% strengthens and ventilates, and gives scattered light, until growing the fatly phospho umbrella sporophore.
For enlarging output, increase bacterial classification quantity in the artificial culture process, also can increase seed tank culture after shake-flask seed is cultivated, the used substratum of substratum and shake-flask seed cultivation is identical.Being 6.5 before PH disappears, is 6.2～6.3 after disappearing, and inoculum size is 10%, 24～26 ℃ of temperature, and air flow quantity 0.5V/V, tank pressure 0.5kg/cm2, incubation time are 3～4 days.
The fatly phospho umbrella polysaccharide obtains by following extracting method:
Method 1
With the fatly phospho umbrella sporophore is raw material, uses decocting 1～3 time, and each time is 1～3 hour, filters, and merging filtrate concentrates, and adds proteolytic enzyme, 45～60 ℃ of temperature adjustments, and pH value 5～7, hydrolysis is except that Deproteinization; Add ethanol after the smart filter and carry out alcohol precipitation, ultimate density is 70%, filters collecting precipitation, and is water-soluble, centrifugal slagging-off, and getting supernatant liquor alcohol, to be sink to determining alcohol be 70%, the fatly phospho umbrella polysaccharide of collecting precipitation.
Method 2
With the fatly phospho umbrella sporophore is raw material, with 20% alcohol reflux 3 times, each time is 3 hours, united extraction liquid, concentrating under reduced pressure, it is 80% to containing the alcohol amount that concentrated solution adds ethanol, leaves standstill suction filtration 24 hours, throw out washs in right amount with dehydrated alcohol, drying is ground into fine powder, is able to the fatly phospho umbrella polysaccharide.
Fatly phospho umbrella nucleic acid obtains by following extracting method:
Get fatly phospho umbrella and add the grinding of chloroform (weight ratio 1: 8～12) back, add isopyknic nucleic acid extraction liquid (100mM Tris-Hcl, 50mM EDTA, 50mM Nacl then, 1.5%SDS), be mixed the back in 10000 rev/mins centrifugal 20 minutes, get supernatant liquor and add ethanol solution, vibrate 2 times, 12000 rev/mins centrifugal 10 minutes, outwell supernatant liquor, add 70% washing with alcohol, get final product after the resulting precipitation drying.
Described method of the present invention can be used for polysaccharide fraction and the nucleic acid component that a large amount of preparations of industrialization have antitumor raising immunizing power isoreactivity.
Press practice of pharmacy, fatly phospho umbrella polysaccharide of the present invention (or nucleic acid) can be prepared into the various clinical pharmaceutical dosage form as the treatment tumor disease, improve the medicine of immunizing power and antalgic and inflammation relieving, said medicament is a said formulation on any pharmaceutics.Wherein, oral preparations is selected from any in tablet, capsule, pill, granule, suspensoid, dripping pill, the oral liquid.
Medicine of the present invention preferably contains the fatly phospho umbrella polysaccharide (or nucleic acid) of 1%-99% and the vehicle (medicine that comprises other compatibility) of 99%-1%, preferably contain the fatly phospho umbrella polysaccharide (or nucleic acid) of 30%-80% and the pharmaceutical excipient (comprising the medicine that other compatibility is used) of 70%-20%, preferably contain the fatly phospho umbrella polysaccharide (or nucleic acid) of 60%-70% and the vehicle (comprising the medicine that other compatibility is used) of 40%-30%.
Auxiliary material in the medicine of the present invention is meant conventional vehicle, as solvent, disintegrating agent, correctives, sanitas, tinting material, tackiness agent etc.The medicine that other compatibility in the medicine of the present invention is used, the fatly phospho umbrella polysaccharide that refers to effective dose is certain medicine material, again compatibility other can allow the Chinese medicine or the pharmaceutical chemicals that share.
Positively effect of the present invention is: the fatly phospho umbrella bacterial classification that provides is a novel bacterial, and is safe, in the enforcement of regulations process, do not jeopardize human health or animal and plant cause of disease or contaminate environment.Go down to posterity through system endurance and not degenerate more than 18 generations, it is stronger to belong to resistance of aging stability and metabolic capacity, the bacterial strain that output and yield are higher, the method of artificial culture disclosed by the invention, its operational path is reasonable, and the time ratio artificial culture method routinely that grows sporophore shifted to an earlier date more than 1 month, had the advantages that growth is fast, output is high, meta-bolites is stable, for rare edible mushrooms development opens up a new way, has higher actual application value.In addition, utilize the fatly phospho umbrella sporophore of this spawn culture and mycelium that the submerged fermentation technology is made to can be used for medicinal way.Fatly phospho umbrella polysaccharide and fatly phospho umbrella nucleic acid that the present invention extracts can be used for being prepared into the treatment tumor disease, improve the medicine of immunizing power and antalgic and inflammation relieving.
Following pharmacological evaluation has confirmed that fatly phospho umbrella polysaccharide medicine preparation has the treatment tumor disease, improves the pharmacologically active of the medicine of immunizing power and antalgic and inflammation relieving:
Summary is a model with lotus S180 knurl mouse and Emhorn ascitic type mouse, has studied the anti-tumor in vivo effect of fatly phospho umbrella polysaccharide.The result shows that the fatly phospho umbrella polysaccharide can not only suppress tumor growth in vivo, can also improve the immunological competence of body.In addition, the fatly phospho umbrella polysaccharide has bacteriostatic action preferably to golden Portugal bacterium and intestinal bacteria, also has tangible strong effect (antifatigue effect)
1 material
1.1 medicine: the fatly phospho umbrella polysaccharide is the polysaccharide material that extracts in the fatly phospho umbrella.Endoxan: Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number 20010304.
1.2 S180 and ehrlich ascites tumor strain: the Cancer Hospital of Chinese Academy of Medical Sciences knurl strain of regularly going down to posterity.
1.3 bacterium: bacterial classification is golden Portugal bacterium, intestinal bacteria, shigella, candida albicans, assorted aspergillus tubigensis, is all provided by microorganism teaching and research room of Bethune medical university.
1.4 animal: Kunming mouse, the male and female dual-purpose, body weight (22 ± 2) g is available from Univ. of Farming and Stockbreeding, PLA's Experimental Animal Center.
2 methods
2.1 mouse-borne tumor:
Get the S of inoculation 6d
180And Emhorn ascitic type mouse ascites, with sterile saline (1: 3) dilution, through right upper extremity keep right scapula place subcutaneous injection (sc) or abdominal injection (ip) 0.2mL (4 * 10
6～6 * 10
6Individual oncocyte, living cell rate＞95%), make solid tumor type or ascitic type bearing mouse model.
2.2 drug dose and route of administration:
By experimental design dosage ip (solid tumor type) or intramuscular injection im (ascitic tumor type) administration, negative control group physiological saline ip or intramuscular injection (im), positive controls endoxan ip or im, every mouse medication every day 1 time.
2.3 the calculating of tumour inhibiting rate, spleen index and thymus index:
Lotus S
180Solid tumor type mouse is in inoculation back 24h random packet, and successive administration 8d weighs behind the 8d, puts to death, and gets knurl, spleen and thymus gland respectively, and body weight is calculated tumour inhibiting rate, spleen index and thymus index and carried out statistical procedures.
2.4 the calculating of survival rate:
Emhorn ascitic type mouse is in inoculation back 24h random packet, and successive administration 8d continues after the drug withdrawal to observe, and up to natural death, calculates its survival rate elongation.
3 results
3.1 to lotus S
180The tumor-inhibiting action of knurl mouse: see Table 1.
Table 1 couple lotus S
180The restraining effect of mouse tumor (x ± s)
Compare with control group:
*P＜0.05
*P＜0.
3.2 the influence to Emhorn ascitic type mouse storaging current: see Table 2.
The influence of table 2 pair Emhorn ascitic type mouse storaging current (x ± s)
Compare with control group:
*P＜0.05
*P＜0.01
Compare with the endoxan group:
P＜0.05
3.3 to lotus S
180The influence of knurl mouse immune index: see Table 3.
Table 3 couple lotus S
180The influence of knurl mouse immune index (x ± s)
| Group | Dosage (mg/kgd) | Number of animals (only) | Spleen index | Ratio # (％) | Thymus index | Ratio ## (％) |
| Normal group | - | 12 | 6.76±0.58 | 2.32±0.43 | ||
| Contrast | - | 18 | 13.39±0.45 | 198.1 | 1.31±0.57 | 56.5 |
| The fatly phospho umbrella polysaccharide | 1000 | 12 | 8.63±0.62 * | 127.8 | 1.70±0.95 | 73.3 |
| 500 | 12 | 9.86±0.52 | 145.9 | 1.92±0.24 □* | 78.6 | |
| 250 | 12 | 10.23±1.36 | 151.3 | 1.55±0.37 | 66.8 | |
| Endoxan | 0.01 | 12 | 9.32±0.87 | 137.9 | 1.61±0.69 | 69.4 |
#Blank group, each medication group and normal group spleen index ratio;
##Blank group, each medication group and normal group thymus index ratio.
Compare with control group:
*P＜0.01; Compare with the endoxan group:
P＜0.01
Table 1～3 results show that fatly phospho umbrella polysaccharide PWAE 100mg/kgd has obvious tumor-inhibiting action (63.2%), and can obviously prolong the survival time (48.0%) of Emhorn ascitic type mouse, and along with dosage reduces, tumour inhibiting rate and survival time also reduce.Fatly phospho umbrella polysaccharide PWAE 100mg/kgd compares with positive controls endoxan group, the tumour inhibiting rate and the latter (58.3%) close (P＞0.05), and the survival time rate elongation is than the high (P＜0.05=of latter's (29.7%).Behind the mouse-borne tumor, cause that immune indexes is undesired, show as spleen index and raise, be 198.1% of normal mice, thymus index descends, and is 56.5% of normal mice, fatly phospho umbrella polysaccharide PWAE various dose group all can make the tumor-bearing mice spleen index reduce, and thymus index raises, and draws close to normal value.Fatly phospho umbrella polysaccharide PWAE 100mg/kgd dosage group reduces spleen index the most remarkable (127.8%), and (no significant difference (P＞0.05) is compared in P＜0.01 with the endoxan group to compare with control group that there were significant differences.500mg/kgd dosage group rising thymus index is the most remarkable, and (P＜0.01, (P＜0.01 illustrates that its effect is stronger than the latter to compare with endoxan group (69.4%) that there were significant differences to compare with control group that there were significant differences.In sum, the fatly phospho umbrella polysaccharide can not only suppress the tumor-bearing mice growth of tumor, prolongs mouse life, and the effect that improves the tumor-bearing mice immunity function is arranged.
3.4 external bacteriostatic experiment:
Get 5 kinds of above-mentioned experimental strains, every kind 2 strain, totally 12 strains.Adopt two times of dilution methods of successively decreasing of test tube, the reagent liquid that is subjected to of serial dilution mixed with bacterium liquid, put 37 ℃ of incubators and cultivate 18～24h, observe bacterial growth situation in vitro, in the pipe limpid person be subjected to for bacterial growth but.White reads the strain bacterium and assorted aspergillus tubigensis is seeded on the husky Bao Shi solid medium that contains soup, and Candida albicans is put 37 ℃ of incubators and cultivated 24～48h observation growing state, and assorted aspergillus tubigensis is put 22 ℃ of incubators and cultivated 48～72h observation growing states.Other establishes blank pipe and does the bacterial growth contrast.Experimental result sees Table 4.
The bacteriostatic action mg/ml of table 4 fatly phospho umbrella polysaccharide
| Bacterial strain strain number | MIC |
| Streptococcus aureus 2 | 9.75 |
| Intestinal bacteria 2 | 4.875 |
| Shigella 2 | ＞78 |
| Candida albicans 2 | ＞78 |
| Assorted aspergillus tubigensis 2 | ＞78 |
Experiment shows that the fatly phospho umbrella polysaccharide is to 2 strain G
+Streptococcus aureus MIC (minimum inhibitory concentration) is 9.75mg/ml, is 4.875mg/ml to intestinal bacteria MIC, shows that Hubei Chinese flowering crabapple leaf decocting liquid has bacteriostatic action preferably to golden Portugal bacterium and intestinal bacteria.This result discloses fatly phospho umbrella polysaccharide intestinal bacteria and golden Portugal bacterium has the pharmacological action that suppresses its growth preferably.
3.5 strong effect (antifatigue effect)
Press table 5 grouping administration, in 7 days, irritate stomach every day once, control group is irritated stomach equal-volume distilled water, and behind the last administration 2h, it is that record mouse swimming time the results are shown in Table 5 in 23～25 ℃ the water that mouse is dropped into temperature.The fatly phospho umbrella polysaccharide can obviously prolong the mouse swimming time, shows that the fatly phospho umbrella polysaccharide has tangible strong effect (antifatigue effect)
The strong effect (antifatigue effect) of table 5 fatly phospho umbrella polysaccharide (x ± s, n=10)
With compare,
*P＜0.05,
* *P＜0.001
4 discuss
With lotus S
180Knurl mouse and Emhorn ascitic type mouse are model, have studied the anti-tumor in vivo effect of fatly phospho umbrella polysaccharide.The fatly phospho umbrella polysaccharide has in the tangible antibody transplanted tumor and prolongs the effect of tumor-bearing mice survival time, and the fatly phospho umbrella polysaccharide can also reduce the tumor-bearing mice spleen index, and the rising thymus index makes it to tend to normal value.Prompting fatly phospho umbrella polysaccharide can not only suppress tumor growth in vivo, can also improve the immunological competence of body.Experimental result explanation fatly phospho umbrella polysaccharide can reach antitumor action by enhance immunity power in the body.In addition, the fatly phospho umbrella polysaccharide also has bacteriostatic action preferably to golden Portugal bacterium and intestinal bacteria, and has tangible strong effect (antifatigue effect).
Following pharmacological evaluation has confirmed that also fatly phospho umbrella nucleic acid drug preparation has the treatment tumor disease, improves the pharmacologically active of the medicine of immunizing power and antalgic and inflammation relieving:
The summary result of study shows that the fatly phospho umbrella nucleic acid component has enhance immunity ability and physical strength reinforcing ability.Its mechanism of action is the protein synthesis that the fatly phospho umbrella nucleic acid component can increase body.
1 material
1.1 medicine: the fatly phospho umbrella nucleic acid component is the nucleic acid substances that extracts in the fatly phospho umbrella.
1.2 animal: Kunming mouse, the male and female dual-purpose, body weight (22 ± 2) g is available from Univ. of Farming and Stockbreeding, PLA's Experimental Animal Center.
2 methods and result
2.1 influence to the mouse immune organ weight
Mouse irritated the stomach medicine respectively every day or with volume distilled water 0.2ml/10g body weight, every day 1 time, continuous 7 days.Model group and drug component be in the 1st, 3,5,6 days intraperitoneal injection of cyclophosphamide 10mg/kg of experiment, on 7th, and 1h after the last administration, all mouse take off cervical vertebra and put to death.Get spleen and thymus gland, peel off totally, weigh, calculate spleen index and thymus index, the results are shown in Table 1 with the mg/10g body weight.
Table 1 fatly phospho umbrella nucleic acid is to mouse immune organ weight's influence (x ± s)
Compare with model group
*P＜0.05,
*P＜0.01.
The result shows that the fatly phospho umbrella nucleic acid component can obviously improve the spleen and the thymic weight of immunosuppressed mice.Point out it to have the effect that improves immune function of mice.
2.2 influence to mouse monokaryon macrophage system phagocytic function
Mouse irritates the stomach medicine respectively or with volume distilled water, once a day, continuous 7 days, 1h tail vein injection india ink 0.1ml/10g after the last administration got blood 20 μ l from the eye socket venous plexus respectively in injection back 2 and 20min, is added to 2ml 0.1%Na
2CO
3Shake up in the solution, measure optical density(OD) at the 680nm place, calculate phagocytic index K and activate the phagocytic capacity α.The results are shown in Table 2
Table 2 fatly phospho umbrella nucleic acid component is to the influence of mouse monokaryon macrophage system phagocytic function (x ± s)
Compare with control group
*P＜0.05
The result shows that the fatly phospho umbrella nucleic acid component has the effect that improves the normal mouse mononuclear-macrophage phagocytic function.Point out it to have the effect that improves the mouse cell immunologic function.
2.3 influence to the generation of mouse hemolytic antibody
Mouse is irritated the stomach medicine respectively every day or with volume distilled water 0.2ml/10g body weight, every day 1 time, continuous 7 days.Model group and drug component be not in experiment the 1st, 3,5,6 days intraperitoneal injection of cyclophosphamide 10mg/kg, administration the 1st day, every mouse carried out immunity with 5% chicken red blood cell 0.2ml abdominal injection, the 7th day eye socket venous plexus of administration got blood 20 μ l, add 1ml and shake up in the physiological saline, add 5% chicken red blood cell 0.5ml again, in ice bath, add 0.5ml complement (1: 10 fresh guinea pig serum), incubation 30min in 37 ℃ of waters bath with thermostatic control, termination reaction 3min in ice bath, the centrifugal 10min of 2000rpm gets supernatant 1ml, add 3ml Dou Shi liquid, after room temperature leaves standstill 10min,, the results are shown in Table 3 at 540nm place photometry density value.
The influence that table 3 fatly phospho umbrella nucleic acid component mouse hemolytic antibody generates (x ± s)
Compare with model group
*P＜0.05,
*P＜0.01.
The result shows that the fatly phospho umbrella nucleic acid component can obviously improve the generation of immunosuppressed mice hemolytic antibody, points out it to have the effect that improves the mouse humoral immune function.
2.4 hypoxia-bearing capability
50 of adult mices in 7 days, are irritated stomach every day once by table grouping administration, and control group is irritated stomach equal-volume distilled water, behind the last administration 24h, it is the wide-necked bottle that 250ml contains the 5g sodica calx that mouse is put into volume, jam-pack, 1/bottle, the record mouse survival time, the results are shown in Table.The fatly phospho umbrella nucleic acid component can obviously prolong the survival time under the mouse normobaric hypoxia.Have resisting oxygen lack.
Fatly phospho umbrella nucleic acid to the influence of mouse normal pressure hypoxia-bearing capability (x ± s, n=10)
With compare,
*P＜0.05
2.5 enhancing body muscle power effect (antifatigue effect)
Influence to the mouse swimming time is pressed table grouping administration with 50 of adult mices, in 7 days, irritates stomach every day once, control group is irritated stomach equal-volume distilled water, and behind the last administration 24h, it is in 23～25 ℃ the water that mouse is dropped into temperature, record mouse swimming time the results are shown in Table.The fatly phospho umbrella nucleic acid component can obviously prolong the mouse swimming time.Has the effect of enhancing body muscle power.
The fatly phospho umbrella nucleic acid component to the influence of mouse swimming time (x ± s, n=10)
With compare,
*P＜0.05,
* *P＜0.001
2.6 promotion protein synthesis
50 of adult mices in 7 days, are irritated stomach every day once by table grouping administration, and control group is irritated stomach equal-volume distilled water, behind the last administration 1h, mouse is put to death, and gets hepatic tissue, surveys protein content, the results are shown in Table.The fatly phospho umbrella nucleic acid component can obviously increase the murine liver tissue protein content.Have the promotion protein synthesis.
The fatly phospho umbrella nucleic acid component to the influence of Mouse Liver protein content (x ± s, n=10)
With compare * P＜0.05
3. discuss
The fatly phospho umbrella nucleic acid component can obviously improve the spleen and the thymic weight of immunosuppressed mice, has the effect that improves the normal mouse mononuclear-macrophage phagocytic function, can obviously improve the generation of immunosuppressed mice hemolytic antibody, point out it to have the effect that improves immune function of mice.The fatly phospho umbrella nucleic acid component can obviously prolong the survival time under the mouse normobaric hypoxia, has resisting oxygen lack; Can obviously prolong the mouse swimming time, have the effect of enhancing body muscle power; Can obviously increase the murine liver tissue protein content, have the promotion protein synthesis.
The above results shows that the fatly phospho umbrella nucleic acid component has enhance immunity ability and physical strength reinforcing ability. its mechanism of action is the protein synthesis that the fatly phospho umbrella nucleic acid component can increase body.
The function assessment research of fatly phospho umbrella nutritive medium:
The mycelium that utilizes fatly phospho umbrella sporophore and submerged fermentation technology to make is mixed with the fatly phospho umbrella nutritive medium, has both kept the inherent color and luster and the bright fragrance of bacterium liquid, can regulate the animal immune function again.The present invention has reported this drink according to the protective foods function assessment assessment process and the method for inspection, respectively selects a project in cellular immunization and humoral immunization, the result who tests.This drink that per os gives the mouse various dose can obviously improve the ability of small white mouse macrophage phagocytic chicken red blood cell after 20 days, and along with dosage increases, this influence is remarkable further, the high dose group of drink can obviously improve the humoral immune function of mouse simultaneously, and there were significant differences with the contrast of distilled water group.Tardy parasexuality reaction of dinitrofluorobenzene inducing mouse (DTH) and serum hemolysin determination experiment show, mouse half hemolysis value and the reaction of tardy parasexuality there is tangible dose-effect relationship, the analysis of effective component of drink shows that polysaccharide content is higher, thereby can present immunologic enhancement preferably, toxicity test is without any side effects, be a kind of mouthfeel novelty, the health promoting beverage that color and luster is bright.
The drink that utilizes fatly phospho umbrella sporophore or mycelium to make, relevant experimental results is as follows:
1, the fatly phospho umbrella nutritive medium is to the influence of mouse macrophage ability.Give the fatly phospho umbrella drink liquid of 3 times, 10 times, 20 times various dose of mouse after 20 days by the visible per os of table 1, high, medium and low dosage group, the phagocytic rate that Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell is higher than control group, learn by statistics and handle, in, low dose group and control group have significant difference, the poor heteropole of high dose group and control group is remarkable.
Table 1, fatly phospho umbrella nutritive medium to the influence of scavenger cell (* ± SD)
| Group | Mouse (only) | Phagocytic rate (%) | The P value |
| The distilled water group | 10 | 21.875±5.60 | |
| Low dose group | 10 | 34.98±10.12 * | ＜0.05 |
| Middle dosage group | 10 | 39.94±9.06 * | ＜0.05 |
| High dose group | 10 | 44.11±18.56 ** | ＜0.01 |
2, the fatly phospho umbrella nutritive medium is to the influence of mouse humoral immune, and by table 2 as seen, per os gave mouse after 20 days, and high dosage and control group relatively have significant difference.(P＜0.05), in, low dosage, two dosage group half hemolysis value though increase than control group, and have dose-response relationship, with control group there was no significant difference relatively.
Table 2, fatly phospho umbrella nutritive medium are to mouse HC
50Measurement result (* ± SD)
| Group | Mouse (only) | HC 50 | The P value |
| The distilled water group | 10 | 270.93±28.09 | |
| Low dose group | 10 | 275.52±21.54 | ＞0.05 |
| Middle dosage group | 10 | 275.81±23.59 | ＞0.05 |
| High dose group | 10 | 281.08±13.91 | ＜0.05 |
Edible and the medicinal research of fatly phospho umbrella:
1, the nutrition of fatly phospho umbrella:
Result of study shows, the fatly phospho umbrella sporophore contains protein, protein content is a kind of high protein, low-fat food between animal and plant, crude protein comprises that water-soluble protein accounts for 23.65%, crude fat 0.32%, carbohydrate account for 65.83%, ash content accounts for 10.1%, 18 seed amino acids, particularly human body that contain A wide selection of colours and designs in the protein can not must account for 42.2% of total amino acid amount from whole 8 kinds of indispensable amino acids of external world's picked-up by synthetic.Contain various carbohydrates in the sporophore carbohydrate, and contain a certain amount of VITAMIN, the VitB1 in the VITAMIN, nicotinic acid, xitix and riboflavin can participate in the redox reaction of human body, regulate the effect of the metabolic function of body.Ash content chats element is higher with Ca, Mg content, also contains a certain amount of zinc, iron.Fatly phospho umbrella is nutritious, and is often edible, can increase people's physique, can be used as protective foods.
2, the anti-inflammatory of fatly phospho umbrella:
Anti-pathogenic bacteria: test-results shows that the fatly phospho umbrella sporophore is removed the material that the polysaccharide aqueous extract has growth such as the pathogenic bacteria that suppresses Pseudomonas aeruginosa, dysentery bacterium, streptococcus aureus, intestinal bacteria.The fatly phospho umbrella antibacterial substance belongs to the small-molecule substance that does not contain polysaccharide.
3, fatly phospho umbrella antitumour activity and immunologic function:
Carried out the experiment of antitumour activity immunologic function with fatly phospho umbrella sporophore water extract and fatly phospho umbrella polysaccharide.Experimental result shows: fatly phospho umbrella polysaccharide (I number) is 7.98～17.6% to the inhibiting rate ♂ of S180 sarcoma, and ♀ is 16～24%, is 10～21% to the ehrlich ascites tumor increase in life span.Greasiness phosphine umbrella vat liquor (II number) all may activate Turnover of Mouse Peritoneal Macrophages, strengthen its phagocytic function, improve immunologic function, suppress the clear speed in mouse IV carbon granules corridor, show the fatly phospho umbrella polysaccharide, fatly phospho umbrella sporophore vat liquor has the function that strengthens the mouse monokaryon scavenger cell, can increase the weight of mouse immune organ (thymus gland and spleen), no matter can think that fatly phospho umbrella is that water extract or polysaccharide all have immuno-potentiation.
Embodiment:
Embodiment 1
Slant strains is cultivated: adopt the PDA synthetic medium, and PH6.5,24～26 ℃ of culture temperature, this bacterium sprouts fast, and it is that about 10～12 days left and right sides bacterium colonies of obvious bacterium colony of basic point cover with pipe that bacterial classification connects that the back forms in 2～3 days with the inoculation piece.Mycelia initial stage white, velveteen shape, branch are many, and the later stage is faint yellow, observe obviously by microscopy that mycelia is sturdy, branch is many, clamp connexion is flourishing, color depth in the methylene blue simple stain, visible raised growth point, no cavity, no living contaminants.
Shake-flask seed is cultivated, and used medium component is peptone 1.5%, yeast extract paste 0.1%, maltose 1%, glucose 1%, wheat bran 5%, KH
2PO
40.15%, MgSO
47H
2O 0.075%, vitamins B
10.001%, PH6.5 (before disappearing), autoclaving pressure 1kg/cm
230min, the inoculation of cooling back, inoculum size is the inclined-plane and the ratio that shakes bottle 1: 6, what promptly inclined-plane met 6 250ml shakes 24～26 ℃ of bottle (loading amount 100ml) culture temperature, 170～200 rev/mins of rotary shaking tables were cultivated 7～10 days, and bacterium ball weight in wet base reaches (weight in wet base measuring method: get 100 milliliters of nutrient solutions and place graduated cylinder more than 20%, leave standstill 15min, observe the settled graduated percentage ratio of bacterium ball) distinguishing the flavor of is fruit aroma, acid slightly, microscopy is observed mycelia and color depth, sturdy, branch is many, and obviously as seen vegetative point has clamp connexion, uncontaminated assorted bacterium, it is standby promptly to can be used as shake-flask seed.
Seed tank culture, substratum is cultivated with shake-flask seed, PH6.5 (before disappearing) PH6.2～6.3 (back disappears), 24～26 ℃ of temperature, air flow quantity 0.5V/V, tank pressure 0.5kg/cm
2Inoculum size is 10～15% of a seeding tank, cultivated about 3～4 days, when being checked through bacterium ball weight in wet base, mode of appearance reaches more than 30%, sense of taste is fruity, microscopy observe mycelia color depth, sturdy, branch is many, as seen vegetative point obviously has clamp connexion not see that living contaminants can carry out fermentation flask (bag) and cultivate.
Fermentation flask (bag) is cultivated used culture medium prescription: hardwood crumbs 30%, soya bean stalk powder 20%, corn cob 20%, Semen Maydis powder 20%, wheat bran 7%, white sugar 1%, gypsum 1%, lime 1%, water content 60%, select 17 * 33cm Polypropylene Bag or low pressure polyethylene bag for use, high 16～17cm feeds, cover neck ring, plug cotton, last bag kraft paper, following 2 hours of autoclaving 1.5kg/cm2, or normal-pressure sterilization kept 10～12 hours for 100 ℃.Cooling back aseptic technique inserts bacterial classification, and 24～26 ℃ of temperature keep dark surrounds, and relative air humidity 50～60% was cultivated 25～35 days.
Visual inspection, mycelia is dense white, is the fine hair shape, tubbiness, close neat, the no beige ponding of bag, not dry shrinkage phenomenon, no living contaminants, growth later stage mycelia is faint yellow, and has part mushroom flower bud to occur, the microscopy mycelia color depth, sturdy, branch is many, clamp connexion is arranged, vegetative point obviously as seen.
Management of producing mushroom: full bag is covered with in bacterium bag cultivation 25～35 days, mycelia, continues to cultivate 7～10 days, opens sack or takes off the bag processing.Bag is taken off in employing, erects to be emitted in the cold bed, and at a distance of the 1cm distance, intermediate gaps fills up with the anti-assorted soil of nutrition that (the anti-assorted soil of nutrition is filled a prescription and is: add lime 2%, plant ash 1%, urea 0.3%, KH in per hundred jin of soil between bag and the bag
2PO
40.1%, MgSO
40.2%), the anti-assorted soil of the thick nutrition of 1～2cm is covered on the bag upper strata, costs and hides the rain sunshade, and widen thermal stimulus, and make temperature remain on 13～34 ℃ (being fit to 15～18 ℃), increase relative air humidity 85～90% simultaneously, add forced ventilation, increase scattered light, can form sporophore in 5～7 days.The general list of sporophore is given birth to or grown thickly, and is general medium big, bacteria cover diameter 3～12cm, first flattened spherical, the edge is involute slightly, after gradually open and flat, very sticking, paddy is yellow, dirty yellow to tawny, the nearly calm scale of brown is arranged, central authorities are empty, bacterial context white is to faint yellow, lamella white is to the rust brown, and is directly living or near curved living, close slightly, not isometric, the long 5cm of stem, thick 0.5～3cm, cylindrical and cap is given birth to together, and the scale of brown warp is arranged, and is sticking or sticking slightly, the bottom is often crooked, cellulosic meat is real, and collarium is faint yellow, and is membranous, give birth on the handle, easily come off.The spore rust, ellipse or oblong, level and smooth, 7.5～9.5um * 5～6.3um.Cystidium is colourless or filbert, the bar-shaped thalamium of not breaking through, 30～42um * 6.3～7.5um.Above-mentioned form and wild fatly phospho umbrella are identical.
Embodiment 2
Take by weighing fatly phospho umbrella sporophore 1Kg, add water 20Kg, 95 ℃ are extracted 3 times repeatedly, each 1 hour.United extraction liquid filters, and reduced vacuum concentrates, and heat is surveyed proportion 1.04, smart filter.Add the edible ethanol alcohol precipitation, ultimate density reaches 70%.Filter, collecting precipitation, water-soluble.Centrifugal slagging-off is got supernatant alcohol precipitation (determining alcohol 70%) 2-3 time, collecting precipitation, many umbrellas phosphatide polysaccharide 120g.
Embodiment 3
Take by weighing fatly phospho umbrella mycelium 1Kg, add water 20Kg, 98 ℃ are extracted each 1 hour three times.United extraction liquid filters, and reduced vacuum concentrates, and heat is surveyed proportion 1.04, smart filter.Add the edible ethanol alcohol precipitation, ultimate density reaches 80%.Filter, collecting precipitation, water-soluble.Centrifugal slagging-off is got supernatant alcohol precipitation (determining alcohol 80%) 2-3 time, collecting precipitation, many umbrellas phosphatide polysaccharide 100g.
Embodiment 4
Take by weighing fatly phospho umbrella sporophore 1Kg, add water 20Kg, 96 ℃ are extracted 3 times repeatedly, each 1 hour.United extraction liquid filters, and reduced vacuum concentrates, and heat is surveyed proportion 1.06, smart filter.Add papoid 2-5g, temperature adjustment 45-60 ℃, pH value 5-7, hydrolysis removes albumen.Smart filter adds the edible ethanol alcohol precipitation, and ultimate density reaches 60%.Filter, collecting precipitation, water-soluble, alcohol precipitation 2-3 time repeatedly, collecting precipitation, many umbrellas phosphatide polysaccharide 120g.
Embodiment 5
Take by weighing fatly phospho umbrella mycelium 1Kg, add water 20Kg, 98 ℃ are extracted 3 times repeatedly, each 1 hour.United extraction liquid filters, and reduced vacuum concentrates, and heat is surveyed proportion 1.02, smart filter.Add papoid 1-3g, temperature adjustment 40-55 ℃, pH value 3-5, hydrolysis removes albumen, smart filter.Add the edible ethanol alcohol precipitation, ultimate density reaches 70%.Filter, collecting precipitation, water-soluble, alcohol precipitation 2-3 time repeatedly, collecting precipitation, many umbrellas phosphatide polysaccharide 100g.
Embodiment 6
The extraction of fatly phospho umbrella nucleic acid:
Get and grind after fatly phospho umbrella 1kg adds chloroform 10kg, add isopyknic nucleic acid extraction liquid (100mM Tris-Hcl, 50mM EDTA, 50mM Nacl then, 1.5%SDS), be mixed the back in 10000 rev/mins centrifugal 20 minutes, get supernatant liquor and add in the dehydrated alcohol, vibrate 2 times, 12000 rev/mins centrifugal 10 minutes, outwell supernatant liquor, add 70% washing with alcohol, get precipitation get final product after the drying fatly phospho umbrella nucleic acid 10g.
Embodiment 7
The fatly phospho umbrella nutritive medium
Add water 20Kg after taking by weighing fatly phospho umbrella mycelium (or sporophore) 1Kg pulverizing, boiled 30 minutes, divide a bottle can, the 10ml/ bottle, other project should meet under Pharmacopoeia of People's Republic of China version oral liquid in 2000 item.
Claims (6)
1. a fatly phospho umbrella kind Pa-IH bacterial strain (Pholiota adipose Fr.Que) CCTCC NO:M203043.
2. cultivate the method for the described fatly phospho umbrella kind of claim 1 Pa-IH bacterial strain, may further comprise the steps and finish:
The described fatly phospho umbrella bacterium of claim 1 Pa-IH bacterial strain is carried out slant culture, liquid submerged fermentation, seed culture, mycelium cultivation and sporophore to be cultivated;
Slant culture, used substratum are the PDA synthetic medium, PH6～6.5,24～26 ℃ of culture temperature, incubation time 10～12 days;
Liquid submerged fermentation adopts shake-flask seed to cultivate, and inoculum size is the inclined-plane and the ratio that shakes bottle 1: 6,24～26 ℃ of culture temperature, and incubation time 7～10 days, the rotating speed of rotary shaking table are 170～200 rev/mins;
Mycelium is cultivated, and the substratum of selection is hardwood crumbs, soya bean stalk powder, corn cob meal, Semen Maydis powder, wheat bran, gypsum, lime mixture, 24～26 ℃ of culture temperature, incubation time 25～35 days, PH6～6.5;
Sporophore is cultivated, and comprises that take off bag handles in good time, 13～34 ℃ of temperature, and humidity 85～90% strengthens and ventilates, and gives scattered light, until growing the fatly phospho umbrella sporophore.
3. Accessory Right requires to extract in the 1 described fatly phospho umbrella kind Pa-IH bacterial strain method of fatly phospho umbrella polysaccharide, may further comprise the steps:
Sporophore with the described fatly phospho umbrella kind of claim 1 Pa-IH strain culturing is a raw material, uses decocting 1～3 time, and each time is 1～3 hour, filters, and merging filtrate concentrates, and adds proteolytic enzyme, 45～60 ℃ of temperature adjustments, and pH value 5～7, hydrolysis is except that Deproteinization; Add ethanol after the smart filter and carry out alcohol precipitation, ultimate density is 70%, filters collecting precipitation, and is water-soluble, centrifugal slagging-off, and getting supernatant liquor alcohol, to be sink to determining alcohol be 70%, the fatly phospho umbrella polysaccharide of collecting precipitation.
4. Accessory Right requires to extract in the 1 described fatly phospho umbrella kind Pa-IH bacterial strain method of fatly phospho umbrella polysaccharide, may further comprise the steps:
Sporophore with the described fatly phospho umbrella kind of claim 1 Pa-IH strain culturing is a raw material, with 20% alcohol reflux 3 times, each time is 3 hours, united extraction liquid, concentrating under reduced pressure, it is 80% to containing the alcohol amount that concentrated solution adds ethanol, leaves standstill suction filtration 24 hours, throw out washs in right amount with dehydrated alcohol, drying is ground into fine powder, is able to the fatly phospho umbrella polysaccharide.
The fatly phospho umbrella polysaccharide that extracts according to claim 3 or 4 methods in preparation treatment tumour, improve the application in the medicine of immunizing power.
6. the application of fatly phospho umbrella polysaccharide in the medicine of preparation analgesia, anti-inflammatory action of extracting according to claim 3 or 4 methods.
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| 滑菇多糖的制备工艺. 宋玉光，李庆章，高学军.中国生化药物杂志，第23卷第4期. 2002 |
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