CN101803531B - China cryptoporus sinensis and solid cultural method and application thereof - Google Patents
China cryptoporus sinensis and solid cultural method and application thereof Download PDFInfo
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Abstract
The invention discloses a china cryptoporus sinensis and application thereof. The bacterial strain is China cryptoporus sinensis Liuzh2009-7523CGMCC No.3575. The new bacterial strain provided by the invention has multiple functions as resisting histamine release, restraining the growth of a plurality of tumor cells and various allergic reactions and treating pain and inflammation. At the aspects of resisting the histamine release and restraining the growth of a plurality of tumor cells, the effect of the bacterial strain or the extract of leavening thereof is better than the effect of the positive control group. Moreover, the method of culturing the China cryptoporus sinensis CGMCC No.3575 has the advantages of simple operation, low cost and high yield and the yield of active extract can reach 1.5 percent (by the solid culture medium dry weight ). Therefore, the bacterial strain, the bacterial strain leavening and the extract of the leavening have wide application foreground.
Description
Technical field
The present invention relates to the latent pore fungi of strain China and solid culture method and application.
Background technology
Latent pore fungi has another name called: Cryptoporus volvatus (peck) Scnear, tree stump, lotus look bacterium, wooden fish bacterium, blue, a bite ear of incense wood belong to Basidiomycotina, Hymenomycetes; Aphyllophorales, Polyporaceae, latent pore fungi belongs to; It is wood decay fungi; Be grown in the shade of pine tree or other seeds trunks or branch, or fall wood earthward, medicinal part is fresh or dry fruit body.Be distributed in province such as Hebei, Sichuan, Yunnan, Jiangsu, Jiangxi, Hubei, Guangxi, Fujian, Hainan, Jilin and Korea, Japan, Europe, the North America of China.The latent pore fungi that distributes in China has two kinds, latent pore fungi Cryptoporus volvatus (Peck) Schear, the latent pore fungi Cryptoporus sinensis Sheng H.Wu & M.Zang of China.2000, Wu Shenghua showed that with the solemn research of a surname the latent pore fungi that China distributes is mainly the latent pore fungi of China.
Latent pore fungi fruit body ecotope is strict, fruit body is gathered difficulty, open-air reserves are few, and the artificial culture difficulty of fruit body is very big, so latent pore fungi fruit body resource scarcity.
Summary of the invention
An object of the present invention is to provide the latent pore fungi of strain China.
China provided by the present invention conceals pore fungi (Cryptoporus sinensis) Liuzh2009-7523, and its deposit number is CGMCC No.3575.
This bacterial strain Liuzh2009-7523 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 01 04th, 2010; Address: Datun Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101), preserving number is CGMCC No.3575, classification called after Cryptoporussinensis.
Another object of the present invention provides a kind of method of cultivating latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China.
Method of cultivating latent pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCCNo.3575 of China provided by the present invention comprises the steps: with following arbitrary said medium culture latent pore fungi (Cryptoporussinensis) Liuzh2009-7523CGMCC No.3575 of China.
In the above-mentioned incubation, said culture condition is: temperature is 25 ℃-30 ℃ and lucifuge.
Another object of the present invention provides the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of a kind of China.
China provided by the present invention conceals the fermentation product of pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575; Be to prepare: cultivate latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China according to above-mentioned arbitrary said method according to the method that comprises the steps; Cultivated 30 days; Collect all materials in the culture vessel, all the material notes in the culture vessel are made fermentation product.
Another object of the present invention provides the extract of the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of a kind of China.
China provided by the present invention conceals the extract of the fermentation product of pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575; Be to prepare: 1) cultivate latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China according to above-mentioned arbitrary said method according to the method that comprises the steps; Cultivated 30 days; Collect all materials in the culture vessel, all the material notes in the culture vessel are made fermentation product;
2) fermentation product in the step 1) and 95% (volumn concentration) ethanol water is mixed, 100 ℃ of refluxing extraction are got organic facies, are the extract of the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China;
The time of each refluxing extraction is 1 hour; The proportioning of fermentation product and said 95% (volumn concentration) ethanol water is (900-1000) g:5 liter in the said step 1).
Latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China also belongs to protection scope of the present invention with/application in the following product of preparation: the product that 1) has the antihistamine release function; 2) has the product that prevents and/or treats the allergic disease function; 3) has the product of analgesia function; 4) has the product that suppresses the growth of tumour cell function; Or 5) has the product that prevents and/or treats the tumour function.
The application of the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of above-mentioned arbitrary said China in the following product of preparation also belongs to protection scope of the present invention: the product that 1) has the antihistamine release function; 2) has the product that prevents and/or treats the allergic disease function; 3) has the product of analgesia function; 4) has the product that suppresses the growth of tumour cell function; Or 5) has the product that prevents and/or treats the tumour function.
The application of the extract of the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of above-mentioned arbitrary said China in the following product of preparation also belongs to protection scope of the present invention: the product that 1) has the antihistamine release function; 2) has the product that prevents and/or treats the allergic disease function; 3) has the product of analgesia function; 4) has the product that suppresses the growth of tumour cell function; Or 5) has the product that prevents and/or treats the tumour function.
Another object of the present invention provides a kind of product with following function: the antihistamine release function, have the allergic disease of preventing and/or treating function, analgesia function, suppress the growth of tumour cell function or prevent and/or treat the tumour function, its active component be the fermentation product of latent pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575 of China and/or latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of above-mentioned arbitrary said China and/or the extract that above-mentioned arbitrary said China conceals the fermentation product of pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575.
In above-mentioned application or the said goods, the pain in the said analgesia is the pain that neuropathic pain, traumatic pain or tumour cause, specifically is the pain that the aqueous solution of 0.6% (volumn concentration) glacial acetic acid causes mouse;
Said tumour cell is human liver cancer cell HepG2, Human Prostate Cancer Cells PC3, human lung cancer cell A549, people's rectum cancer cell HCT-15, human cervical carcinoma cell Hela, gastric carcinoma cells SGC7901, mouse mastopathy cell EMT6 or mouse prostate cancer RM-1 cell;
Said tumour is liver cancer, prostate cancer, lung cancer, the carcinoma of the rectum, cervical carcinoma, cancer of the stomach or breast cancer;
Said allergic disease is bronchial astehma, allergic rhinitis or allergic dermatitis.
Last purpose of the present invention provides a kind of medium that is used for cultivating latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China.
The present invention is used for cultivating the medium of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China, comprises rice, mineral salt and water; Said rice is long-grained nonglutinous rice, polished rice and/or glutinous rice; Said mineral salt are at least a in phosphate, sodium chloride, magnesium sulfate and the potassium chloride; Said phosphate is K
2HPO
4, K
3PO
4, KH
2PO
4, NaH
2PO
4And Na
2HPO
4In at least a.
The medium that is used for latent pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCCNo.3575 of cultivation China of the present invention also can comprise carbon source and nitrogenous source;
Said carbon source is at least a in glucose, fructose, galactose, starch, cellulose and the sucrose;
Said nitrogenous source is at least a in peptone, malt extract and the yeast extract;
The proportioning of said rice, carbon source, nitrogenous source, mineral salt and water is rice (80-100) g: carbon source (0-12) g: nitrogenous source (0-6) g: mineral salt (0.01-2) g: water (100-150) ml.
The medium specific as follows 1 that is used for cultivating latent pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCCNo.3575 of China of the present invention), 2), 3), 4) or 5) shown in:
1) said medium is made up of rice, mineral salt and water; The proportioning of said long-grained nonglutinous rice, mineral salt and water is the 100g long-grained nonglutinous rice: the 0.35g mineral salt: 130 ml waters;
2) said medium is by long-grained nonglutinous rice, magnesium sulfate, K
2HPO
4Form with water; Said long-grained nonglutinous rice, magnesium sulfate, K
2HPO
4With the proportioning of water be the 100g long-grained nonglutinous rice: 0.15g magnesium sulfate: 0.2g K
2HPO
4: 130 ml waters;
3) said medium is made up of long-grained nonglutinous rice, carbon source, nitrogenous source, mineral salt and water; The proportioning of said long-grained nonglutinous rice, carbon source, nitrogenous source, mineral salt and water is (90-95) g long-grained nonglutinous rice: (4-6) g carbon source: (0.9-3.9) g nitrogenous source: 0.1g mineral salt: (130-150) ml water;
4) said medium is made up of long-grained nonglutinous rice, glucose, yeast extract, NaCl and water; The proportioning of said long-grained nonglutinous rice, glucose, yeast extract, NaCl and water is the 90g long-grained nonglutinous rice: 6g glucose: the 3.9g yeast extract: 0.1g NaCl:150 ml water;
5) said medium is made up of long-grained nonglutinous rice, cellulose, malt extract, magnesium sulfate and water; The proportioning of said long-grained nonglutinous rice, cellulose, malt extract, magnesium sulfate and water is the 95g long-grained nonglutinous rice: the 4g cellulose: the 0.9g malt extract: 0.1g magnesium sulfate: 130 ml waters.
In the above-mentioned medium, peptone, malt extract and yeast extract can obtain from commercial sources.Peptone is available from Oxoid Ltd., and lot number is 806586; Malt extract is available from Oxoid Ltd., and lot number is 1069878; Yeast extract is available from Oxoid Ltd., and lot number is 1074139.
The said goods specifically can be medicine; Said medicine can be tablet, capsule, oral liquid, emulsion, injection, supensoid agent, tincture, granule or aerosol.
The said goods also can be health products; Said health products can be tablet, capsule, oral liquid or granule.
New bacterial strain provided by the present invention has multiple function, discharges, suppresses the kinds of tumor cells growth, suppresses various allergy, treats pain and treatment inflammation like antihistamine; Method of cultivating latent pore fungi (Cryptoporussinensis) the CGMCC No.3575 of China of the present invention, simple to operate, with low cost, output is high, the activity extract yield can reach 1.5% (calculating according to the solid culture medium dry weight).Therefore, the extract of strain fermentation thing of the present invention and fermentation product thereof has broad application prospects.
Description of drawings
Fig. 1 is the latent pore fungi solid culture bacterium fermentation product high performance liquid chromatogram collection of illustrative plates of embodiment 2 China.
Fig. 2 is the latent pore fungi solid culture bacterium fermentation product high performance liquid chromatogram collection of illustrative plates of embodiment 3 China.
Fig. 3 is the latent pore fungi solid culture bacterium fermentation product high performance liquid chromatogram collection of illustrative plates of embodiment 4 China.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Separation of embodiment 1, bacterium and evaluation
One, the separation of bacterium
China's cryptoporus volvatus kind obtains from the new fresh sporophore separation of the latent pore fungi of picking up from the Wuyi Mountain, Fujian Province; Latent new fresh sporophore of pore fungi of China and basic thing are taken back the laboratory; In aseptic, use aseptic manipulation, cut the xylem part of fruit body bacterial context part and basic thing respectively, put on the inclined-plane of potato glucose agar medium; 25～28 degrees centigrade of cultivations, the pure culture of acquisition bacterial strain.With a strain bacterium called after Liuzh2009-7523 wherein.
Two, the evaluation of bacterium
The evaluation of 1 fruit body
Cultivate bacterial strain Liuzh2009-7523,, observe the form of thalline and fruit body to obtaining fruit body.
The cap of original sub entity has velum, and the fruit body layer is wrapped in the velum.This characteristic is unique in Polyporaceae.Size 7～10 * 4～5 μ m of basidiospore during microcosmic detects, this characteristic meets the scope of the latent pore fungi of China.Appraisal basis is publication in 1998 " Chinese fungi will the 3rd volume Polyporaceae " and Mycotaxon LXXIV volume in 2000 415-422 page or leaf Cryptoprus sinensis sp.nov., A new polyporue found in China.
2 identification of strains
White mycelium, the fine hair shape, mycelia is through cotton blue dyeing visible a plurality of clamp connections under high-power microscope, and clamp connection is the exclusive structure of basidiomycetes.
Comprehensive above-mentioned qualification result; Show that this bacterial strain Liuzh2009-7523 belongs to the latent pore fungi (Cryptoporussinensis) of China; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC, address: No. 3, No. 1 institute in North Star West Road, Chaoyang, Beijing, Institute of Microorganism, Academia Sinica on 01 04th, 2010; Postcode 100101), preserving number is CGMCC No.3575.
The cultivation of embodiment 2, bacterium, the preparation of extract and application
One, bacterial strain activation
Bacterial classification inoculation to the inclined-plane of potato agar glucose, is cultivated in advance; Pre-incubated condition is: 25 degrees centigrade, lucifuge was cultivated 7 days.
The PDA medium is formed: potato 200g, glucose 20g, agar powder 16g, pure water are settled to 1L (pH nature).
Two, seed culture
In cultivating in advance, treat that mycelia is covered with whole inclined-plane after, under aseptic condition, mycelium is transferred in the liquid nutrient medium and cultivates, obtain seed culture fluid.Culture condition is: 25 degrees centigrade, lucifuge was cultivated 7 days.
Liquid nutrient medium: glucose 4.0g/L, Malt Extract (malt extract) 10.0g/L, YeastExtract (yeast) 4.0g/L, water is settled to 1L.
Three, fermented and cultured
Getting the seed culture fluid that 10 milliliters of step 2 obtain is inoculated into respectively in the 500 milliliters of triangular flasks that following solid culture medium is housed through sterilization; Inoculate 10 bottles; 25 ℃, lucifuge was cultivated 30 days; Material notes all in the triangular flask is made fermentation product, and fermentation product is made up of by the material that bacterium utilizes the back to produce metabolite, medium and the medium of thalline, bacterium.
Solid culture medium: form by 90g long-grained nonglutinous rice, 6g glucose, 3.9g Yeast Extract (yeast extract), 0.1g NaCl and 150 ml waters.
Yeast Extract (yeast extract) is available from Oxoid Ltd., and lot number is 1074139.
Four, the preparation of extract
1, experimental group: the preparation of the extract of fermentation product
With cultivating the fermentation product freeze drying that obtains in the experiment three, to weigh, weight is 900 grams.With 5 liter of 95% (volumn concentration) ethanol water refluxing extraction 3 times, each 1 hour, merge ethanol extract, the concentrating under reduced pressure drying obtains 60 gram extracts, and the extract note is made CS-E1.
The refluxing extraction step is following:
Mixes with 5 liter of 95% (volumn concentration) ethanol water cultivating the fermentation product obtain in the experiment three, 100 ℃ of refluxing extraction 1 hour are got organic facies and residue, organic facies is remembered made ethanol extract I, residue is remembered made residue I;
Residue I is mixed with 5 liter of 95% (volumn concentration) ethanol water, and 100 ℃ of refluxing extraction 1 hour are got organic facies and residue, and the organic facies note is made ethanol extract II, and the residue note is made residual II;
Residual II is mixed with 5 liter of 95% (volumn concentration) ethanol water, and 100 ℃ of refluxing extraction 1 hour are got organic facies and residue, and the organic facies note is made ethanol extract III, and the residue note is made residual II I;
Merge ethanol extract I, ethanol extract II and ethanol extract III, note is made ethanol extract.
2, high performance liquid chromatography detects
Extract C S-E1 high performance liquid chromatography is detected.
Colleges and universities' liquid phase chemical fingerprint map analyzing condition is following:
Use An Jielun Agilent 1200 high performance liquid chromatographs, quaternary gradient pump, DAD detector, Agilent chromatographic work station.Chromatographic column YMC C8 analytical column (4.6mm * 150mm, 5 μ m), flowing phase are that methyl alcohol-0.04% (percent by volume) trifluoroacetic acid aqueous solution carries out gradient elution (elution program is following), flow velocity 1.00mL/min.Sample is used dissolve with methanol, is made into the solution of 10mg/mL.Sample size 10 μ L, ultraviolet detection wavelength 210nm.
The linear gradient elution step is following:
Time (min) methyl alcohol 0.04% (percent by volume) trifluoroacetic acid aqueous solution
0 50％ 50％
40 100％ 0％
60 100％ 0％
Under as above chromatographic condition, the main metabolites chromatographic peak of extract C S-E1 appears in the retention time 25-40 minute scope (Fig. 1).
Five, the function of extract
Below the used animal of experiment is all available from Guangdong Medical Lab Animal Center (credit number SCXK (Guangdong) 2003-0002)
(1) the antihistamine release action of extract C S-E1:
PCA (perchloric acid, Xing Jin chemical plant, Beijing, 070523), Compound 48/80 is available from Alexis, and catalog number is LSALX-420-008-M100.
After 8 age in week SD rat broken end sacrificed by exsanguination, with MCM (+) matrix liquid (50ml～100ml) be injected into rat abdominal cavity, about abdomen massage 120s, the whole peritoneal fluids of sucking-off, 4 ℃ of centrifugal 7min of 680rpm.Abandon supernatant, wash deposition, 4 ℃ of centrifugal 7min of 680rpm with 35ml MCM (+) matrix liquid.Abandoning supernatant again, same 35ml MCM (+) matrix liquid is washed deposition, 4 ℃ of centrifugal 7min of 680rpm, collecting precipitation.The gained deposition is a mast cell.Add MCM (-) matrix liquid 5ml again, trypan blue dyeing.Dyeing liquor is the mixed solution of MCM liquid and 4% trypan blue liquid, MCM liquid: 4% trypan blue liquid=0.25: 0.75 (volume ratio), use preceding 37 ℃ of constant-temperature shaking 3min.The microscopically cell counting, cell concentration is 1 * 10 in the adjustment mast cell suspension
5Cell/mL.Cell is divided into nature discharges the histamine group, Compound 48/80 stimulates release histamine group and sample sets three parts.
Naturally discharge the histamine group: establish three parallel pipes, every pipe adds mast cell suspension 500 μ L; Add 20 μ L60%PCA, room temperature is placed 20min ,-4 ℃ of preservations;
Stimulate and discharge the histamine group: establish three parallel pipes, every pipe adds mast cell suspension 500 μ L, and 37 ℃ of vibrations add 4 μ L250mM CaCl after hatching 5min
25min adds physiological saline 5 μ L10min, adds ice bath cooling behind the 5 μ L0.05mg/mL Compound 48/8010min again, gets supernatant 400 μ L behind 4 ℃ of centrifugal 2.5min of 6000rpm and adds 20 μ L60%PCA, and room temperature is placed 20min.
Sample sets: establish extract C S-E1 high (1000 μ g/mL), low (500 μ g/mL) two dosage, each dosage is respectively established three parallel pipes.Every pipe adds mast cell suspension 500 μ L, and 37 ℃ of vibrations add 4 μ L250mM CaCl after hatching 5min
25min adds and to add ice bath cooling behind the 0.05mg/mL Compound 48/8010min behind the extract C S-E15 μ L 10min again, and 4 ℃ of centrifugal 2.5min of 6000rpm get supernatant 400 μ L then and add 20 μ L 60%PCA, and room temperature is placed 20min.
Above-mentioned three groups after room temperature is placed 20min, all add 400 μ L physiological saline, 4 ℃ of centrifugal 2.5min of 13000rpm.Supernatant is stored in-20 ℃ with 0.2 μ m filtering with microporous membrane, is used for HPLC and analyzes, and detects the amount of the histamine of each group release, is calculated as follows sample histamine release inhibiting rate (%).
Sample histamine release inhibiting rate=(1-sample histamine release rate) * 100%.
Experimental result is seen table 1, and latent pore fungi solid culture fermentation product (CS-E1) has shown the effect that good antihistamine discharges under test dose.
Table 1 conceals the release action of pore fungi solid culture fermentation product (CS-E1) antihistamine
The preparation of MCM (-) matrix liquid: precision takes by weighing NaCl 8.766g, KCl 0.2758g, Na
2HPO
41.07g, KH
2PO
40.48g, D-Glucose 1g, BSA 1g, gelatin (Gelatin) 1g adds water and is settled to 1000ml, shakes up, and transfers pH to 6.8 with the Na0H aqueous solution, promptly gets MCM (-) solution, and-4 ℃ of preservations are subsequent use.
The preparation of MCM (+) matrix liquid: add heparin to MCM (-) matrix liquid, the concentration of heparin in MCM (-) solution is 10U/mL.
MCM matrix liquid preparation: the mixed solution of MCM (-) solution and MCM (+) solution, MCM (-) solution: MCM (+) solution=29: 1 (V: V).
(2) anti-dinitrofluorobenzene (DNFB) allergic contact dermatitis of bringing out
Dexamethasone (DEX) is available from Tianjin Pharmaceutical Group Xinzheng Co., Ltd..Dinitrofluorobenzene (CAS numbers 70-34-8) is available from Shanghai Ru Ji biotechnology Development Co., Ltd.
Laboratory animal KM male mice; Be divided into model control group (giving the distilled water of equivalent), latent pore fungi solid culture fermentation product (CS-E1) 500mg/kg of China, latent pore fungi solid culture fermentation product (CS-E1) 1000mg/kg of China and dexamethasone 1mg/kg group at random; Totally 4 groups, 10 every group.Animal subject arrived DNFB in preceding 1 day in sensitization and attacks gastric infusion on continuous 7 days of the same day.Test the first day DNFB ethanolic solution with 15mg/mL (being the mixed solution of DNFB and ethanol, DNFB15mg, ethanol 1mL) 50 μ L as antigen in mouse back hypodermic injection sensitization.Sensitization is set up behind the 5d; 6d is coated with 10mg/mL DNFB on mouse right ear (acetone: olive oil 4: 1) solution (is the mixture of DNFB, acetone and olive oil; DNFB is 10mg in the mixture; Acetone 0.8mL and olive oil 0.2mL) 25 μ L are as antigen challenge, cause scorching 24h, make mouse produce the ear contact dermatitis.With 8mm diameter card punch respectively about two ear same area lay round auricle, measure left and right sides ear tissue piece weight difference, calculate administration group ear swelling inhibiting rate (%), the result is as shown in table 2.Latent pore fungi solid culture fermentation product (CS-E1) has shown the effect of good anti-DNFB induced allergy dermatitis under test dose.
In the table 2, the auricle weight difference is the poor of auris dextra auricle weight and left ear auricle weight;
Ear swelling inhibiting rate=[1-(administration group weight difference/model group weight difference)] * 100%.
The latent anti-DNFB induced allergy of pore fungi solid culture fermentation product (CS-E1) the dermatitis effect of table 2 China
(3) anti-xenogenesis passive cutaneous anaphylaxis:
DPT vaccine: Chengdu Inst. of Biological Products provides, lot number 20020403.Ovalbumin: Shanghai chemical reagents corporation of Chinese Medicine group produces, lot number F20010702.
The male cleaning level of 18～22g in 7 ages in week Kunming mouse is divided into 5 groups, 10 every group at random; 7 the week age 180～220g male SD rat be divided into 5 groups at random, 10 every group; Establish the latent pore fungi solid culture fermentation product (CS-E1) 500 of blank group, positive controls (Ketotifen 5mg/kg, sigma chemical Co.) and China, 1000mg/kg dose groups respectively, continuous 7 days mouse oral administrations.(concrete grammar is: healthy rat to utilize ovalbumin and DPT vaccine to prepare rat anti ovalbumin serum (anti-OVA) with reference to people's methods such as Tada; Body weight 100-200g; With 1% ovalbumin normal saline solution (the 1g ovalbumin joins in the 100ml physiological saline) according to the 10mg/kg intramuscular injection in the back leg both sides, simultaneously the lumbar injection DPT vaccine 2 * 10
10U/ only.After 12-14 days (IgE rush hour), put to death the animal blood sampling, the blood low-speed centrifugal, separation of serum places refrigerator to preserve.)。The xenogenesis passive cutaneous anaphylaxis be with 10 μ l rat anti ovalbumin serum injections to the mouse right ear intracutaneous, left ear intracutaneous injection equivalent physiological saline.In mouse tail vein, injecting 1% ovalbumin Yi Wensilan normal saline solution by 0.1ml/10g body weight dosage behind the sensitization 48h attacks.Take off neck behind the 30min and put to death, with 8mm diameter card punch respectively about the same position of two ears lay round auricle, measure left and right sides ear tissue piece weight difference, calculating administration group ear swelling inhibiting rate (%), the result is as shown in table 3.Latent pore fungi solid culture fermentation product (CS-E1) has shown the effect of good anti-xenogenesis passive cutaneous anaphylaxis under test dose.
1% ovalbumin Yi Wensilan normal saline solution is formed: the 1g Evans blue, physiological saline 100ml includes ovalbumin 1g.
In the table 3, the auricle weight difference is the poor of auris dextra auricle weight and left ear auricle weight;
Inhibiting rate=[1-(administration group weight difference/model group weight difference)] * 100%.
The effect of the latent anti-xenogenesis passive cutaneous anaphylaxis of pore fungi solid culture fermentation product (CS-E1) of table 3
(4) anti-SRBC brings out delayed hypersensitivity:
The preparation of SRBC (sheep red blood cell (SRBC)): adopt the jugular vein blood of healthy sheep, inject immediately in the aseptic triangular flask that bead arranged,,, obtain anti-freezing sheep whole blood to remove fibrin through fully shaking 15～20min.(Beijing Eastwin Scientific, Inc. 02-045-1B) was mixed with 1: 2 with whole blood and Alsever liquid.Whole blood is put 4 ℃ of preservations and can be used about 3 weeks with after Alsever liquid fully mixes.Get an amount of anticoagulation before the use in centrifuge tube, with SPSS wash cell 2～3 times (each 2000rpm, 10min).At last, use with SPSS dilution back.
The 7 week male cleaning level Kunming mouses of 18～22g in age (Guangdong Medical Lab Animal Center (credit number SCXK (Guangdong) 2003-0002)) are divided into 4 groups at random; Every group 10; Establish blank group, the latent pore fungi solid culture fermentation product (CS-E1) 500 of China, 1000mg/kg dose groups respectively, positive drug Ketotifen 5mg/kg.Test 1d in mouse back hypodermic injection 1 * 10
8Individual SRBC is as antigen sensibilization.Behind the 5d that mouse left side lower limb are stretching, self-sustaining sole of the foot intracutaneous middle and lower part is upwards injected equivalent SRBC and is attacked, and equivalent physiological saline is upwards injected in right sufficient sole of the foot intracutaneous middle and lower part.Ketotifen and cryptoporus volvatus filament extract 1d before sensitization begin to attack continuous 6d administration on the same day to sufficient sole of the foot skin SRBC.Behind the sensitization 24h, with sufficient sole of the foot thickness about Digimatic slide calliper rule (resolution is 0.01mm) measurement, calculate edema inhibiting rate (%), the result is as shown in table 4.In the table 4, the pedal swelling difference is the poor of left footpad thickness and right side foot sole of the foot thickness.Latent pore fungi solid culture fermentation product (CS-E1) has shown that under test dose good anti-SRBC brings out the effect of delayed hypersensitivity.
Edema inhibiting rate (%) computing formula is following:
Edema inhibiting rate=[1-(swelling of administration group poor/model group swelling is poor)] * 100%.
The latent anti-SRBC of pore fungi solid culture fermentation product (CS-E1) of table 4 brings out the effect of delayed hypersensitivity
(5) to the therapeutic action of allergic rhinitis:
With 50 the 7 male cleaning level of 18～22g in age in week Kunming mouses, be divided into 5 groups at random: (1) blank group (A group); (2) model control group (B group); (3) experiment medicine small dose group (C group); (4) the experiment heavy dose of group of medicine (D group); (5) positive controls (BIYANKANG group E group), 10 every group.Laboratory animal A group is given the physiological saline injection, and B, C, D, E group all are aided with aluminium hydroxide 30mg with ovalbumin 0.3mg (sigma chemical Co., grade V) and make adjuvant, add physiological saline 1ml formation suspension; The row lumbar injection; The next day 1 time, totally 7 times, be basic sensitization.Attack two nasal cavities, every side 0.05ml, every day 1 time, totally 7 times with the physiological saline of 0.5% ovalbumin again.All observe 30min after each the attack, content comprises sneeze, scratches, and makes up rat in allergic rhinitis model.A, B group are irritated physiological saline, and C, D, E group are irritated latent pore fungi solid culture fermentation product (CS-E1) 0.5 gram/kilogram of medicine China, 1.0 gram/kilograms and positive control medicine BIYANKANG 1.8g/kg by body weight respectively.The animal 7d that takes medicine behind the perfusion 4h, attacks two nasal cavities with 0.1ml/ ovalbumin physiological saline, and every side 0.05ml observes sniffle behind the 0.5h.Measure skin mound diameter behind the 20-30min all above 1cm; And cut the schneiderian membrane tissue and be soaked in 4% formalin subsequent use; Optional 10 high power fields are observed the plasmacytic number of acidophic cell, neutrophil(e) cell and lymph in the schneiderian membrane tissue, and the result is as shown in table 6.The latent pore fungi solid culture fermentation product (CS-E1) of China has shown fine therapeutic action to allergic rhinitis under test dose, suitable with positive drug.
Table 6 schneiderian membrane histocyte is observed
*P＜0.01. "-" do not see the cell that will observe; "+" cell that is scattered of minority; " ++ " visible more cell; " +++" accounts for more than 2/3 of whole epitheliums
(6) variation of inflammatory cell in the broncho-pulmonary perfusate after the sensitization rat antigen challenge
Ovalbumin is produced by Shanghai chemical reagents corporation of Chinese Medicine group, lot number F20010702.50 of the male cleaning level of 18～22g in 7 ages in week Kunming mouses are divided into 5 groups: (1) blank group (physiological saline 0.5ml/kg) at random; (2) model control group (B group); (3) experiment medicine small dose group (latent pore fungi solid culture fermentation product (CS-E1) 0.5g/kg of China); (4) the heavy dose of group of experiment medicine (latent pore fungi solid culture fermentation product (CS-E1) 1.0g/kg of China); (5) positive controls (DEX 1mg/kg).The mouse etherization; 0.1% ovalbumin gel aluminum hydroxide (0.1% ovalbumin gel aluminum hydroxide: the 0.1g ovalbumin joins in the 100mL physiological saline that contains 10g aluminium hydroxide) is 8 hypodermic injection 0.4mL in two back volas, two groins, two oxters, neck and back; The 10th day lumbar injection 0.1% ovalbumin gel aluminum hydroxide (0.1% ovalbumin gel aluminum hydroxide: the 0.1g ovalbumin joins in the 100mL physiological saline that contains 10g aluminium hydroxide) of sensitization 0.2mL is sensitization 1 time once more; The 2nd sensitization is after 11 days; Ovalbumin with 1% (the 1g ovalbumin joins 100mL physiological saline) atomizing sucks attacks; Each 30 minutes, attacked continuously 6 days.Each attack preceding 1 hour according to the grouping administration, last is attacked and is taken out mouse bronchial after back 24 hours and carry out alveolar wass.Mouse is put to death in dislocation, and skin of neck is cut off in stringer, separates to expose tracheae, and the promoting the circulation of qi cannula is with irrigating solution (Thornwell-Mcdowell solution: NaCl 6.6g, KCl 0.46g, CaCl
20.05g, NaHCO
32.52g, MgCl
20.09g, Na
2HPO40.1g adds water to 1000mL) lavation, each 0.5mL; Totally 3 times; Collect BAL fluid, shake up the back and take out 50 microlitres, with 4 times of leucocyte dilution (the big Parkson of perseverance biological Beijing development in science and technology Co., Ltd) dilutions; Take a morsel and drip on cell counting count board, at microscopically counting total white blood cells.Behind the centrifugal 10min of BAL fluid low temperature 1000rpm, abandoning supernatant is played even back smear with pipettor.The smear drying at room temperature, dyeing 6-8min redyed 1 minute.With running water dyestuff is rinsed well, under 40 times of mirrors of light microscope, carried out Arneth's count.Count 200 cells, calculate the percentage of plymphomonocyte and eosinophil.Result such as table 7 show that BA inflammatory cell gathering has the obvious suppression effect to latent pore fungi solid culture fermentation product (CS-E1) to mouse asthmatic model.
The work sun of latent pore fungi solid culture fermentation product (CS-E1) the mouse asthmatic model bronchovesicular inflammation cell of table 7 China
(7) latent pore fungi solid culture hypha extract analgesic activity
With the male cleaning level of 18～22g in 7 ages in week Kunming mouse, be divided into model control group, the latent pore fungi solid culture fermentation product (CS-E1) 500 of China at random, 1000mg/kg and aspirin 200mg/kg group, totally 4 groups, 10 every group.Behind the gastric infusion 1 hour, the aqueous solution 0.2ml of every mouse peritoneal injection 0.6% (volumn concentration) glacial acetic acid causes pain.The record injection causes the number of times of each mouse writhing in the 30min of pain back, and the result is as shown in table 8.Latent pore fungi solid culture fermentation product (CS-E1) has shown good analgesic activity under test dose.
The computing formula of analgesia rate %=(1-administration group is turned round body number of times/model group and turned round the body number of times) * 100%
Latent pore fungi solid culture fermentation product (CS-E1) analgesic activity of table 8 China
(8) suppress the growth of tumour cell effect:
Human liver cancer cell HepG2 (ATCC HB-8065), Human Prostate Cancer Cells PC3 (ATCC CRL-1435), human lung cancer cell A549 (ATCC CCL-185), people's colorectal cancer cell HCT-15 (ATCC CCL-225), human cervical carcinoma cell Hela (ATCCCCL-2), gastric carcinoma cells SGC-7901 (ATCC SGC-7901), mouse mastopathy cell EMT6 (ATCC CRL-2755), mouse prostate cancer RM-1 cell (ATCC RM-1) are all available from Zhongshan Medical Univ.'s Animal Lab..
The human hepatoma HepG2 cell; Human prostata cancer PC3 cell is with the DMEM culture fluid that contains 10% hyclone; People's lung cancer A549 cell, people's rectum cancer cell HCT-15, human cervical carcinoma Hela cell, people's cancer of the stomach SGC-7901 cell, mouse breast cancer EMT6 cell, mouse prostate cancer RM-1 cell are with the RPMI RPMI-1640 that contains 10% hyclone, in 37 ℃, 5%CO
2The conventional cultivation gone down to posterity every other day in the incubator.Use the MTT method, the various tumour cells in the vegetative period of taking the logarithm are used trypsinization, process every milliliter and contain 1 * 10
5The single cell suspension of individual cell is inoculated in 96 orifice plates (every hole 200 μ L), establishes 3 parallel holes for every group.The latent pore fungi solid culture fermentation product (CS-E1) of China that adds variable concentrations after 24 hours was cultivated 48 hours, established blank group and positive controls (cis-platinum) simultaneously, carried out MTT and measured the tumour cell number, calculated inhibiting rate (%).The result shows that the latent pore fungi solid culture fermentation product (CS-E1) of China has the activity of good restraining growth of tumour cell in 12.5-100 mcg/ml scope.
The computing formula of inhibiting rate (%): (1-administration group OD value/blank group OD value) * 100%.
The latent pore fungi solid culture fermentation product (CS-E1) of table 9 is to the inhibitory action of growth of tumour cell
The cultivation of embodiment 3, bacterium, the analysis of extract
One, bacterial strain activation and seed culture are with embodiment 2.
Two, fermented and cultured
Liquid nutrient medium was cultivated after 10 days, got the seed culture fluid that 10 milliliters of step 1 obtain and was inoculated into respectively in the 500 milliliters of triangular flasks that following solid culture medium is housed through sterilization, inoculated 10 bottles, and 30 ℃, lucifuge was cultivated 30 days, obtained fermentation product.
Solid culture medium: form by 95g long-grained nonglutinous rice, 4g cellulose, 0.9g malt extract, 0.1g magnesium sulfate and 130 ml waters.
Malt extract is available from Oxoid Ltd., and lot number is 1069878.
Three, the preparation of extract
1, the preparation of the extract of fermentation product
With the fermentation product freeze drying, to weigh, weight is 950 grams.Carry out refluxing extraction according to method described in the embodiment 2, merge ethanol extract, the concentrating under reduced pressure drying obtains 65 gram extracts, and the extract note is made CS-E2.
2, high performance liquid chromatography detects
The extract C S-E2 for preparing among the embodiment 3 is carried out high performance liquid chromatography to be detected.Analysis condition is with embodiment 2.The result is as shown in Figure 2, and the main metabolites chromatographic peak of extract C S-E2 appears in the retention time 25-40 minute scope, and is similar with CS-E1.
Four, functional verification
CS-E2 is carried out each item functional verification described in embodiment 2 experiments five, and result and CS-E1 do not have significant difference.
The cultivation of embodiment 4, bacterium, the preparation of extract
One, bacterial strain activation and seed culture are with embodiment 2.
Two, fermented and cultured
Liquid nutrient medium was cultivated after 10 days, got the seed culture fluid that 10 milliliters of step 1 obtain and was inoculated into respectively in the 500 milliliters of triangular flasks that following solid culture medium is housed through sterilization, inoculated 10 bottles, and 28 ℃, lucifuge was cultivated 30 days, obtained fermentation product.
Solid culture medium: by 100g long-grained nonglutinous rice, 0.15g magnesium sulfate, 0.2g K
2HPO
4Form with 130 ml waters.
Peptone is available from Oxoid Ltd., and lot number is 806586.
Three, the preparation of extract
1, the preparation of the extract of fermentation product
With the fermentation product freeze drying, to weigh, weight is 1000 grams.Extract 3 times with 5 liter of 95% alcohol reflux, each 1 hour, merge ethanol extract, the concentrating under reduced pressure drying obtains 63 gram extracts, and the extract note is made CS-E3.
2, high performance liquid chromatography detects
The extract C S-E3 for preparing among the embodiment 4 is carried out high performance liquid chromatography detect, analysis condition is with embodiment 2.The result is as shown in Figure 3, and the main metabolites chromatographic peak of extract C S-E3 appears in the retention time 25-40 minute scope, and is similar with CS-E1.
Four, functional verification
CS-E3 is carried out each item functional verification described in embodiment 2 experiments five, and result and CS-E1 do not have significant difference.
Claims (10)
1. a medium that is used for cultivating latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China comprises rice, mineral salt, water, carbon source and nitrogenous source;
Said rice is long-grained nonglutinous rice, polished rice and/or glutinous rice;
Said mineral salt are at least a in phosphate, sodium chloride, magnesium sulfate and the potassium chloride; Said phosphate is K
2HPO
4, K
3PO
4, KH
2PO
4, NaH
2PO
4And Na
2HPO
4In at least a;
Said carbon source is at least a in glucose, fructose, galactose, starch, cellulose and the sucrose;
Said nitrogenous source is at least a in malt extract and the yeast extract;
The proportioning of said rice, carbon source, nitrogenous source, mineral salt and water is rice 80-100g: carbon source 0-12g: nitrogenous source 0-6g: mineral salt 0.01-2g: water 100-150ml.
2. medium according to claim 1 is characterized in that: said medium is following 1), 2), 3), 4) or 5) shown in:
1) said medium is made up of long-grained nonglutinous rice, mineral salt and water; The proportioning of said long-grained nonglutinous rice, mineral salt and water is a 100g rice: the 0.35g mineral salt: 130 ml waters;
2) said medium is by long-grained nonglutinous rice, magnesium sulfate, K
2HPO
4Form with water; Said long-grained nonglutinous rice, magnesium sulfate, K
2HPO
4With the proportioning of water be the 100g long-grained nonglutinous rice: 0.15g magnesium sulfate: 0.2g K
2HPO
4: 130 ml waters;
3) said medium is made up of long-grained nonglutinous rice, carbon source, nitrogenous source, mineral salt and water; The proportioning of said long-grained nonglutinous rice, carbon source, nitrogenous source, mineral salt and water is the 90-95g long-grained nonglutinous rice: the 4-6g carbon source: the 0.9-3.9g nitrogenous source: the 0.1g mineral salt: the 130-150 ml water;
4) said medium is made up of long-grained nonglutinous rice, glucose, yeast extract, NaCl and water; The proportioning of said long-grained nonglutinous rice, glucose, yeast extract, NaCl and water is the 90g long-grained nonglutinous rice: 6g glucose: the 3.9g yeast extract: 0.1g NaCl: 150 ml waters;
5) said medium is made up of long-grained nonglutinous rice, cellulose, malt extract, magnesium sulfate and water; The proportioning of said long-grained nonglutinous rice, cellulose, malt extract, magnesium sulfate and water is the 95g long-grained nonglutinous rice: the 4g cellulose: the 0.9g malt extract: 0.1g magnesium sulfate: 130 ml waters.
3. cultivate the method for latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China, comprise the steps: with claim 1 or 2 said medium culture latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China.
4. method according to claim 3 is characterized in that: said culture condition is: temperature is 25 ℃-30 ℃ and lucifuge.
5. China conceals the fermentation product of pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575; Be to prepare: cultivate latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China according to claim 3 or 4 said methods according to the method that comprises the steps; Cultivated 30 days; Collect all materials in the culture vessel, all the material notes in the culture vessel are made fermentation product.
6. right is wanted the application of 5 said fermentation products in the following product of preparation: the product that 1) has the antihistamine release function; 2) has the product that prevents and/or treats the allergic disease function; 3) has the product of analgesia function; 4) have the product that suppresses the growth of tumour cell function, or 5) have a product that prevents and/or treats the tumour function.
7. China conceals the extract of the fermentation product of pore fungi (Cryptoporus sinensis) Liuzh2009-7523CGMCC No.3575; Be to prepare: a), cultivate latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China according to claim 3 or 4 said methods according to the method that comprises the steps; Cultivated 30 days; Collect all materials in the culture vessel, all the material notes in the culture vessel are made fermentation product; B) using volumn concentration is that 95% ethanol water carries out refluxing extraction to fermentation product in the step a), gets organic facies, is the extract of the fermentation product of latent pore fungi (Cryptoporus sinensis) the Liuzh2009-7523CGMCC No.3575 of China; The time of each refluxing extraction is 1 hour, and temperature is 100 ℃; Fermentation product and said volumn concentration are that the proportioning of 95% ethanol water is 900-1000g in the said step a): 5 liters.
8. right is wanted the application of 7 said extracts in the following product of preparation: the product that 1) has the antihistamine release function; 2) has the product that prevents and/or treats the allergic disease function; 3) has the product of analgesia function; 4) have the product that suppresses the growth of tumour cell function, or 5) have a product that prevents and/or treats the tumour function.
9. one kind has following 1)-5) product of arbitrary function, its active component is claim 5 or 6 said fermentation products and/or claim 7 or 8 said extracts;
1) antihistamine release function;
2) prevent and/or treat the allergic disease function;
3) analgesia function;
4) suppress the growth of tumour cell function;
5) prevent and/or treat the tumour function.
10. according to the said product of claim 9, it is characterized in that: the pain in the said analgesia is the pain that neuropathic pain, traumatic pain or tumour cause;
Said tumour cell is human liver cancer cell HepG2, Human Prostate Cancer Cells PC3, human lung cancer cell A549, people's rectum cancer cell HCT-15, human cervical carcinoma cell Hela, gastric carcinoma cells SGC7901, mouse mastopathy cell EMT6 or mouse prostate cancer RM-1 cell;
Said tumour is liver cancer, prostate cancer, lung cancer, the carcinoma of the rectum, cervical carcinoma, cancer of the stomach or breast cancer;
Said allergic disease is bronchial astehma, allergic rhinitis or allergic dermatitis;
The delayed hypersensitivity that xenogenesis passive cutaneous anaphylaxis that to be dinitrofluorobenzene allergic contact dermatitis that mouse is brought out, rat anti ovalbumin serum bring out mouse of said allergic dermatitis or sheep red blood cell (SRBC) are brought out mouse;
The allergic contact dermatitis that said dinitrofluorobenzene brings out mouse is the ear swelling that dinitrofluorobenzene brings out mouse; The xenogenesis passive cutaneous anaphylaxis that said rat anti ovalbumin serum brings out mouse is the ear swelling that rat anti ovalbumin serum brings out mouse, and the delayed hypersensitivity that said sheep red blood cell (SRBC) is brought out mouse is the pedal swelling that sheep red blood cell (SRBC) is brought out mouse.
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Non-Patent Citations (3)
| Title |
|---|
| 杨祝良等.中国南部高等真菌的热带亲缘.《云南植物研究》.2003,第25卷(第2期),第136页2.9. * |
| 肖崇厚 陆蕴如.《中药化学》.《中药化学》.1987,第432-433页. * |
| 赵航.隐孔菌胶囊的制备工艺和质量标准研究.《浙江大学》.2004,第9-10页. * |
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