CN102242069A - Paecilomycescicadae (Miq.)Samson and application thereof - Google Patents
Paecilomycescicadae (Miq.)Samson and application thereof Download PDFInfo
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- CN102242069A CN102242069A CN2011101206031A CN201110120603A CN102242069A CN 102242069 A CN102242069 A CN 102242069A CN 2011101206031 A CN2011101206031 A CN 2011101206031A CN 201110120603 A CN201110120603 A CN 201110120603A CN 102242069 A CN102242069 A CN 102242069A
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- miq
- samson
- paecilomyces cicadae
- cicada fungus
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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 the product after some cicada nymph is subjected to Paecilomyces cicadae bacterium parasitism, is a kind of bacterium worm complex body.This bacterium was named by Miquel in 1838 and is the mould Isaria cicadae of cicada Isaria.After this multiple synonym appears.As cicada grass Cordyceps cicadae, base is given birth to Isaria Isaria 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 or the like.
The perfect stage of Paecilomyces cicadae is considered to big cicada grass Cordyceps cicadae, and what widely distribute at nature is Paecilomyces cicadae, and big cicada grass is rare.What often adopt is complex body behind Paecilomyces cicadae and the host mountain cicada nymph parasitism thereof.According to the investigation of 1467 duplicate samples in Zhejiang, historical producing region, the medicinal material cicada fungus mainly is the entomogenous fungi complex body behind the Paecilomyces cicadae parasitism.Paecilomyces cicadae belongs to the imperfect fungi fungi, and its coremium grows from the polypide head, single giving birth to or the 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, D-N.F,USP MANNITOL, multiple alkaloid, ergosterol isoreactivity composition, to Cordyceps sinensis similar activeconstituents is arranged.The tradition traditional Chinese medical science thinks that cicada fungus has dispelling wind and heat pathogens, promoting eruption, relievng spasm by subduing liver-wind, item are moved back the effect of screen, modern medicine study finds, that cicada fungus has is antitumor, it is immune, hypoglycemic to regulate, reducing blood-fat, hypotensive, make eye bright, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve effect such as renal function.
The wild cordyceps resource-constrained because its personal value costliness has formed coyoting and plucked excessively, brings havoc to vegetation and ecotope for many years.In view of cicada fungus and Cordyceps sinensis have similar activeconstituents, also have immunomodulatory, antipyretic-antalgic, improve multiple pharmacological effect such as renal function, tranquilizing soporific, illustrate that cicada fungus is a kind of medicinal entomogenous fungi that has a extensive future that has, and can do 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 common micro-organisms center, preserving number is CGMCC No.3453.
Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] adopts following method to separate:
Gather pure pollution-free wild cicada fungus sample from Yunnan sources of three rivers head, the transplanting tweezer of crossing with flame sterilization on the clean work station take a morsel from sample conidium in the PSA flat board that adds rose-bengal put 22 ℃-25 ℃ cultivate 120h down after again picking list bacterium colony insert slant medium, 22 ℃-25 ℃ cultivate down and through purifying repeatedly get final product Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson].
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 morphological specificity that biological study is carried out on September 10th, 2009 and is identified that qualification result is:
Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson], its main morphological specificity is:
On the PDA substratum, cultivated 10 days for 25 ℃, the bacterium colony circle, open and flat, first cotton-shaped, back 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.The conidiophore multi-branched, wide 3.0～5.5 μ m are taken turns 2～5 conidiogenous cells and are formed the colyliform branch by every.The conidiogenous cell doleiform, base portion is spherical to be expanded, and it is narrow upwards to attenuate, 4.5～6.5 * 2.5～3.5 μ m.Conidium is cylindrical, and is unicellular, colourless, level and smooth, and chain is given birth to, and is straight or crooked, 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) puts 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 begins 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, the fine hair shape.Late stage of culture because of producing a large amount of conidiums, causes the bacterium colony outward appearance to show the 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) the 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) and 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, pure growth is handled 30h through 40 ℃, handles inactivation behind the 2.5h for 50 ℃;
2. humidity: under saturated humidity, conidia germination rate 24h is 86%, and 96h is 99%; During RH 90%, the 24h germination rate is 0%, and 96h is 78.3%; During RH 76%, the 72h germination rate is 0%, and 96h is 18%;
(5) PH: this bacterium is at the equal energy of PH 4～12 scopes normal growth, but PH 5～6 mycelium production height, the slant culture bacterium colony is difficult for aging;
(6) C, N source utilize: this bacterium can utilize carbon sources such as glucose, fructose, maltose, seminose, glycerine, but does not utilize synanthrin, sorbose, rhamnosyl and lactose.As carbon source, conidium output is significantly higher than above-mentioned several carbon source with glucose, and as carbon source, mycelium production is higher than other carbon sources with fructose.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, but can not utilize NaNO than amino N
2And thiocarbamide.This bacterium is quite responsive to the carbon source required amount.For example, carbon source maintained on 2% the normal growth developmental level, improve the N source or reduce the N source not quite coremium growth and sporulation quantity influence; But the N source is fixed on the normal growth level, carbon source is reduced to 0.5% from 2%, find not produce coremium, or little coremium produces.Otherwise, carbon source is improved 10 times (20%), then coremium is many and difficult aging, but generation of conidium is later;
(7) light: this bacterium has obvious phototaxis, and coremium growth is tilted towards light source direction, and it is prosperous secretly to cultivate mycelial growth, only produces sclerotium and does not form coremium.Natural light and light all can stimulate coremium growth and conidium to form;
(8) this bacterium has strong radioprotective phenomenon, and flat-plate bacterial colony is from 30Wt ultraviolet source 0.5m place irradiation 281h, moves and does not see inactivation after growing, and does not also see growth failure;
(9) solid fermentation: do substratum with cereal such as wheats, inoculum size 3%～5% (weight percent), inoculation back 3～5 days, visible canescence fine hair shape mycelium covers stromal surface.Formed sclerotium in 20～25 days 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, the afterwards withered gradually lodging of coremium.
2, Paecilomyces cicadae bacterial classification sequencing:
Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson] send in Institute of Microorganism, Academia Sinica on September 11st, 2009 and carries out strain identification, and receive and detect No. the 249th, the little searching of probation report (2009) that 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 shown in sequence in the sequence table 1; Its ITS1 gene order is shown in sequence in the sequence table 2; Its 5.8S rRNA gene order is shown in sequence in the sequence table 3; Its ITS2 gene order is shown in sequence in the 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, characteristics that output is high, that its cultured products has is antitumor, it is immune, hypoglycemic to regulate, reducing blood-fat, hypotensive, make eye bright, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve function such as renal function, so have the extensive industrialized using value.
Description of drawings
Fig. 1: Cordyceps polysaccharide typical curve
Fig. 2: N.F,USP MANNITOL typical curve
Fig. 3: adenosine cordycepin correction graph
Embodiment
Further describe technical scheme of the present invention below by specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The separation of embodiment 1, Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453
1, prepares 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 sources of three rivers head, the transplanting tweezer of crossing with flame sterilization on the clean work station take a morsel from sample conidium in the PSA flat board that adds rose-bengal put 22 ℃-25 ℃ cultivate 120h down after again picking list bacterium colony insert slant medium, 22 ℃-25 ℃ cultivate down and through purifying repeatedly get final product Paecilomyces cicadae bacterial strain of the present invention [Paecilomyces cicadae (Miq.) Samson].
3, spawn culture:
CGMCC No.3453 is connected on the bell potato sucrose nutrient agar with Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson], 24 ℃ of incubations 14 days, and diameter reaches 60～72mm, grow prolifically, at the cultivation initial stage, mycelium is greyish white to pale yellow, the fine hair shape; Late stage of culture because of producing a large amount of conidiums, causes the bacterium colony outward appearance to show the opaque shape.
CGMCC No.3453 is connected on the peptone sucrose nutrient agar with Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson], 24 ℃ of incubations 14 days, and diameter 54～70mm, it is good to grow, local crowning or subside yellowish pink; Later stage is covered with conidium, back side Vandyke brown.
4, bacterial classification sequencing:
Prepare the potato sucrose nutrient solution by following proportioning: potato 200g, sucrose 20g, water 1000ml.
With 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 and grind fragmentation, extract total DNA of bacterial classification with the Benzyl Chloride method.
With total DNA is 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 zone.
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, 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 the PCR instrument: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ kept 4 minutes, and moved 35 circulations altogether; 72 ℃ were extended 10 minutes.
After the PCR reaction finishes, the gel electrophoresis product is reclaimed the recovery of test kit purifying with gel institute's calling sequence and ncbi database are carried out sequence comparing analysis, the result shows that bacterial classification of the present invention is Paecilomyces cicadae [Paecilomyces cicadae (Miq.) Samson].
The solid culture of bacterial classification:
(1) strain preparation: embodiment 1 gained bacterial classification;
(2) batching dress box: prepare solid medium, the substratum for preparing is packed into cultivate in the box, charge amount is 15%, seals with sealing film; The described solid medium proportioning of 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 are 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: earlier grain is boiled, sucrose, saltpetre are dissolved in water, proportionally mix with bagasse, oyster shell whiting, dried silkworm chrysalis meal.
(3) sterilization: will cultivate box and put into Autoclave, sterilization 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 was cultivated 50 days for 80% time, and later stage coremium growth phase requires daylighting, and envrionment temperature is compared, and the mycelial growth stage need be hanged down 2～3 ℃ in earlier stage, the ventilation of need in good time windowing every day, it is fresh to keep cultivating room air;
(6) gather: when the coremium height of cicada fungus culture reaches about 9cm, can gather when the surface has a small amount of conidium to occur; When gathering, remove and seal film, with instrument the culture medium culturing thing is taken out and separate coremium, the oven dry classification, the cooling back uses bag film vacuum packaging, mycoplasma to place-20 ℃ of storages.
One: the polysaccharide detection method
1 experimental principle
Adopt phenol sulfuric acid colorimetry that the sample polysaccharide is detected.Its principle is: polyose is hydrolyzed into the monose molecule earlier under vitriol oil effect, and dehydration generates the alditol derivative rapidly, the alditol derivative generates colored compound with phenolic substance such as phenol reactant again, at wavelength 490nm place maximum absorption is arranged, and satisfy Law of Lambert-Beer, by the light absorption value of test sample under this wavelength, can calculate the content of polysaccharide in the sample.
2 instruments and material
2.1 key instrument ultraviolet-visible pectrophotometer; Electronic balance; Adjustable closed electric furnace; Whizzer H-1650.
2.2 experiment reagent dextrose anhydrous (AR); Phenol (AR); The vitriol oil (AR); Watson distilled water.
Reagent is prepared 5% phenol solution: accurately take by weighing 5g phenol, water is settled to 100ml.
The preparation of glucose reference liquid: compound concentration is the glucose reference liquid of 0.25mg/mL, accurately takes by weighing the glucose after 0.2500g is dried, and adds water and is settled to 1000mL.
2.3 the cultured products of material embodiment 3
Natural cicada fungus (originating from Yunnan)
3 experimental techniques
3.1 the preparation of sample Crude polysaccharides extracting solution accurately takes by weighing the 1g sample and places in the exsiccant 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 over make its constantly boiling 15min on the electric furnace after, be put in rapidly and be cooled to room temperature in the cold water, take out, after drying the globule of bottle outer wall, to wherein adding distilled water to final quality is ml+100g, adds water and shakes up after finishing, and filters, access filtered liquid, dilute 2 times standby, be testing sample ,-20 ℃ of preservations.
3.2 the mensuration of sample Crude polysaccharides
3.2.1 the drafting precision of typical curve takes by weighing anhydrous glucose 0.2500g, be mixed with the glucose standard 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, place tool plug test tube respectively, each adding distil water, making volume is 2.0ml, adds 5% phenol solution 1ml again, shakes up, drip 98% sulfuric acid 5ml rapidly, shake up the back and placed 5 minutes, put in the boiling water bath and heated 15 minutes, take out cold water and be cooled to room temperature; Adding distil water 2ml in addition, blank is done in the same operation, in 490nm place mensuration light absorption value (Abs).With standard substance glucose concn (mg/ml) is X-coordinate, and Abs is an ordinate zou, gets regression equation: Y=0.6622X+0.0088, R
2=0.9997, description standard product glucose amount is good linear relationship with Abs in 0～2.75mg/ml scope.
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: the 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 the working sample.
2 instruments and material
2.1 key instrument ultraviolet-visible pectrophotometer; Electronic balance; Whizzer H-1650; Electric heating constant temperature 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.
The reagent preparation
Potassium periodate solution: 15mmol (being 3.45g) potassium periodate is dissolved in the 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.
2.3 the cultured products of experiment material embodiment 3
Natural cicada fungus (originating from Yunnan)
3 experimental techniques
3.1 the preparation of sample N.F,USP MANNITOL extracting solution accurately takes by weighing the 1g sample and places in the exsiccant 500ml Erlenmeyer flask, the total mass of weighing bottle and sample is designated as ml, adds the about 70ml of boiling distillated water in triangular flask, be positioned over make its constantly boiling 15min on the electric furnace after, be put in rapidly and be cooled to room temperature in the cold water, take out, dry the globule of bottle outer wall after, to wherein adding distilled water to final quality is ml+100g, add water and shake up after finishing, filter, access filtered liquid, be testing sample ,-20 ℃ of preservations.
3.2 the mensuration of sample N.F,USP MANNITOL
3.2.1 the preparation precision of standardized solution takes by weighing D-N.F,USP MANNITOL standard substance 0.1g in beaker, it is complete to add a small amount of dissolved in distilled water, be transferred to constant volume in the 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 above-mentioned each 1mL of concentration standard product mannitol solution, split in the different test tubes, add the 1mL sodium periodate solution then respectively, mixing, room temperature is placed 10min, add 2mL0.1%L-rhamnosyl solution to remove too much periodate, the vibration mixing 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, the place measures its absorbancy at the 412nm wavelength.Replace the N.F,USP MANNITOL standardized solution with distilled water, use the same method operation in contrast, measure its absorbancy.With the concentration of standard solution is X-coordinate, and absorbancy is an ordinate zou, the drawing standard curve.With standard substance mannitol concentration (mg/ml) is X-coordinate, and Abs is an ordinate zou, gets regression equation: Y=0.0826X+0.0019, R
2=0.9998, description standard is tasted with discrimination the N.F,USP MANNITOL amount in 0～21.25mg/L scope, 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: specimen amount (g)
Three: the adenosine content measuring 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 grams, Zhejiang industry and trade company limited 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.
1.3 the cultured products of sample embodiment 3
Natural cicada fungus (originating from Yunnan)
2 methods
2.1 chromatographic condition
Chromatographic column: XBridge C18 chromatographic column (Waters, 4.6mm * 250mm, 5 μ m);
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 adenosine cordycepin typical curve is drawn
Precision takes by weighing 50mg adenosine reference substance, puts in the 50mL volumetric flask, adds the ultrapure water dissolving and be diluted to scale to make 1mg/mL solution.Precision pipettes an amount of 1mg/mL adenosine reference substance solution, is mixed with the adenosine reference liquid of 1,5,10,30,50,100 μ g/mL respectively, and is standby.
2.3 the preparation of need testing solution
Get about 1.0g sample powder, the accurate title, decide, and puts in the 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 puts in the lump in the tool plug bottle together with filter paper, adds the 20mL ultrapure water in bottle, claim gross weight (comprising Erlenmeyer flask weight), ultrasonic 30min.Weigh after the cooling fast, mend to gross weight with ultrapure water, centrifugal behind the mixing, get supernatant liquor, cross 0.22 μ m millipore filtration, standby.
2.4 typical curve is drawn
With 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 chromatographic condition is measured under, is X-coordinate with sample concentration (μ g/mL), peak area (microvolt second) is an ordinate zou, the drawing standard curve, and getting 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 the trial-product adenosine content is measured
Get 2.3 need testing solutions of preparation down,, make liquid to be measured with the mixed of ultrapure water with 2: 1.Get liquid to be measured by 2.1 following chromatographic condition sample introductions, get liquid adenosine concentration x to be measured (μ g/mL), obtain trial-product adenosine content (mg/g) according to following formula by regression equation.
Detected result
The equal ratio content of various effective constituents in the cicada fungus cultured products that makes in the present embodiment is than natural cicada fungus height, and product quality is stable, is of very high actual application value.
Tried thing: the culture of embodiment 2 (artificial cicada fungus is called for short in the back).
Positive control drug: losartan, Mo Shadong pharmaceutical Co. Ltd in Hangzhou produces; Natural cicada fungus, the place of production are 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:
After SD rat adaptability fed for 1 week, be divided into 2 groups at random: 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 the experimental rat model: the 1st operation, operation group rat left side kidney always excises renal cortex weight and accounts for 2/3 of left kidney weight; Carry out the 2nd operation behind the 7d, 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 the 2nd operation 2 weeks of back, all animal row inner canthus arteriovenous clump blood samplings are carried out serum creatinine (Scr) and are detected.And, be divided into 5 groups: 12 of model control group (model group), 10 of losartan groups, 11 of natural cicada fungus groups, the heavy dose of group of artificial cicada fungus (A group) 10,11 of artificial cicada fungus small dose group (B group) according to the Scr stratified random with 54 operation group rats.The 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 is at the 2nd operation back 4 week beginning medicine feed, and treatment time is 42d.Survival of rats was 44 when experiment finished, each 8 of sham operated rats, the artificial heavy dose of groups of cicada fungus (A group), respectively 7 of model group, losartan group, natural cicada fungus group, artificial cicada fungus small dose group (B group).
Dosage:
Natural cicada fungus group, the heavy dose of group of artificial cicada fungus (A group) rat are pressed 4gkg
-1D
-1Consumption, artificial cicada fungus small dose group (B group) is pressed 2gkg
-1D
-1Consumption adds administration in the feed, and irritate stomach 1 time with physiological saline every day; Losartan group rat is adopted administration by gavage, presses 30mgkg
-1D
-1Consumption is dissolved in the physiological saline, irritates stomach every day 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 when finishing; Collect the 24h urine, it is quantitative to measure urine protein.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 the glomerular sclerosis degree.Under light microscopic (* 200) visual field, 40 renal glomeruluss are observed in every section at least, be divided into 0-++++ according to the shared renal glomerulus ratio of glomerular sclerosis kitchen range, 0 expression renal glomerulus does not have sclerosis, 1 renal glomerulus of+expression 25% impaired, ++ 26%-50% is impaired in expression, ++ 51%-75% is impaired in+expression, ++ ++ 76%-100% is impaired in expression.Calculate glomerular sclerosis index (GSI) then.
Adopt SPSS Version 11.0 for Windows to carry out data statistics, with mean ± standard deviation
Expression relatively adopts One-Way ANOVA to analyze between group, the 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) than model group, each group of artificial cicada fungus (heavy dose of group, small dose group) is compared no difference of science of statistics (P＞0.05) with natural cicada fungus, 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) significantly raise (P＜0.05), and the artificial heavy dose of group of cicada fungus (A group) is compared no difference of science of statistics (P＞0.05) with natural cicada fungus group and sham operated rats; The 24h urine albumen amount of rat, each group is 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) compares with model group, and obvious minimizing (P＜0.05) is then arranged.See the following form.
Rat model hematology and urine detection result are relatively
Annotate: compare with sham operated rats,
△P＜0.05; Compare with model group,
*P＜0.05; Compare with natural cicada fungus group,
#P＜0.05
Conventional H E and PAS dyeing, light microscopic are observed the nephridial tissue pathology down and are changed: sham operated rats kidney of rats bead vessel open, and clear in structure, PAS dyeing back renal glomerulus collagen is thin-line-shaped distribution; Severe increases severe hyperplasia in companion's mesangial cell in the model group kidney of rats bead mesangial region matrix, part capillary vessel button loop pressurized obturation, and hyaloid pathology, and matter lymphocytic infiltration between the companion are seen protein cast in the part uriniferous tubules.Each treatment group glomerular sclerosis degree alleviates (P＜0.05) than model group is obvious, be followed successively by B group＞A group＞losartan group＞natural cicada fungus group, and 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 organizes the glomerular sclerosis index and matrix positive area ratio value compares
Annotate: compare with sham operated rats,
△P＜0.05; Compare with model group,
*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 by weighing sample 10000mg, adding distil water is to 40ml, fully behind the mixing 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) 2007-0005.20-25 ℃ of receptacle temperature, relative humidity: 40-70%.Laboratory animal occupancy permit number: SYXK (Shanghai) 2007-0008.Mouse feed provides registration card number by the two lion laboratory animal feed technology Services Co., Ltd in Suzhou: the E of Soviet Union raises new word (2002) 006.
D. experimental technique:
1. animal fasting (can't help water) was selected each 10 of female, male mouse for use by the body weight requirement after 16 hours, divided to be put in two mouse cages, was no more than 3g with body weight difference between the sex mouse.
2. given the test agent is adopted the maximum tolerated dose method that laboratory animal is contaminated, mouse is irritated gastric capacity by each 0.4ml/20g batheroom scale by only weighing, and secondary is to mouse stomach in 24 hours, and irritating stomach pitch time is 6 hours.
3. after the contamination, observe general state, body weight change, toxicity symptom and the death condition etc. of animal.Observation period is a week.
4. weigh to animal once more in the experiment end.The dead animal and the animal of putting to death that expires are carried out necrotomy, and the visual inspection general pathology changes situation.
5. experiment whole process and observed content are all done detail record, press maximum tolerated dose method test-results, try to achieve chmice acute per os MTD.
E. result:
Male and female chmice acute per os toxicity test result
1. main physical signs performance:
Each treated animal of duration of test is movable normal, and the hair color glossiness is good, does not see any poisoning sign and death; Expire and put to death animal, 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 to the maximum tolerated dose MTD of male and female chmice acute per os toxicity test all greater than 10000mg/kg.Belong to actual nontoxic level.
The pharmacological action of embodiment 5, Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture
Pharmacological action to Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] the CGMCC No.3453 culture of 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, Hu bean English, artificial culture of cicada fungus and pharmacological research thereof, fungi journal, 1993,12 (2): 138; Wang Haiying, Chen Yiping, professor Chen Yiping skilfully use the cicada fungus experience, China's Chinese materia medica information magazine, 2000,7 (10): 71 ", detected result proof Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture has good antipyretic effect;
2, to the Immune Function effect: concrete detection method is referring to " Song Jiemin; Chen Ling; Chen Wei etc.; cicada fungus is to the experimental study of Immune Function; Chinese Chinese materia medica science and technology, 2007,14 (1): 371 ", detected result proof Paecilomyces cicadae bacterial strain [Paecilomyces cicadae (Miq.) Samson] CGMCC No.3453 culture can significantly improve serum hemolysin level and macrophage activity, promotes immunologic 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, make eye bright, radioprotective, antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics, improve function such as renal function.
Claims (2)
1. a Paecilomyces cicadae bacterial strain Paecilomyces cicadae (Miq.) Samson CGMCC No.3453.
2. the application of Paecilomyces cicadae bacterial strain Paecilomyces cicadae (Miq.) Samson CGMCC No.3453 in food, healthcare products, medicine and makeup preparation.
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| 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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