CN102242069B - Paecilomycescicadae (Miq.)Samson and application thereof - Google Patents
Paecilomycescicadae (Miq.)Samson and application thereof Download PDFInfo
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- CN102242069B CN102242069B CN201110120603.1A CN201110120603A CN102242069B CN 102242069 B CN102242069 B CN 102242069B CN 201110120603 A CN201110120603 A CN 201110120603A CN 102242069 B CN102242069 B CN 102242069B
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- samson
- paecilomyces cicadae
- cicada fungus
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Images
Abstract
The invention belongs to the field of microbes, and in particular relates to a Paecilomycescicadae (Miq.)Samson and application thereof. The Paecilomycescicadae (Miq.)Samson has the collection number of CGMCCNo.3453. The Paecilomycescicadae (Miq.)Samson with the collection number of CGMCCNo.3453 has the characteristics of simple culture and high yield; and a culture product has effects of resisting tumors, regulating immunity, reducing blood pressure, blood sugar and blood fat, improving eyesight, resisting radiation, clearing away heat and easing pain, calming and promoting sleep, nourishing and strengthening, improving kidney function and the like, so the invention has high industrial application value.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of Paecilomyces cicadae bacterial strain and application thereof.
Background technology
Cicada fungus, another name worm flower, is that some cicada nymph is subject to the product after Paecilomyces cicadae bacterium parasitism, is a kind of bacterium worm complex body.This bacterium was named as the mould Isaria cicadae of cicada Isaria by Miquel in 1838.After this there is multiple synonym.As cicada grass Cordyceps cicadae, the raw Isaria Isaria of base basili, Xin Kelai spherical shell spore Sphaeria sinclairii, the worm shell bacterium Torrubia caespitosa of growing thickly, Xin Kelai Chinese caterpillar fungus Cordyceps sinclairii, Harry's Isaria Isariahariottii, the fine and soft Cordyceps sobolifera of little cicada, Xin Kelai Isaria Isaria sinclairii, the Munch Isaria Isaria mokanshawii of Soviet Union and racemosus Isaria Isaria arbuscula etc.
The perfect stage of Paecilomyces cicadae is considered to large cicada grass Cordyceps cicadae, and what widely distribute at nature is Paecilomyces cicadae, and large cicada grass is rare.What often adopt is the complex body after Paecilomyces cicadae and host mountain cicada nymph parasitism thereof.According to the investigation of 1467 duplicate samples in Zhejiang, historical producing region, medicinal material cicada fungus is mainly the entomogenous fungi complex body after Paecilomyces cicadae parasitism.Paecilomyces cicadae belongs to imperfect fungi fungi, and its coremium grows from polypide head, single raw or clustering bunchy, and light yellow, long 1.5～8.0 centimetres, front end expands, and is fusiform or is cockscomb flower-shaped.
Cicada fungus contains polysaccharide, adenosine, cordycepic acid, multiple essential amino acid, PEARLITOL 25C, multiple alkaloid, ergosterol isoreactivity composition, has similar activeconstituents to Cordyceps sinensis.Traditional Chinese Medicine thinks that cicada fungus has dispelling wind and heat pathogens, promoting eruption, relievng spasm by subduing liver-wind, item are moved back effect of screen, modern medicine study finds, that cicada fungus has is antitumor, it is immune, hypoglycemic to regulate, reducing blood-fat, hypotensive, improving eyesight, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve the effects such as renal function.
Wild cordyceps resource-constrained, due to its personal value costliness, has formed coyoting and has plucked excessively for many years, brings havoc to vegetation and ecotope.In view of cicada fungus and Cordyceps sinensis have similar activeconstituents, also have immunomodulatory, antipyretic-antalgic, improve the multiple pharmacological effect such as renal function, tranquilizing soporific, illustrate that cicada fungus is a kind of medicinal entomogenous fungi having a extensive future that has, and can do the research and development such as food, healthcare products, medicine, makeup.
Summary of the invention
The object of the present invention is to provide a kind of Paecilomyces cicadae bacterial strain and application thereof.
The present invention solves the problems of the technologies described above by following technical solution:
A kind of Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson], registration preservation that this bacterial strain (is called for short CGMCC) on November 18th, 2009 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.3453.
Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] is adopted with the following method and is separated:
Gather pure pollution-free wild cicada fungus sample from Yunnan Head streams, the transplanting tweezer of crossing with flame sterilization on clean work station from sample take a morsel conidium in the PSA flat board that adds rose-bengal put at 22 ℃-25 ℃, cultivate 120h after picking list bacterium colony access slant medium again, at 22 ℃-25 ℃, cultivate and get final product to obtain Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] through purifying repeatedly.
Paecilomyces cicadae bacterial strain of the present invention belongs to Deuteromycotina Deuteromycotina, hyphomycetes Hyphomycetes, bundle stalk spore order Stilbellales, Stilbaceae Stilbellaceae, paecilomyces Paecilomyces.
Paecilomyces cicadae bacterial strain of the present invention is delivered Chinese Academy of Sciences's identification by morphological characters that biological study is carried out on September 10th, 2009, and qualification result is:
Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson], its Main Morphology is characterized as:
On PDA substratum, cultivate 10 days for 25 ℃, bacterium colony circle, open and flat, first cotton-shaped, rear powdery, white is to light yellow, and the back side is light yellow, 5～6 centimetres of diameters.Hyphae colorless, branch, tool barrier film, wide 1.2～2.5 μ m.Conidiophore multi-branched, wide 3.0～5.5 μ m, are taken turns 2～5 conidiogenous cells and are formed colyliform branch by every.Conidiogenous cell doleiform, base portion is spherical to be expanded, upwards attenuate narrow, 4.5～6.5 × 2.5～3.5 μ m.Conidium is cylindrical, unicellular, colourless, level and smooth, and chain is raw, straight or bending, 6.5～8.8 (～11.0) × 2.5～3.5 μ m.
Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson], has following microbial characteristic:
1, cultivate the feature of learning:
(1) put conidium in 24 ℃, 1% C
6h
12o
6solution is made slide glass and is cultivated, and 4h is sprouted, and the 8h long mycelia that sprouts, starts to need 36h to inferior for generation of conidium from conidia germination;
(2) bacterium colony is on potato-sucrose-agar, grow prolifically, and 24 ℃ of incubation 14d, diameter reaches 60～72mm.Mycelium is greyish white to pale yellow, fine hair shape.Late stage of culture, because producing a large amount of conidiums, causes bacterium colony outward appearance to show opaque shape.Bacterium colony is grown good on peptone-sucrose-agar, 14d diameter 54～70mm, and local crowning or subside, yellowish pink, the later stage is covered with conidium, back side Vandyke brown;
(3) liquid submerged fermentation synthetic medium is with Richard substratum (KNO
310g, KH
2pO
45g, MgSO
4.7H
2o 2.5g, sucrose 50g, FeCl
30.03g, water 1000ml) be good;
(4) humiture:
1. temperature: 6 ℃ of this bacteria growing origin temps, 24～26 ℃ of optimum growth temps, but coremium temperature in vegetative period is on the low side, and pure growth is processed 30h through 40 ℃, inactivation after 50 ℃ of processing 2.5h;
2. humidity: under saturated humidity, conidia germination rate 24h is that 86%, 96h is 99%; When RH 90%, 24h germination rate is that 0%, 96h is 78.3%; When RH 76%, 72h germination rate is that 0%, 96h is 18%;
(5) PH: this bacterium is at all energy normal growths of PH 4～12 scopes, but PH 5～6 mycelium productions are high, and slant culture bacterium colony is difficult for aging;
(6) C, N source utilize: this bacterium can utilize the carbon sources such as glucose, fructose, maltose, seminose, glycerine, but does not utilize synanthrin, sorbose, rhamnosyl and lactose.With glucose, as carbon source, conidium output is significantly higher than above-mentioned several carbon source, and with fructose, as carbon source, mycelium production is higher than other carbon sources.To 9 kinds of inorganic N cultivation situations such as nitro, nitroso-group, amino, this bacterium is to KNO
3make good use of, and utilize nitro N better than amino N, but can not utilize NaNO
2and thiocarbamide.This bacterium is quite responsive to carbon source required amount.For example, carbon source is maintained on 2% normal growth developmental level, improve N source or reduce N source on coremium growth and sporulation quantity impact not quite; But N source is fixed in normal growth level, carbon source is reduced to 0.5% from 2%, find not produce coremium, or rarely coremium produces.Otherwise, carbon source is improved to 10 times (20%), coremium is many and be difficult for agingly, but generation of conidium is more late;
(7) light: this bacterium has obvious phototaxis, coremium growth is tilted towards light source direction, secretly cultivates mycelial growth prosperous, only produces sclerotium and does not form coremium.Natural light and light all can stimulate coremium growth and illumination;
(8) this bacterium has strong radioprotective phenomenon, and flat-plate bacterial colony irradiates 281h from 30Wt ultraviolet source 0.5m place, move after growing and have no inactivation, also has no growth failure;
(9) solid fermentation: do substratum with cereal such as wheats, inoculum size 3%～5% (weight percent), latter 3～5 days of inoculation, visible canescence fine hair shape mycelium covers stromal surface.Within 20～25 days, form sclerotium and grow with pin main body on similar coremium.Coremium is yolk yellow, upright, column or palmate branch, and 20～30 (～80) × 3～5mm, produce a large amount of conidiums after common 30 days, afterwards coremium withered lodging gradually.
2, Paecilomyces cicadae bacterial classification sequencing:
Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] sent in Institute of Microorganism, Academia Sinica and carries out strain identification on September 11st, 2009, and receive and detect No. 249th, the micro-searching of probation report (2009), qualification result shows that the bacterial classification of censorship is Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson].The 18S rRNA gene order of Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] is as shown in sequence in sequence table 1; Its ITS1 gene order is as shown in sequence in sequence table 2; Its 5.8S rRNA gene order is as shown in sequence in sequence table 3; Its ITS2 gene order is as shown in sequence in sequence table 4.
Paecilomyces cicadae bacterial strain of the present invention can be cultivated under above-mentioned substratum and culture condition, have be easy to cultivate, feature that output is high, that its cultured products has is antitumor, it is immune, hypoglycemic to regulate, reducing blood-fat, hypotensive, improving eyesight, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve the functions such as renal function, therefore has industrial applications value widely.
Accompanying drawing explanation
Fig. 1: Cordyceps polysaccharide typical curve
Fig. 2: N.F,USP MANNITOL typical curve
Fig. 3: Cordycepin correction graph
Embodiment
Further describe technical scheme of the present invention below by specific embodiment.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The separation of embodiment 1, Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453
1, prepare substratum according to following proportioning;
Potato sucrose nutrient agar: potato 200g, sucrose 20g, agar 20g, water 1000ml;
Peptone sucrose nutrient agar: peptone 20g, sucrose 10g, agar 20g, water 1000ml.
2, strain separating:
Gather pure pollution-free wild cicada fungus sample from Yunnan Head streams, the transplanting tweezer of crossing with flame sterilization on clean work station from sample take a morsel conidium in the PSA flat board that adds rose-bengal put at 22 ℃-25 ℃, cultivate 120h after picking list bacterium colony access slant medium again, at 22 ℃-25 ℃, cultivate and get final product to obtain Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] through purifying repeatedly.
3, spawn culture:
Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 is connected on bell potato sucrose nutrient agar, 24 ℃ of incubations 14 days, diameter reaches 60～72mm, grow prolifically, Initial stage of culture, mycelium is greyish white to pale yellow, fine hair shape; Late stage of culture, because producing a large amount of conidiums, causes bacterium colony outward appearance to show opaque shape.
Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 is connected on peptone sucrose nutrient agar, 24 ℃ of incubations 14 days, diameter 54～70mm, grows good, local crowning or subside yellowish pink; Later stage is covered with conidium, back side Vandyke brown.
4, bacterial classification sequencing:
Prepare potato sucrose nutrient solution by following proportioning: potato 200g, sucrose 20g, water 1000ml.
By Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 at 3 days (140rpm of potato sucrose nutrient solution shake-flask culture, 24 ℃), filter and collect mycelium, add liquid nitrogen grinding fragmentation, extract total DNA of bacterial classification with Benzyl chloride method.
Take total DNA as template, reference literature " Eiji Yokoyama, Kenzo Yamagishi, Akira Hara, Development of a PCR-based mating-type assay for Clavicipitaceae, FEMS MicrobiologyLetters 237 (2004) 205-212 " method amplification Paecilomyces cicadae ITS1-5.8S-ITS2rDNA region.
The primer is Foreword primer:5 ' GTCATATGCTTGTCTCAAAG and Reverse primer:5 ' TTACTGGGGCAATCCCTGTT.PCR reaction system (25 μ L) is: 2.5 μ L10 × Taq enzyme buffer liquid, the each 200 μ mol/L of dNTP, MgCl
21.5mmol/L, primer 0.2ng, Taq enzyme 1.5U, template DNA 20ng～1 μ g.
The response procedures of amplified reaction on PCR instrument: 95 ℃ of denaturations 5 minutes; 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ keep 4 minutes, move altogether 35 circulations; 72 ℃ are extended 10 minutes.
PCR after completion of the reaction, gel electrophoresis product is purified to reclaim with gel recovery test kit institute's calling sequence and ncbi database are carried out to sequence comparing analysis, result shows that bacterial classification of the present invention is Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson].
The solid culture of bacterial classification:
(1) bacterial classification preparation: embodiment 1 gained bacterial classification;
(2) batching mounted box: preparation solid medium, the substratum preparing is packed into and cultivated in box, charge amount is 15%, seals with sealed membrane; Solid medium proportioning described in present embodiment is matrix 46 weight parts, bagasse 10 weight parts, and oyster shell whiting 0.5 weight part, dried silkworm chrysalis meal 5 weight parts, saltpetre 0.5 weight part, water is 44 weight parts; Its mesostroma is grain and sucrose (in the gross weight of grain and sucrose, the weight percent of grain is 98%, and the weight percent of sucrose is 2%).Its preparation method is: first grain is boiled, sucrose, saltpetre are dissolved in water, more proportionally mix with bagasse, oyster shell whiting, dried silkworm chrysalis meal.
(3) sterilizing: cultivation box is put into Autoclave, sterilizing 30min under 0.15MPa pressure;
(4) inoculation: after solid medium is cooled to below 20 ℃, inoculate under aseptic condition, every 300ml one-level kind connects 80 and cultivates box;
(5) solid fermentation: postvaccinal cultivation box moves to culturing room at 25 ℃, relative humidity is cultivated 50 days for 80% time, later stage coremium growth phase requires daylighting, and envrionment temperature is compared the mycelial growth stage in early stage need be low 2～3 ℃, the ventilation of need in good time windowing every day, keeps cultivating room air fresh;
(6) gather: can gather when the coremium height of cicada fungus culture reaches when about 9cm, surface have a small amount of conidium to occur; While gathering, remove sealed membrane, by culture medium culturing thing taking-up separation coremium, dry classification with instrument, the cooling rear bag film vacuum packaging of using, mycoplasma is placed in-20 ℃ of storages.
One: polysaccharide detection method
1 experimental principle
Adopt phenol sulfuric acid colorimetry to detect sample polysaccharide.Its principle is: polyose is first hydrolyzed into monose molecule under vitriol oil effect, and dehydration generates alditol derivative rapidly, alditol derivative generates colored compound with phenolic substance as phenol reactant again, there is maximum absorption at wavelength 490nm place, and meet Law of Lambert-Beer, by detecting the light absorption value of sample under this wavelength, can calculate the content of polysaccharide in sample.
2 instruments and material
2.1 key instrument ultraviolet-visible pectrophotometers; Electronic balance; Adjustable closed electric furnace; Whizzer H-1650.
2.2 experiment reagent dextrose anhydrouss (AR); Phenol (AR); The vitriol oil (AR); Watson distilled water.
Reagent is prepared 5% phenol solution: accurately take 5g phenol, water is settled to 100ml.
The preparation of glucose reference liquid: the glucose reference liquid that compound concentration is 0.25mg/mL, accurately take the glucose after 0.2500g is dried, add water and be settled to 1000mL.
The cultured products of 2.3 material embodiment 3
Natural cicada fungus (originating from Yunnan)
3 experimental techniques
The preparation of 3.1 sample Crude polysaccharides extracting solutions accurately takes 1g sample and is placed in dry 500ml Erlenmeyer flask, the total mass of weighing bottle and sample is designated as ml, in triangular flask, add the about 70ml of boiling distillated water, be positioned on electric furnace and make after its constantly boiling 15min, be put in rapidly and in cold water, be cooled to room temperature, take out, dry after the globule of bottle outer wall, adding wherein distilled water to final quality is ml+100g, and complete the shaking up afterwards that add water, filters, access filtered liquid, dilute 2 times for subsequent use, be testing sample ,-20 ℃ of preservations.
The mensuration of 3.2 sample Crude polysaccharides
3.2.1 the drafting precision of typical curve takes anhydrous glucose 0.2500g, be mixed with the dextrose standard sample solution of 0.25mg/ml with distilled water, draw glucose reference liquid 0,0.1,0.3,0.5,0.7,0.9,1.1ml, be placed in respectively tool plug test tube, each adding distil water, making volume is 2.0ml, then adds 5% phenol solution 1ml, shakes up, drip rapidly 98% sulfuric acid 5ml, shake up rear placement 5 minutes, put in boiling water bath and heat 15 minutes, take out cold water and be cooled to room temperature; Another adding distil water 2ml, blank is done in the same operation, measures light absorption value (Abs) in 490nm place.Take standard substance glucose concn (mg/ml) as X-coordinate, Abs is ordinate zou, obtains regression equation: Y=0.6622X+0.0088, R
2=0.9997, description standard product glucose amount, within the scope of 0～2.75mg/ml, is good linear relationship with Abs.
3.2.2 Data Processing in Experiment
M: polysaccharide content (mg/g); A: extension rate; C: liquid polysaccharide concentration to be measured (mg/mL);
V: volume (mL); W: sample quality (g)
Two: N.F,USP MANNITOL detection method
The color reaction that 1 experimental principle utilizes polyol compound N.F,USP MANNITOL composition and sodium periodate and Nash reagent to produce, the content of N.F,USP MANNITOL in working sample.
2 instruments and material
2.1 key instrument ultraviolet-visible pectrophotometers; Electronic balance; Whizzer H-1650; Electric heat constant temp. water tank CU600 type; Adjustable closed electric furnace.
2.2 experiment reagent N.F,USP MANNITOL standard substance; Ammonium acetate, methyl ethyl diketone, Glacial acetic acid, potassium periodate, L-rhamnosyl.
Reagent preparation
Potassium periodate solution: 15mmol (being 3.45g) potassium periodate is dissolved in 1L 0.12mol/L hydrochloric acid soln.
Nash reagent: 150g ammonium acetate+2mL Glacial acetic acid+2mL methyl ethyl diketone, with distilled water diluting to 1L (matching while using).
L-rhamnosyl solution: L-rhamnosyl 100mg, is settled to 100mL with distilled water.
The cultured products of 2.3 experiment material embodiment 3
Natural cicada fungus (originating from Yunnan)
3 experimental techniques
The preparation of 3.1 sample N.F,USP MANNITOL extracting solutions accurately takes 1g sample and is placed in dry 500ml Erlenmeyer flask, and the total mass of weighing bottle and sample is designated as ml, in triangular flask, adds the about 70ml of boiling distillated water, be positioned on electric furnace and make after its constantly boiling 15min, be put in rapidly in cold water and be cooled to room temperature, take out, dry after the globule of bottle outer wall, adding wherein distilled water to final quality is ml+100g, add water after complete and shake up, filter, access filtered liquid, be testing sample ,-20 ℃ of preservations.
The mensuration of 3.2 sample N.F,USP MANNITOL
3.2.1 the preparation precision of standardized solution takes PEARLITOL 25C standard substance 0.1g in beaker, adding a small amount of distilled water dissolves completely, be transferred to constant volume in 100ml volumetric flask, be mixed with the mannitol solution of 1mg/ml, after diluting, obtain that mass concentration is respectively 10,50,90,130, the N.F,USP MANNITOL standardized solution of 170mg/L.Get the each 1mL of above-mentioned concentration standard product mannitol solution, split in different test tubes, then add respectively 1mL sodium periodate solution, mix, room temperature is placed 10min, add 2mL0.1%L-rhamnosyl solution to remove too much periodate, vibration mixes, and adds the freshly prepared Nash reagent of 4mL, and 53 ℃ of heating in water bath 15min make its colour developing, be quickly cooled to room temperature, at 412nm wavelength, place measures its absorbancy.Replace N.F,USP MANNITOL standardized solution with distilled water, use the same method operation in contrast, measure its absorbancy.Take concentration of standard solution as X-coordinate, absorbancy is ordinate zou, drawing standard curve.Take standard substance mannitol concentration (mg/ml) as X-coordinate, Abs is ordinate zou, obtains regression equation: Y=0.0826X+0.0019, R
2=0.9998, description standard is savored N.F,USP MANNITOL amount within the scope of 0～21.25mg/L, is good linear relationship with Abs.
3.2.2 Data Processing in Experiment
M: mannitol content (mg/g); A: extension rate; C: liquid mannitol concentration to be measured (mg/mL);
V: extract liquor capacity (mL); W: test sample size (g)
Three: Determination of Adenosine method
1 instrument and reagent
11 instruments
Waters?1525?Binary?HPLC?Pump；
Waters?2998?Photodiode?Aarry?Detector；
Ultrasonic washing instrument SB25-12DT, NingBo XinZhi Biology Science Co., Ltd;
Electronic balance AL104 type, Mettler-Toledo Instrument (Shanghai) Co., Ltd.;
Two liang of dress high speed Universalpulverizer QE-100 gram, the Zhejiang Trade Co., Ltd. that stands erect;
Simplicity，Millipore；
Syringe-type strainer 0.22 μ m, Pall.
1.2 reagent
Adenosine 068K0691, Sigma;
Cordycepin 2007914, Sigma;
Acetonitrile HPLC, Lot100606, Fisher;
Methyl alcohol HPLC, Lot096964, Fisher;
Sherwood oil AR level, 60-90 ℃, Chemical Reagent Co., Ltd., Sinopharm Group;
Potassium primary phosphate AR, 10017618, Chemical Reagent Co., Ltd., Sinopharm Group;
Watson distilled water, Guangzhou Watson food-drink company limited.
The cultured products of 1.3 sample embodiment 3
Natural cicada fungus (originating from Yunnan)
2 methods
2.1 chromatographic condition
Chromatographic column: (5 μ m) for Waters, 4.6mm × 250mm for XBridge C18 chromatographic column;
Moving phase: acetonitrile-0.04mol/L potassium primary phosphate (5: 95);
Flow velocity: 1.0mL/min;
Detect wavelength: 260nm;
Column temperature: 35 ℃;
Sample size: 20 μ L.
2.2 Cordycepin Specification Curve of Increasings
Precision takes 50mg adenosine reference substance, puts in 50mL volumetric flask, adds ultrapure water and dissolves and be diluted to scale and make 1mg/mL solution.Precision pipettes appropriate 1mg/mL adenosine reference substance solution, is mixed with respectively the adenosine reference liquid of 1,5,10,30,50,100 μ g/mL, for subsequent use.
The preparation of 2.3 need testing solutions
Get about 1.0g sample powder, accurately weighed, put in tool plug 100mL tool plug Erlenmeyer flask, add sherwood oil (60-90 ℃) 10mL, close plug, ultrasonic 30 minutes, filters, discard sherwood oil, the slag of getting it filled volatilizes, and in filter paper one juxtaposition tool plug bottle, adds 20mL ultrapure water in bottle, claim gross weight (comprising Erlenmeyer flask weight), ultrasonic 30min.After fast cooling, weigh, mend to gross weight with ultrapure water, mix rear centrifugally, get supernatant liquor, cross 0.22 μ m millipore filtration, for subsequent use.
2.4 Specification Curve of Increasing
By reference substance 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 30 μ g/mL, 50 μ g/mL, 100 μ g/mL solution sample introductions, press
2.1 lower chromatographic conditions are measured, and take sample concentration (μ g/mL) as X-coordinate, peak area (microvolt second) is ordinate zou, drawing standard curve, and obtaining regression equation is y=7.08e+004x-1.05e+004, R
2=0.999978, good at 1～100 μ g/mL and peak area linear relationship.
2.5 trial-product Determination of Adenosines
The need testing solution of getting preparation under 2.3, mixes with the ratio of 2: 1 with ultrapure water, makes liquid to be measured.Get liquid to be measured by 2.1 lower chromatographic condition sample introductions, obtain liquid adenosine concentration x to be measured (μ g/mL) by regression equation, obtain trial-product adenosine content (mg/g) according to following formula.
Detected result
Various effective constituents in the cicada fungus cultured products making in the present embodiment are all high than natural cicada fungus than content, and product quality is stable, is of very high actual application value.
The culture (being called for short afterwards artificial cicada fungus) of tested material: embodiment 2.
Positive control drug: losartan, Hangzhou Mo Shadong pharmaceutical Co. Ltd produces; Natural cicada fungus, the place of production is Zhejiang.
Laboratory animal:
70 of SPF level male SD rats, at 2 monthly ages, body weight 200 ± 10g, is provided by Shanghai Univ. of Traditional Chinese Medicine's Experimental Animal Center.
Grouping and modeling:
SD rat adaptability was fed after 1 week, was divided at random 2 groups: 62 of blank group (sham operated rats) 8 and experimental group (operation group).Adopt the most of cutting method of 5/6 kidney to set up experimental rat model: the 1st operation, the left kidney of operation group rat always excises renal cortex weight and accounts for 2/3 of left kidney weight; After 7d, carry out the 2nd operation, right kidney is cut entirely.Two operations is for 5/6 of excision two kidney gross weights.Sham operated rats SD rat adopts same step to expose kidney, separates kidney peplos, but refuses the nephrectomy.54 of operation treated animal survivals, sham operated rats is all survived 8.In latter 2 weeks of the 2nd operation, all animal row inner canthus arteriovenous clumps blood samplings, carried out serum creatinine (Scr) and detect.And by 54 operation group rats, be divided into 5 groups according to Scr stratified random: 12 of model control group (model group), 10 of losartan groups, 11 of natural cicada fungus groups, heavy dose of group (the A group) 10 of artificial cicada fungus, 11 of artificial cicada fungus small dose group (B group).Losartan group, natural cicada fungus group, the artificial heavy dose of group of cicada fungus (A group), artificial cicada fungus small dose group (B group) rat starts medicine feed in 4 weeks after the 2nd operation, and treatment time is 42d.44 of survival of rats when experiment finishes, sham operated rats, heavy dose of group (the A group) each 8 of artificial cicada fungus, each 7 of model group, losartan group, natural cicada fungus group, artificial cicada fungus small dose group (B group).
Dosage:
Natural cicada fungus group, heavy dose of group (A group) rat of artificial cicada fungus are pressed 4gkg
-1d
-1consumption, artificial cicada fungus small dose group (B group) is pressed 2gkg
-1d
-1consumption, adds administration in feed, and every daily physiological saline gavage 1 time; Losartan group rat adopts administration by gavage, by 30mgkg
-1d
-1consumption, is dissolved in physiological saline, every day gavage 1 time.All rat grouping sub-cage rearings, every cage 4-5 rat, the whole rats of experimental session freely drink water, the commercially available blocky-shaped particle normal diet of taking food.
Testing tool and method:
Experiment detects Scr, blood urea nitrogen (BUN), the albumin (Alb) of all rats while end; Collect 24h urine, measure quantity of proteinuria.Above index adopts Hitachi's 7170 type automatic biochemistry analyzers to measure.
With HE dyeing and PAS dyeing nephridial tissue pathology sheet inspection glomerular sclerosis, wherein, glomerular sclerosis is defined as that capillary lumen subsides and/or vitreous degeneration.The semi-quantitative method of employing Rajj etc. is only assessed glomerular sclerosis degree.Under light microscopic (× 200) visual field, 40 renal glomeruluss are at least observed in every section, be divided into 0-++++ according to the shared renal glomerulus ratio of glomerular sclerosis kitchen range, 0 represents that renal glomerulus is without sclerosis, + represent the 25% impaired of 1 renal glomerulus, ++ represent that 26%-50% is impaired, +++ represent that 51%-75% is impaired, ++++represent that 76%-100% is impaired.Then calculate glomerular sclerosis index (GSI).
Adopt SPSS Version 11.0 for Windows to carry out data statistics, with mean ± standard deviation
represent, between group, relatively adopt One-Way ANOVA to analyze, statistical significance level is P < 0.05.
Test-results:
Found that, the Scr of rat, BUN value, each treatment group significantly reduces (P < 0.05) compared with model group, each group (heavy dose of group of artificial cicada fungus, small dose group) no difference of science of statistics compared with natural cicada fungus (P > 0.05), the seralbumin value of rat, sham operated rats, losartan group, natural cicada fungus group, the artificial heavy dose of group of cicada fungus (A group) is rising (P < 0.05) significantly, and the heavy dose of group (A group) of artificial cicada fungus no difference of science of statistics (P > 0.05) compared with natural cicada fungus group and sham operated rats, the 24h urine albumen amount of rat, each group compared with sham operated rats, all obviously increase (P < 0.05), but losartan group, natural cicada fungus group, the artificial heavy dose of group of cicada fungus (A group) are compared with model group, have obvious minimizing (P < 0.05).See the following form.
Rat model hematology and the comparison of urine detection result
Note: with sham operated rats comparison,
△p < 0.05; With model group comparison,
*p < 0.05; Compare with natural cicada fungus group,
#p < 0.05
Conventional H E and PAS dyeing, light Microscopic observation nephridial tissue pathology change: rats in sham-operated group renal glomerulus vessel open, clear in structure, after PAS dyeing, renal glomerulus collagen is thin-line-shaped distribution; In model group Renal Glomeruli In Rats mesangial region matrix, severe increases severe hyperplasia in companion's mesangial cell, part Capillary loops pressurized obturation, and hyaloid pathology, and accompany interstitial lymphocytic infiltration, in part uriniferous tubules, see protein cast.Each treatment group glomerular sclerosis degree alleviates (P < 0.05) than model group is obvious, be followed successively by the natural cicada fungus group of B group > A group > losartan group >, A group, B group and natural cicada fungus group are compared no difference of science of statistics (P > 0.05).See the following form
Each group glomerular sclerosis index and the comparison of matrix positive area ratio
Note: with sham operated rats comparison,
△p < 0.05; With model group comparison,
*p < 0.05; Compare with natural cicada fungus group,
#p < 0.05
Embodiment 4 acute oral toxicity tests
A. the culture of sample title: embodiment 2
B. sample preparation: take sample 10000mg, adding distil water, to 40ml, fully mixes rear as given the test agent.
C. laboratory animal: 20 of Kunming mouses, male and female half and half, body weight 19-22 gram, is provided by Shanghai Slac Experimental Animal Co., Ltd..Production licence number: SCXK (Shanghai) 2007-0005.Receptacle temperature 20-25 ℃, relative humidity: 40-70%.Laboratory animal occupancy permit number: SYXK (Shanghai) 2007-0008.Mouse feed is provided by Suzhou two lion laboratory animal feed technology Services Co., Ltd, registration card number: the E of Soviet Union raises new word (2002) 006.
D. experimental technique:
1. animal fasting (can't help water), after 16 hours, is selected each 10 of female, male mouse by body weight requirement, divides and is put in two mouse cages, with the poor 3g that is no more than of body weight between sex mouse.
2. given the test agent is adopted maximum tolerated dose method to contaminate to laboratory animal, mouse is by only weighing, and gavage capacity is by each 0.4ml/20g batheroom scale, and in 24 hours, secondary is to mouse stomach, and gavage interval time is 6 hours.
3. after contamination, observe general state, body weight change, toxicity symptom and the death condition etc. of animal.Observation period is one week.
4. weigh to animal again in experiment end.Dead animal and the animal of putting to death that expires are carried out to necrotomy, and visual inspection general pathology changes situation.
5. experiment whole process and observed content all do detailed record, by maximum tolerated dose method test-results, try to achieve chmice acute per os MTD.
E. result:
Male and female chmice acute Oral toxicity test-results
1. main physical signs performance:
The each treated animal of duration of test is movable normal, and hair color glossiness is good, has no any poisoning sign and death; Expire and put to death animal, the each internal organs situation of gross anatomy visual inspection, no abnormality seen.
2. female mice: MTD > 10000mg/kg
Male mice: MTD > 10000mg/kg
F. conclusion:
Sample is all greater than 10000mg/kg to the maximum tolerated dose MTD of male and female chmice acute Oral toxicity test.Belong to actual nontoxic level.
The pharmacological action of embodiment 5, Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture
The pharmacological action of Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] the CGMCC No.3453 culture to separation and Culture in the present embodiment detects, and detected result is as follows:
1, antipyretic effect: concrete detection method is referring to " Chen Zhuan, Liu Guangyu, bean English recklessly, the artificial culture of cicada fungus and pharmacological research thereof, fungi journal, 1993,12 (2): 138; Wang Haiying, Chen Yiping, professor Chen Yiping skilfully uses cicada fungus experience, China's Chinese materia medica information magazine, 2000,7 (10): 71 ", detected result proves that Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture has good antipyretic effect;
2, to Immune Function effect: concrete detection method is referring to " Song Jiemin; Chen Ling; Chen Wei etc.; the experimental study of cicada fungus to Immune Function; Chinese Chinese materia medica science and technology, 2007,14 (1): 371 ", detected result proves that Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture can significantly improve serum hemolysin and macrophage activity, Promote immunity function.
To sum up, visible Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture have antitumor, regulate immune, hypoglycemic, reducing blood-fat, hypotensive, improving eyesight, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve the functions such as renal function.
Claims (2)
1. a Paecilomyces cicadae bacterial strain [Paecilomyces cicadae(Miq.) Samson], the preserving number of described Paecilomyces cicadae bacterial strain is CGMCC No.3453.
2. the application of Paecilomyces cicadae bacterial strain [Paecilomyces cicadae(Miq.) Samson] in food, healthcare products and medicine preparation, the preserving number of described Paecilomyces cicadae bacterial strain is CGMCC No.3453.
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| CN102813179B (en) * | 2012-08-26 | 2014-06-11 | 浙江泛亚生物医药股份有限公司 | Harvesting production process for cordyceps sobolifera culture materials and special equipment for process |
| CN104756753B (en) * | 2015-01-27 | 2017-04-12 | 罗福仲 | Artificial cultivation method of cordyceps sobolifera |
| CN106912293B (en) * | 2015-12-24 | 2020-08-25 | 浙江泛亚生物医药股份有限公司 | Artificial culture method of cordyceps sobolifera |
| CN108685952A (en) * | 2017-04-10 | 2018-10-23 | 浙江泛亚生物医药股份有限公司 | The new application of cicada fungus |
| CN108315266B (en) * | 2018-03-06 | 2020-06-30 | 杭州师范大学 | Paecilomyces cicadae and application thereof |
| CN110295245B (en) * | 2018-03-23 | 2023-05-23 | 浙江泛亚生物医药股份有限公司 | SCAR molecular marker identification method of cordyceps sobolifera strain |
| CN109266556B (en) * | 2018-10-15 | 2021-08-17 | 贵州大学 | Artificial cultivation method of paecilomyces cicadae and analgesic aqueous extract cultivated by same |
| CN111388386B (en) * | 2020-04-02 | 2022-04-26 | 南京定宏医药科技有限公司 | Rice fermented extract and preparation method and application thereof |
| CN111494273A (en) * | 2020-04-02 | 2020-08-07 | 南京定宏医药科技有限公司 | Rice fermentation extracting solution and preparation method and application thereof |
| CN111996129B (en) * | 2020-09-07 | 2022-06-17 | 广东省微生物研究所（广东省微生物分析检测中心） | New strain of cicada fungus and its use in anti-tumor and bacteriostasis |
| CN113209143A (en) * | 2021-05-26 | 2021-08-06 | 贵州大学 | Paecilomyces cicadae extract with blood sugar reducing effect and application thereof |
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