CN1614003A - Culture of Chansi mould strains and extraction and use of anagentic compound therefrom - Google Patents

Abstract

An artificial culture method of new balm cricket Penicillium notatum and extraction and application of delivery pains compounds are disclosed. This invention involves declutching and breeding quality new balm cricket Penicillium notatum APC-20 from nature, researching production of the strain, further, defining method of extracting delivery pains compounds to be medicine. The compound is N6-(2- hydroxyethyl) adenosine. Molecular weight is 311.297. Molecular formula is C12H17N5O5. Delivery pains ratio is 99%by the method of mouse test. Principle of delivery pains is particular and cannot induce addiction.

Description

The artificial culture of the new bacterial strain of Paecilomyces cicadae and the extraction of analgesic compounds and utilization

Technical field

The present invention relates to Cordyceps fungus fermentation, efficient part extracts and utilize technical field, especially relates to artificial solid and/or the liquid fermenting of the new strains A PC-20 of a kind of Paecilomyces cicadae, therefrom extract the compound of providing analgesic activities and and then be prepared into analgesic.

Background technology

Cicada fungus is the very high precious Chinese medicine of a kind of pharmaceutical use since ancient times, and it is the complex body that is parasitized the cicada nymph by Paecilomyces cicadae (Paecilomyces cicadae) (genus Cordyceps).Past people gathers mainly that rare natural cicada fungus is used for various healthcare products or as combination drug material and tonic medicated food.But natural cicada fungus is fewer and feweri, and yields poorly, and can only gather once in 1 year, and quality product is affected by environment and uneven simultaneously.The existing Paecilomyces cicadae bacterium of nature has growing way, output, spotty problem, can not adapt to need of industrial production; And have article report to think (Feng immediately, 2002 02 phases of Shanghai Institute Of Technology's journal (nature)) this natural bacterial classification directly to be used for carrying out the general sporophore (coremium) of can not growing of artificial solid culture fermentation; And existing technology not match when being used for the industrialization liquid fermenting, mycelium production is low, problems such as bad.This can not satisfy the heavy demand of tcm clinical practice medication to coremium, has also restricted the industrialization deep processing development utilization to Paecilomyces cicadae bacterium meta-bolites.

Chinese caterpillar fungus is the important insect pathogenic fungus of a class, its meta-bolites has characteristics such as diversity and multiple pharmacological effect, report Chinese caterpillar fungus the earliest to effective Brewster of being of central nervous system and Alsberg, they suppress to have effect to rabbit and mouse vein or subcutaneous injection Cordyceps sinensis (Cordyceps sinensis (Berk.) Sscc.) vat liquor proof to maincenter.Domestic Zhang Shishan etc. also reported the Cordyceps sinensis preserved material have calmness and syngignoscism (Zhang Shishan Acta Pharmaceutica Sinica, 1991,26[5]: 326-330).The Chen Zhu of this study group peace waits report cicada fungus and artificial culture thing Paecilomyces cicadae (Paecilomycec Cicadae) thereof, can suppress by the writhing response due to the acetic acid with 25g crude drug/Kg dosage injection mouse peritoneal, the analgesia rate reaches more than 95%, its effect is equivalent to 10mg/Kg morphine (Chen Zhuan etc., artificial culture of cicada fungus and pharmacological action fungi journal thereof, 1993,12[2]: 138-144).In addition, paecilomyces tenuipes (Paecilomyces teniupes (Peck) Samson) also has similar effect.Reports such as Li Shufang arrive with the mouse writhing method experimental observation, at examination Cordyceps gunnii (Berk.) Berk. (Cordyceps Gunnii (Berk.) Berk.) mycelium extract, the hot plate stimulus method threshold that eases pain after 15 hours is extended for 79%, similar (the Li Shufang of its analgesia intensity to dolantin, the refined beautiful Guiyang Medical College journal of Bao, 1990,15 (1): 25～28).Have analgesic activity though reported Chinese caterpillar fungus and anamorph thereof both at home and abroad, do not disclose the chemical ingredients of its analgesic activity as yet.Researchs such as Zhang Shishan think that the sedative effect of Cordyceps sinensis is by amino acid, and especially tryptophane causes.The active substance of Zhu Zhenyuan report paecilomyces gunniliang (Paecilomyces Gunnii Liang) mycelium analgesic activity is that molecular weight is not higher than 12000 polypeptide (Zhu Zhenyuan, Liang Zongqi, the biologically active substance I of Cordyceps gunnii (Berk.) Berk.s such as an invincible army contain the separation and the character microorganism journal 2002 of peptide analgesia component.42(6)：722～726)。Abroad also have report from FENBEICHONGCAO (Cordyceps pruinosa Petch) separation and Extraction to N ⁶-(2-hydroxyethyl) adenosine and structural formula thereof, because the peculiar property of this compound, used it as one of Cordyceps sinensis Products Quality controlling index, its biological applications is generally as Ca2+ antagonist and Muscle contraction activity, (the Tsutomu FuruyaN but the analgesic activity of this compound does not appear in the newspapers ⁶-(2-HYDROXYETHYL) ADENOSINE, A BIOLOGICALLY ACTIVECOMPOUND FROM CULTURED MYCELIA OF CORDYCEPS AND ISARIASPECIES, Phytochemistry, Vol.22.No.11, pp.2509-2512,1983).In sum, about analgesic activities material of Paecilomyces cicadae and preparation method thereof and utilize and do not appear in the newspapers as yet, therefore the chemical ingredients and the extracting method thereof of clear and definite Paecilomyces cicadae tool analgesic activity, and then the analgesic that is developed to new type of safe has important society and economic benefit.

Summary of the invention

The present invention is directed to above-mentioned deficiency, propose following goal of the invention, the one, separate and the artificially breeding vitality is strong, metabolic active substance output height, the new bacterial strain of the measured Paecilomyces cicadae of matter from nature; The 2nd, research and propose and utilize the new bacterial strain of this Paecilomyces cicadae to carry out industrialization solid culture and/or liquid culture, produce high yield, fine coremium, mycelium and meta-bolites and substratum preparation process of mixture; The 3rd, the characteristic and the structure thereof of contained analgesic compounds in the clear and definite Paecilomyces cicadae bacterium tunning; The 4th, propose from Paecilomyces cicadae bacterium tunning comprises the mixture of coremium, mycelium and meta-bolites thereof and substratum, progressively to extract each efficient part of tool analgesic activities and the method for pure product thereof; The 5th, further above extract is prepared into the analgesic of multiple formulation.

The present invention seeks to be achieved by following proposal.

The separation of bacterial strain, seed selection, evaluation and preservation:

Different land occupation conditions, different ecological environment collect specimen from Wenzhou, Zhejiang, therefrom the chooser entity is big, and coremium is sturdy, and pollution-free bacterium person carries out separation and purification, is numbered respectively, and further by the monospore separation method, has set up 25 strain bacterial strains and preserved.Wherein from No. 022009 starter bacteria, isolation and selection is to the excellent strain APC-20 of monospore bacterial strain, through the solid-substrate fermentation mycelial growth potential, the test of liquid nutrient medium mycelium dry weight and three indexs of polysaccharide content behaves oneself best, this bacterial strain is checked through Institute of Microorganism, Academia Sinica and is accredited as Paecilomyces cicadae (Paecilomyces cicadae), and be the non-toxic and safe bacterial strain by Ministry of Health's bacterial strain security virulence experiment, this bacterial classification is preserved in 4 ℃ of refrigerators and was not seen inactivation in 544 days at the sterile soil of 40% water content.

Paecilomyces cicadae APC-20 bacterial strain of the present invention on 03 03rd, 2004 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC NO:1104.Paecilomyces cicadae APC-20 bacterial strain (CGMCC NO:1104) has following Microbiological Characteristics:

1, morphological specificity:

(1), coremium (cicada fungus) is orange-yellow, single giving birth to or Cheng Cong, 30-50 * 2-5mm, top or repeatedly branch become the head of cauliflower shape, and produce a large amount of powdery conidiums.

(2), the strain morphology feature on the PDA substratum, mycelia is greyish white to pale yellow, the fine hair shape.The conidiophore branch, 78-203 * 2.9-50mm, 2-5 bottle stalk clusters on brachyplast usually, becomes colyliform to arrange.Subsphaeroidal the expanding of bottle metulae portion, 4.2-7 (13.5) * 2.3-3.5 (5.2) mm, the stalk neck is elongated, and basipetal produces conidia chain. and conidium is cylindric or long avette, colourless monospore, wall is smooth, 3.5-9 * 1.5-3.7mm, minority bending.

2, cultural characteristic:

Growth was cultivated colony diameter 60-72mm 14 days for 24 ℃ soon on the PSA substratum.Mycelium is greyish white or pale yellow, the fine hair shape.Sometimes wheel line or radioactive rays appear in the surface.After producing spore, the bacterium colony appearance shows the opaque shape, and the back side is colourless, and transudate is colourless, globule sample.

Growth is limited to slowly on the Czapek substratum, and 14 days diameters are 49-55mm.Bacterium colony is open and flat, and lawn is thin, and greyish white, the fine hair shape is dense, and the back side is colourless, does not see that transudate produces.

On peptone sucrose agar, grow 14 days diameter 54-70mm.There is the protuberance loop wire on the bacterium colony surface, and there is radial rill at the edge, yellowish pink, and back side Vandyke brown is not seen transudate.

On wheat or wheat bran+corn+natural substratum such as cavings, mycelium fine hair shape is to cotton-shaped, and is greyish white to pale yellow.Form sclerotium in 15 days, and produce coremium.Coremium palmate or cylindric, orange-yellow or yolk yellow, 20-30 * 3-5mm, long reached at 80cm.Produce a large amount of conidiums after general 30 days, the withered gradually lodging of later coremium.

3, carbon, the nitrogenous source utilization:

(1) utilization of carbon source has been measured 10 kinds of C sources such as glucose, sucrose, fructose, maltose, N.F,USP MANNITOL, synanthrin, sorbose, rhamnosyl, lactose, glycerine, and except that synanthrin, sorbose, rhamnosyl, lactose can not utilize, all the other all can utilize.But do the C source with glucose, the spore output utmost point is significantly higher than other C source (t＞t _0.01), do the C source with fructose, mycelium production is significantly higher than other C source (t＞t _0.05).

(2) KNO has been measured in the nitrogenous source utilization ₃, NaNO ₃, (NH ₄) ₂SO ₄, (NH ₄) ₂HPO ₄, NH ₄NO ₃, (NH ₂) ₂CO, NH ₄CL, NaNO ₂, H ₂NCSNH ₂Contain 9 kinds of N sources such as nitro, amino, nitroso-group, with KNO ₃Be utilized as.But can not utilize NaNO ₂And thiocarbamide, the utilization of nitrate there is the trend better than amino nitrogen.

This bacterium is to the KNO in sucrose, glucose and the N source ₃Utilize and use up, but responsive to the requirement of C source amount.Maintain when C source amount 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, and the C source is reduced to 0.5% from 2%, finds not produce coremium or only few coremium.Otherwise, the C source is improved 10 accompany (20%), then coremium is many and difficult aging, and conidium has to postpone leads to a phenomenon.

4, artificial culture product determination of polysaccharide:

Natural cicada fungus polysaccharide content is 4.40%, and liquid culture product polysaccharide content is 11.4%, and solid culture product polysaccharide content is 12.8%.

In addition, APC-20 bacterial strain and parental plant 22009 (contrast) are tested through solid culture and liquid culture, in growing way, coremium output, there is bigger difference in aspects such as mycelium dry weight:

Upgrowth situation on table 1 solid medium

Bacterial strain	Upgrowth situation	Average coremium length (mm)	Start the coremium time (my god)	Coremium wear out the lodging time (my god)
					APC-20 contrast (parental plant) (22009, growth is general)	Bacterium is sturdy by thick coremium, white or pale yellow, growing way is prosperous, no transudate coremium growing way is poor, weak, sporulation quantity is more, water breakthrough strain shape transudate	????78.0×3.51 ? ? ????55.47×3.10	????7 ? ? ????15	????40 ? ? ????22

Mycelium dry weight on table 2 liquid nutrient medium

Bacterial strain	Mycelium dry weight (g/200ml)
		APC20 contrast (022009, growth is general)	????2.76 ????1.51

Annotate:

Shaking table is cultivated rotating speed 110r/min, and potato-sucrose nutrient solution was cultivated 10 days.

Mycelium dry weight is measured and to be got fermented liquid 100ml with 100 order yarn strainer filterings, takes a sample at every turn and repeats for 3 times, get wherein filter residue mycelium in CT-C-O type Hotaircirculatingoven 70 ℃ dry to constant weight, be converted into the dry weight of mycelia in per 100 milliliters of fermented liquids.

Use bacterial strain of the present invention to carry out the method for artificial solid fermentation and/or liquid submerged fermentation, be included in and contain carbon source, nitrogenous source, cultivate step, the technology of Paecilomyces cicadae APC-20 bacterial strain in inorganics and other nutraceutical substratum, until top layer at solid medium, form sporophore (coremium), layer has also formed a large amount of mycelium and effective meta-bolites thereof simultaneously within it; Form the mycelial while in the liquid medium within, also accumulated mycelial effective meta-bolites, become cultured products with further exploitation value with substratum.

A, solid culture fermentation process:

1, culture condition

(1) strain preparation:

1., the APC-20 bacterial strain preserved in 4 ℃ of refrigerators of preparation of slant strains is transplanted on the PSA substratum and cultivated 7 days, activates stand-by.

2., the preparation of the preparation first order seed of I and II seed: every is shaken bottle (the bottled 200ml seed culture medium of 500ml triangle) and moves conidial powder 3 rings that insert on the slant strains, puts in the DHZ-C vibrator 25 ℃, and the 120r/min rotating speed was cultivated 4 days down.The preparation of secondary seed: adorn 7.5L in the 10L fermentor tank (with 75% volume calculation, secondary seed medium down together), 121 ℃ of sterilization 30min postcooling to 24 ℃, insert first order seed, inoculum size 10% (down together), the control fermentation condition (24 ℃, the about 0.01-0.05Mpa of tank pressure, the about 0.75m of air flow quantity ³/ h), fermentation 48h.

(2) substratum:

Slant medium: adopt PSA or Richard substratum;

First order seed substratum: PSA solid medium (potato 200g, sucrose 20g, agar 20g, water 1000ml) or Richard substratum (KNO ₃10.0g, KH ₂PO ₄5.0g, MSO ₄.7H ₂O2.5g, FeCl ₃0.02g, sucrose 50.0g, water 1000ml);

Secondary seed medium: PSA liquid nutrient medium (potato 200g, sucrose 20g, water 1000ml);

Solid state fermentation substratum: pure wheat or wheat bran (2-4): corn (1-3): the weight ratio preparation of cavings (1-3), screening formulation is a wheat bran 2: corn 1: cavings 1 also adds 2% fructose;

(3) the closed cultivation of inoculum size, inoculum size 1%-5% is preferably 3%; Open cultivation, inoculum size is 5%-20%, is preferably 10%;

(4) kind incubation time in incubation time inclined-plane was advisable with 5-7 days.1-2 level seed adopts liquid shaking table or seed tank culture, and inoculate back and grow prosperous for well the 2-3 days length of time.20-30 days solid culture time, coremium begins aging lodging after general 35 days, and mycelia produces autolysis.

(5) culture temperature inclined-plane kind and seed culture stage, all can grow for 6-38 ℃, wherein the suitableeest is 24-25 ℃.The mycelial growth phase temperature of solid fermentation is advisable with 24-25 ℃, and the coremium phase temperature that starts should be controlled at 20-22 ℃.

(6) about pH pH6.5. this bacterium requires slant acidity, and under alkaline condition, the aging easily look of mycelia becomes.

(7) light modulation light, natural light all can stimulate the coremium growth, and dark cultivation does not produce coremium, or the coremium amount is few, and weak, look becomes.

(8) the closed culture vessel of incubator and culturing room can be cultivated with pester bottle (500mL) or plastics ZP4 bottle (500ml) or polypropylene plastics pocket.Open culture container available metal or ceramic tray, but requirement for height is about 6cm.Open culturing room requires to have equipment such as warm and humid regulation and control and ventilation (sterile filtration), illumination, and is convenient to sterilization, and cleanliness factor reaches 100 grades.

2, cultural method and step

With inclined-plane kind inoculation first order seed, and the cultivation of 500ml triangular flask shaking table (72h, 110-120r/min); The preparation of secondary seed employing 10L seeding tank (24 ℃, tank pressure 0.04-0.05Mpa, the about 0.75m of air flow quantity ³/ h cultivates 36h); Adopt the preparation of 70L seeding tank (24 ℃ of about 0.05Mpa of tank pressure, air flow quantity 1.3-1.6m ³/ h) cultivate 36h, just can be used to inoculation.Inoculum size is that 1-5% puts indoor cultivation then, and early stage, the culture temperature may command was 24-25 ℃, should be controlled at 20-22 ℃ after coremium starts, and cultivated 20-30 days, can gather.

When doing open cultivation with koji tray, sterilization back culture material controllable thickness is built in about 1.5-3.0cm, and inoculum size is 5-20%.The wet gauze of two layers of sterilization of inoculation back charge level lid covers the polypropylene film that one deck was sterilized again on the gauze.Whole culturing process will be noted preserving moisture and keeping clean.Coremium growth beginning after date can be opened gauze and polypropylene film (inoculation one week of back), and 20-25 days beginnings gathered, and the back coremium of gathering dries or 80 ℃ of oven dry.

B, liquid culture fermentation process

1, culture condition:

(1) bacterial classification seed preparation: the same solid culture of method for making;

(2) substratum:

Inclined-plane mother culture media: PSA or PDA substratum: potato 200g, sucrose (or glucose) 20g, agar 20g, water 1000ml PH nature;

First order seed substratum: PSA cultivates liquid nutrient medium: potato 200g, sucrose 20g, water 1000ml;

Secondary seed medium: PSA nutrient solution: potato 200g, sucrose 20g, water 1000ml;

Produce fermention medium: potato 20%, fructose 2%, peptone 0.1%, aspartic acid 0.01%, KH ₂PO ₄0.15%, MgSO ₄0.05%, vegetables oil 0.1-0.15%, water 77.54-77.59%;

(3) inoculum size of inoculum size first order seed or secondary seed access secondary medium or production fermented liquid is 10%;

(4) the leavening temperature variation of temperature is more obvious to mycelial growth effect, all can grow for 18-34 ℃, and wherein 23-26 ℃ of growth is better, and preferably 24-25 ℃, the best is 24 ℃, and mycelial growth is the fastest, dry weight height (seeing Table 3);

Table 3 leavening temperature is to the influence of Paecilomyces cicadae mycelial growth

(5) pH value APC-20 bacterial strain all can be grown by normal growth at PH 4-12, and preferred PH is 6.5, and this scope mycelium dry weight output is the highest; Also can adopt the nature pH value, be convenient to production operation;

(6) fermentation time is an index with the mycelia yield, puts jar selection of time and be optimum about 84h.

2, cultural method and step:

Adopt the 70L canned 52.5L fermentation culture that ferments, wherein add defoamer vegetables oil 0.1-0.15%, 121 ℃ of sterilization 30min postcooling to 24 ℃ insert cultured secondary seeds, control fermentation condition (the about 0.05Mpa of tank pressure, the about 1.3-1.6m of air flow quantity ³/ h, about 24 ℃ of temperature), ferment and put jar after 84 hours.

C, above solid culture coremium and liquid culture mycelium and natural cicada fungus major ingredient are done following comparative analysis:

1, the glycitols composition is relatively through silica gel G TLC chromatography, and with Virahol: ethyl acetate: water (83: 11: 6) be developping agent, and the pure liquid of 0.5% potassium permanganate and 0.5% p-diaminodiphenyl is developer, and the macula lutea of the blue end of the two appearance is selected consistent with the Rf value of N.F,USP MANNITOL.

2, the organic acid composition is relatively through the ply of paper chromatography, and with propyl carbinol: Glacial acetic acid: water (3: 1: 1) be developping agent, and tetrabromo-mcresolsulfonphthalein is a developer, and culture yellow the putting than natural product in the blue end occur and Duos 3 spots.If with propyl carbinol: ethanol: ammoniacal liquor: (4: 1: 2: 2) be developping agent, tetrabromophenol sulfonphthalein was a developer to water, the spot that both occur, Rf value unanimity.

3, alkaloids is relatively through silica gel G TLC chromatography, and with chloroform: methyl alcohol (9: 1) is developping agent, orange red spot than natural product few 1 occurs.If with propyl carbinol: ethanol: water: (4: 1: 2: 2) be developping agent, both spots of appearance were consistent with the Rf value for ammonia.

4, the sterols composition is relatively through silica gel G TLC layer, and with the rearmounted 110 ℃ of heating of aubepine acetic acid sulfuric acid liquid colour developing 10 minutes, both showed that color spot is no less than 10, its spot colors and the equal basically identical of Rf value.

In addition, the both is contained 15 kinds of same hydrolysis amino acid and 12 kinds of trace elements.This shows that Paecilomyces cicadae artificial culture thing and natural cicada fungus Chemical Composition are basic identical, all contain effective ingredients such as fats, protein, amino acid, sterols, organic acid and alkaloid.

A kind of analgesic compounds that from the new strains A PC-20 artificial fermentation of Paecilomyces cicadae product, extracts, comprise and from coremium, mycelium and the meta-bolites thereof of solid and/or liquid culture fermentative production and solid culture based mixtures and/or mycelium and meta-bolites and liquid culture based mixtures, extract the compound that obtains with analgesic activities, the pure product CYDd-2-A of this compound through RP-HPLC analyze that (Shanghai Pharmaceutical Inst., Chinese Academy of Sciences), INFRARED SPECTRUM, ESI high resolution, ESI electron spray(ES) are analyzed, H composes, ¹³C nuclear magnetic resonance spectrum, hydrocarbon relation (HMQC) nuclear magnetic resonance spectrum (Shanghai Organic Chemistry Institute, Chinese Academy of Sciences) and fourier transformation (FTMS) interpretation of mass spectra, clearly this compound chemistry name is called N ⁶-(2-hydroxyethyl) adenosine [N ⁶-(2-hydroxyethyl)-adenosine], 99% analgesia rate (measuring with the small white mouse writhing method) is arranged, have following physicochemical property:

(1) molecular weight: 311.297;

(2) accurate molecular weight: 311.12297;

(3) molecular composition: C 46.30%; H 5.50%; N 22.50%; O 25.70%;

(4) molecular formula: C ₁₂H ₁₇N ₅O ₅

(5) outward appearance: white powder;

(6) UV spectrum: λ max=213.5nm, 267nm (log ε=4.19,4.20) [in the ethanol];

(7) infrared spectra: IR bands, 3300cm ^-1(OH, NH) and 1625cm- ¹(-C=N-);

(8) fusing point: 193 ℃;

(9) proterties: methyl alcohol is cultivated needle;

(10) solvability: water-soluble, methyl alcohol;

(11) acid, neutral and alkaline distinguishing: weakly alkaline material;

(12) color reaction: sulfuric acid-4-hydroxyl-3-methoxylbenxaldehyde colour developing; Sulfuric acid: 5% phenol water (volume ratio 5: 1) colour developing; Triketohydrindene hydrate does not develop the color;

(13) thin-layer chromatography: Rf value 0.38, launch solvent: chloroform: methyl alcohol (volume ratio)=5: 1;

(14) ph stability: PH=3 is active constant;

(15) thermostability: 100 ℃ following 10 minutes, activity stabilized;

(16) enzyme stability: do not degraded by stomach en-;

(17) structural formula:

C ₁₂H ₁₇N ₅O ₅Exact?Mass：311.12Mol.Wt：311.29C，4630；H，5.50；N，22.50；O，25.70

2-[6-(2-Hydroxy-ethylamino)-purin-9-yl]-5-hydroxymethy

1-tetrahydro-furan-3，4-diol

In extracting above-mentioned analgesic compounds process, its each efficient part comprises that CYD, CYDd and CYDd-2 also all contain this compound, adopts small white mouse acetic acid twisting method to record the analgesia rate and reaches 95.25%, 97.81% and 98.20% respectively.

A kind of method of from the new strains A PC-20 artificial fermentation of Paecilomyces cicadae product, extracting analgesic compounds, comprise from coremium, mycelium and the meta-bolites thereof of solid culture and/or liquid culture fermentative production and solid culture based mixtures and/or mycelium and meta-bolites and liquid culture based mixtures and extract CYDd-2-A, step in the following order, condition is carried out:

(1) raw material pulverizing: solid feed or liquid raw material are ground into 200 order particle diameter particles after 80 ℃ of ventilation oven dry, standby;

(2) ethanol lixiviate: will pulverize raw material and 40% ethanol by 1: 3 (weightmeasurement ratio) dipping, and behind ultrasonic extraction and 85 ℃ of water reflux suction filtrations repeatedly, mix the filtrate of carrying, 70 ℃ of Rotary Evaporators are concentrated;

(3) solvent distributes: concentrated solution in 2: 1 ratios (volume), is used methylene dichloride (CH respectively ₂Cl ₂) ethyl acetate (Ethyl Acetate), propyl carbinol (n-Butyl Alcohel) distribute, CYA methylene dichloride position, CYB ethyl acetate extract, CYC n-butanol portion and CYD water position, wherein water position evaporate to dryness gets CYD;

(4) CYD removes polysaccharide: CYD is mixed by 1: 3 volume ratio with 95% ethanol, 3600 γ/min4 ℃ of centrifugal 15min, after getting supernatant liquor 1, in throw out, add the suitable quantity of water dissolving, precipitation is removed in centrifugal back, and the same method of supernatant liquor is repeated 2 times, gets supernatant liquor 2,3, merge supernatant liquor 1,2,3, the rotation evaporate to dryness is the CYDd behind the removal polysaccharide;

(5) CYDd resin isolation: adopt CDA-40 type macroporous resin, after 9cm * 1.0m pillar absorption, add water successively, 50% ethanol, 95% ethanol elution, wherein 50% ethanol eluate evaporate to dryness gets CYDd-2;

(6) CYDd-2 post layer separates: adopt Art.10626  post, elutriant: methyl alcohol: water=2: 8, get CYDd-2-A, CYDd-2-B, CYDd-2-C, and wherein 59～77 pipe amalgamation liquids are faint yellow (for the big peak of 3.932min) to be CYDd-2-A behind the evaporate to dryness;

(7) CYDd-2-A column chromatography purification Sephedex LH-20 Φ 2cm, long 50cm, solvent are methyl alcohol, get white powder behind the purifying evaporate to dryness, the final extraction yield that is this compound of the pure product CYDd-2-A. of analgesic compounds is 0.36%.(seeing example 7).

In extracting above-mentioned analgesic compounds process, its each efficient part can adopt same raw material, after 60～80 ℃ of oven dry, pulverizing, ethanol lixiviate, solvent can get CYD after distributing, remove polysaccharide, can get CYDd, reclaim through resin isolation, 50% ethanol again and promptly get CYDd-2.

Beneficial effect of the present invention, the one, it is strong that seed selection has obtained growth potential, artificial fermentation's culture (coremium, mycelium) output height, the new bacterial strain of Paecilomyces cicadae APC-20 of quality better; The 2nd, the present invention proposes and use optimization technology and the matching method that the APC-20 bacterial strain carries out artificial culture solid fermentation production sporophore (coremium), thereby realized artificial, anniversary, the target of producing the traditional famous and precious fungi Chinese medicine cicada fungus of (the highest individual plant coremium dry weight reaches 9.77 grams, and mean length reaches 18.92mm) best in quality uniform quality in enormous quantities; The 3rd, the present invention proposes and use optimization technology and the matching method that this bacterial strain carries out artificial culture liquid fermenting production mycelium and meta-bolites and nutrient solution mixture, make mycelium dry weight output bring up to the 2.76mg/200ml of APC-20 bacterial strain by the 1.51g/200ml of close bacterial strain, the highest 3.03mg/200ml that reaches, and liquid fermenting also has (3-5 days) with short production cycle, advantages such as quality is convenient to control (industrialized standard production), and is easy to operate; The contained glycitols of product, organic acid, alkaloids and the sterols that aforesaid method is produced all with natural cicada fungus basically identical; The 4th, the present invention first from Paecilomyces cicadae separation and Extraction go out N ⁶-(2-hydroxyethyl) adenosine [N ⁶-(2-hydroxyethyl)-adenosine], though it is identical that this compound and structural formula thereof and Tsutomu are reported, but the production bacterial classification difference of extracting, and found that first this compound has analgesic activity, this compound is to discharge the generation analgesic activity by having suppressed neurotransmitter with the d receptors bind, its mechanism is different from opioid analgesic commonly used at present, do not have habituation, pure product analgesia rate is up to 99.00%, each efficient part can be made into multiple formulation in these pure product and the leaching process, the analgesic of safety has bigger development prospect; The 5th, the Paecilomyces cicadae culture condition is 23-26 ℃, is low than the requirement of Cordyceps sinensis mushroom low temperature, high height above sea level, so a whole set of method of fermenting and extracting to analgesic compounds from spawn culture proposed by the invention, tool is reasonable in design, practical, characteristics such as yield is higher, and is with low cost.

Description of drawings

Fig. 1 analgesic compounds CYDd-2-A and each efficient part extraction process schematic flow sheet

Fig. 2 feed ethanol lixiviate schematic flow sheet

Fig. 3 CYD measures synoptic diagram to stomach en-stability

Fig. 4 CYD removes the polysaccharide schematic flow sheet

Fig. 5 CYDd-2-A RP-HPLC analysis of spectra

Fig. 6 CYDd-2 RP-HPLC analysis of spectra

Fig. 7 CYDd-2-A infrared spectrum

The ESI high resolution spectrogram of Fig. 8 CYDd-2-A

The ESI electron spray(ES) analysis of spectra of Fig. 9 CYDd-2-A

Figure 10 H spectrogram-1

Figure 11 H spectrogram-2

Figure 12 ¹³C nmr spectrum-1

Figure 13 ¹³C nmr spectrum-2

The hydrocarbon relation of Figure 14 (HMQC) nmr spectrum-1

The hydrocarbon relation of Figure 15 (HMQC) nmr spectrum-2

Figure 16 fourier transformation matter (FTMS) spectrogram

Embodiment

To be described in more detail the present invention by following examples, following examples only are illustrative, and the present invention is not subjected to the restriction of these embodiment.

Embodiment 1:(solid culture fermentation process " 1 ")

Get the APC-20 bacterial strain of preserving in 4 ℃ of refrigerators and be transplanted to and (annotate 1) on the slant medium and cultivated 7 days, activate stand-by; Every the bottled 200ml first order seed of 500ml triangle substratum (annotating 2) inserts slant strains spore powder 3 rings, puts interior 25 ℃ of DHZ-C vibrator, the 120r/min rotating speed, and cultivating 4 days is first order seed; Dress 7.5L secondary medium (annotating 3) in the 10L fermentor tank, 121 ℃ of sterilization 30min are cooled to 24 ℃, insert first order seed, 24 ℃, the about 0.04Mpa of tank pressure, the about 0.75m of air flow quantity by 10% inoculum size ³/ h, ferment 48 hours be secondary seed.After solid medium (annotate 4) and water mixed thoroughly by 1: 2, a kind of mode can directly be packed in the culture vessel, and charging accounts for 2/5 of capacity and is advisable, and puts in the high pressure steam pot, through 121 ℃, after 1.5 hours, be cooled to 20 ℃ 0.11Mpa sterilize, the inoculum size by 1.5% inserts carries out closed indoor cultivation after secondary seed is mixed thoroughly, early stage, temperature kept 24 ℃, treat to reduce to 21 ℃ after coremium starts, cultivated 20-30 days, can gather; Another kind of mode can be with in the high about 6 centimeters koji tray in limit of packing into the solid medium after the method sterilization, (expecting thick 3 centimeters), after mixing thoroughly by 20% inoculum size inoculation, topped two layers of sterilization wet gauze and one deck polypropylene film are put through sterilization, cleaning with the vaporization prevention of preserving moisture on charge level, the bright indoor open cultivation of carrying out, temperature is the same, and coremium starts after the week, opens gauze and film, cultivate and to gather in 20-25 days, dry after gathering or 80 ℃ of oven dry.

Annotate 1: slant medium, adopt PSA or PDA substratum: potato 200g, sucrose 20g, agar 20g, water 1000ml PH nature;

Annotate 2: first order seed PSA nutrient solution, potato 200g, sucrose 20g, water 1000ml;

Annotate 3: secondary seed PSA nutrient solution, potato 200g, sucrose 20g, water 1000ml add vegetables oil 1-1.5ml;

Annotate 4: the solid fermentation substratum, wheat bran, corn, cavings were by 3: 2: 2 composition by weight proportionings.

Embodiment 2:(solid culture fermentation process " 2 ")

The solid fermentation substratum of present embodiment is a wheat bran: corn: cavings was by 2: 1: 1 proportionings; Solid medium siccative and water are mixed thoroughly in 1: 2.9 composition by weight ratio;

The second class inoculum inoculum size that the container closure formula is cultivated is 3%; The second class inoculum inoculum size of the open cultivation of koji tray is 10%;

The substratum of present embodiment and all the other technology and step all are same as embodiment 1.

Embodiment 3:(solid culture fermentation process " 3 ")

With the wheat wheat is solid medium, wheat is soaked in water spends the night earlier, cooks (until the wheat cracking), and pack into container or koji tray account for all that holding accumulates score of three one is advisable; Secondary or three-class strain inoculum size that the container closure formula is cultivated are 3%; The bacterial classification inoculation amount of the open cultivation of koji tray is 10%; All the other culture process and step all are same as embodiment 1.

Embodiment 4:(liquid culture fermentation process " 1 ")

The inclined-plane of present embodiment strain preparation, I and II seed culture medium and method are same as embodiment 1 substantially, only add vegetables oil 0.1% as defoamer in secondary seed medium; Its fermention medium also is same as secondary seed medium;

Present embodiment adopts the 70L canned 52.5L fermentation culture that ferments, PH4.5, and 121 ℃ of sterilization 30min postcooling to 24 ℃ insert cultured secondary seed by 10% inoculum size, the about 0.05Mpa of control tank pressure, air flow quantity 1.3m ³/ h, 24 ℃ of temperature are fermented and are put after 84 hours jar.

Embodiment 5:(liquid culture fermentation process " 2 ")

All add 0.15% vegetables oil in present embodiment secondary seed medium and the production fermentation culture and make defoamer; PH is 6.5; Temperature is controlled at 26 ℃; Air flow quantity is 1.6m ³/ h;

All the other technologies and the step of present embodiment all are same as embodiment 4.

Embodiment 6:(liquid culture fermentation process " 3 ")

The inclined-plane of present embodiment strain preparation, I and II seed culture medium and method are same as embodiment 1 substantially, and producing fermention medium is potato 20%, fructose 2%, peptone 0.1%, aspartic acid 0.01%, KH ₂PO ₄0.15%, MgSO ₄0.05%, vegetables oil 0.1-0.15%, water 77.54-77.59%.

All the other technologies and the step of present embodiment all are same as embodiment 4.

The pure product extracting method 1 of embodiment 7:(analgesic compounds)

The entire method step is undertaken by shown in Figure 1, and the Paecilomyces cicadae product (coremium or mycelium and meta-bolites thereof and substratum mixture) with solid and/or liquid fermenting through 60～80 ℃ of oven dry, is ground into 200 order particles (525g weighs);-→ will pulverize raw material and 40% ethanol is in ratio lixiviate in 1: 3, (lixiviate flow process such as Fig. 2), merging filtrate 1,2,3,70 ℃ of simmer down to 1800ml of Rotary Evaporators;-→ with concentrated solution used methylene dichloride (CH respectively in 2: 1 ratios (volume) ₂Cl ₂) ethyl acetate (Ethyl Acetate), propyl carbinol (n-ButylAlcohel) distribute, CYA methylene dichloride position, CYB ethyl acetate extract, CYC n-butanol portion and CYD water position, evaporate to dryness carries out the analgesia rate and measures respectively;-→ water position CYD is removed polysaccharide, instrument: TH-1 type gradient mixer (the dynamo-electric factory of Shanghai Hu Xi), LC-6 whizzer (Shanghai centrifugal machine institute), reagent: ethanol, ether and acetone are homemade analytical pure (flow process is seen Fig. 3), CYDd after getting CYDc (polysaccharide) and removing polysaccharide carries out the analgesia rate and measures; → CYDd is carried out macroporous resin to be separated, adopt CDA-40 type macroporous resin Nanjing University of Chemical Technology to produce, application of sample 90g is after 9cm * 1.0m pillar absorption, add water successively, 50% ethanol, 95% ethanol elution, get CYDd-1 65.904g behind the evaporate to dryness respectively, CYDd-211.094g, CYDd-3 0.494g, and carry out analgesic activities and measure;-→ CYDd-2LOBAR the post is separated: adopt Art.10626  post, the automatic Fraction Collector of BSZ-100,1 pipe/3min, the 15ml/ pipe, pressure 500psi, flow velocity 8ml/min, elutriant: methyl alcohol: water=2: 8, making RP-HPLC every 10 pipes detects, identical merging gets CYDd-2-A, CYDd-2-B, CYDd-2-C, and wherein CYDd-2-A is 59～77 pipe amalgamation liquids, be faint yellow behind the evaporate to dryness, be the big peak of 3.932min; CYDd-2-B is backlash Xian Zhu subdivision; CYDd-2-C is 1～56 pipe, and the big peak of 3.932min fore portion is measured CYDd-2-A through the analgesia rate and risen to 99.0%;-→ with CYDd-2-A Sephedex LH-20 purifying Sephedex LH-20 Φ 2cm, long 50cm, solvent are methyl alcohol gets white powder 1.892 grams behind the purifying evaporate to dryness, be the pure product of analgesic compounds, and it extracts yield is 0.36%.

Respectively the ease pain extracting method of efficient part of embodiment 8:()

The extracting method of each efficient part that eases pain adopts embodiment 7 same raw materials, after 60～80 ℃ of oven dry, pulverizing, ethanol lixiviate, solvent distribute CYD, get CYDd after removing polysaccharide, reclaim through resin isolation, 50% ethanol and promptly get CYDd-2, each step concrete grammar is with embodiment 7 (seeing accompanying drawing 1).

The embodiment 9:(capsular manufacturing of easing pain)

With CYD, CYDd, CYDd-2 or CYDd-2-A is raw material, and 100 orders were pulverized in 80 ℃ of oven dry, evenly add appropriate amount of starch (100 order), behind the survey content, with capsule machine packing (No. 5 capsule shell), make and make the analgesia capsule and contain CYDd-2-A 50mg/ grain, the qualified back packing of inspection after construction.

The manufacturing of embodiment 10:(tablet)

With bulk drug CYDd-2-A, and Microcrystalline Cellulose, the lactose separated pulverizing is crossed 100 orders, progressively increases above-mentioned three component mixings by equivalent, make 16 order particles with 8% starch slurry mixing, air seasoning 3-6 hour (in the moisture 5%), be the whole grain of 14 orders after, add sodium starch glycolate (80 order), Magnesium Stearate (80 order) mixes, survey the content backlash and be pressed into tablet, gross weight 1.5g/ grain contains effective composition 50mg/ grain, the qualified back of inspection after construction packing.

The manufacturing of embodiment 11:(injectable powder)

Under class 100 clean environment: CYDd-2-A is dissolved in water with medicine material, adds the pin bioactive peptide, sterile filtration, and spraying drying was pulverized 300 mesh sieves, was sub-packed in the aseptic vial of removing thermal source, and the 100mg/ bottle is jumped a queue immediately, the aluminium lid sealing.Use the Injectable sterile water dissolution during use.

Test example 1 (each efficient part of analgesic activities and compound are to the test of animal analgesia rate)

Laboratory animal: Kunming small white mouse male and female respectively account for 50%, and about body weight 25g/ head, the unified raising 3-4 days provided by the Shanghai Pharmaceutical Inst., Chinese Academy of Sciences animal testing center.

Analgesia measuring method: adopt writhing method, treatment dosage is 5g crude drug/Kg body weight, drug administration by injection (Sc), and pneumoretroperitoneum was injected 0.7% acetate in 30-40 minute, turn round the body number in the record small white mouse 10 minutes, calculate the inhibiting rate of the pain writhing response that 0.7% acetate is produced.

(1) the extract solvent distributes analgesia rate in back to measure:

By table 4 as seen, solvent distributes the back analgesic activities mainly to concentrate on the CYD position.

Table 4 different solvents position is to the analgesic activity (writhing method) of small white mouse

Sample	Dosage (g crude drug/K body weight)	Small white mouse number (head)	Analgesia inhibiting rate (%)
				Physiological saline CYA (methylene dichloride position) CYB (ethyl acetate extract) CYC (n-butanol portion) CYD (water position)	????-Sc ????5Sc ????5Sc ????5Sc ????5Sc	????18 ????8 ????8 ????8 ????8	?????- ????23.21 ????33.33 ????58.40 ????95.25 **

Annotate: ^*P＜0.01 level is remarkable; Physiological saline is contrast;

(2) the analgesia rate behind the CYD removal polysaccharide is measured:

By table 5 as seen, it is the highest to go CYD behind the polysaccharide to be that CYDd analgesia rate shows, reach 97.81%, and polysaccharide CYDc analgesia rate is 0.

Table 5CYDa～d is to the analgesic activity of small white mouse

Sample	Dosage (g crude drug/K body weight)	Small white mouse number (head)	Analgesia rate inhibiting rate (%)
				Physiological saline CYDa (pepsin) CYDb (Buffer processing) CYDc (polysaccharide) CYDd (removal polysaccharide)	????-Sc ????5Sc ????5Sc ????5Sc ????5Sc	????18 ????8 ????8 ????8 ????8	?????- ????95.10 **????96.30 **????0.00 ????97.81 **

Annotate: ^*P＜0.01 level is extremely remarkable

(3) mensuration of CYDd analgesia rate after resin isolation:

CYDd after resin isolation, CYDd-2 (50% ethanol position) analgesia rate the highest (seeing Table 6) wherein.

(4) the CYDd-2 post is analysed the mensuration of analgesia rate behind the purifying:

Table 6CYDd-2～CYDd-2-C is to the analgesic activity (writhing method) of small white mouse

Sample	Dosage (the g crude drug/Kg)	Small white mouse number (head)	On average turn round the body number	Analgesia presses down agent rate (%)
					Physiological saline CYDd-2 (after the resin isolation) CYDd-2-A (behind the purifying) CYDd-2-B CYDd-2-C	????-Sc ????5Sc ????5Sc ????5Sc ????5Sc	????18 ????8 ????8 ????8 ????8	????25.80 ????4.33 ????0.25 ????23.70 ????33.70	?????- ????98.20 **????99.00 **????8.10 ????0.00

Annotate: ^*P＜0.01 level is remarkable; Physiological saline is contrast.

Table 6 shows that the CYDd-2-A analgesia rate behind the purifying reaches 99%, illustrates that this analgesic compounds purity is high more, and activity is high more.

Test example 2 (analgesic compounds is to stomach en-, trypsinase and acid stable test)

Stomach en-is purchased the HyClone-PIERCE company in the U.S..

The Buffer preparation of PH=3: citric acid (0.1M 18.6ml)+Trisodium Citrate (0.1M 1.4ml).

Experiment process such as Fig. 4.

By table 5 as seen, CYD is not subjected to pepsic the influence, Pepsin PH4, and 30 ℃ of reaction 12h do not change analgesic activities, and CYDa analgesia rate still reaches 95.10%, and acid (PH3) down is stable, i.e. and CYDb analgesia rate reaches 96.30%.

Claims (8)

1, the new strains A PC-20 of a kind of Paecilomyces cicadae (CGMCC NO:1104) is characterized in that the artificial culture thing and the natural cicada fungus Chemical Composition that produce by solid and/or liquid fermenting are basic identical, and can improve its coremium and/or mycelial output.

2, Paecilomyces cicadae APC-20 bacterial strain carries out the method for artificial solid and/or liquid culture fermentation, it is characterized in that cultivating Paecilomyces cicadae strains A PC-20 (CGMCC NO:1104) through inclined-plane and I and II seed in solid and/or liquid nutrient medium, wherein

Solid fermentation is cultivated with pure wheat or wheat bran (2-4): corn (1-3): cavings (1-3) weight part ratio is mixed with substratum; closed container bacterial classification inoculation amount is 1-5%; the open container inoculum size is 5-20%; the mycelial growth stage is 24-25 ℃; coremium starts the back for 20-22 ℃; PH4.5-6.5, light or natural light grow 20-30 days down until the coremium of gathering.

Liquid fermentation and culture inserts fermentation tank culture medium by 10% bacterial classification amount: potato 20%, fructose 2%, peptone 0.1%, aspartic acid 0.01%, KH ₂PO ₄0.15%, MgSO ₄0.05%, surplus is a water, leavening temperature 23-26 ℃, and the about 0.05Mpa of tank pressure, air flow quantity 1.3-1.6m ³/ h, PH4-12, the 48-120 hour mycelium of gathering ferments.

3, according to the described method of claim 2, it is characterized in that described solid fermentation culture medium prescription is a wheat bran 2: corn 1: cavings 1 also adds 2% fructose, and closed container bacterial classification inoculation amount is 3%, and the open container inoculum size is 10%, and PH is 5.8; Described liquid fermentation and culture, culture temperature are 24-25 ℃, and the best is 24 ℃, and PH is 4.5-6.5, and best fermentation time is 84 hours.

4, a kind of Accessory Right requires the analgesic compounds that extracts in the 2 described method cultured products, it is characterized in that this compound extracts from coremium, mycelium and the meta-bolites thereof of artificial solid of Paecilomyces cicadae strains A PC-20 and/or liquid culture fermentative production and solid culture based mixtures and/or mycelium and meta-bolites and liquid culture based mixtures obtains, and this compound name is called N ⁶-(2-hydroxyethyl) adenosine, its pure product CYDd-2-A molecular weight is 311, molecular formula is C ₁₂O ₅H ₁₇N ₅, be not subjected to pepsicly to influence, acid stable (PH=3), thermally-stabilised (100 ℃).

5, by the described analgesic compounds of claim 4, it is characterized in that the pure product CYDd-2-A of this compound, each efficient part in leaching process comprises that CYD, CYDd and CYDd-2 also all contain this compound and possess the analgesic activities that does not wait.

6, the extracting method of the described analgesic compounds of a kind of claim 4, it is characterized in that with the artificial solid of Paecilomyces cicadae strains A PC-20 and/or liquid culture tunning coremium, mycelium and meta-bolites thereof and solid culture based mixtures and/or mycelium and meta-bolites thereof and liquid culture based mixtures be raw material, distribute, remove polysaccharide, resin isolation recovery, column chromatography purification through 60～80 ℃ of oven dry, pulverizing, ethanol lixiviate, solvent, obtain the pure product of white powdery of analgesic compounds CYDd-2-A.

7, press the extracting method of the described analgesic compounds of claim 6, it is characterized in that described each efficient part adopts same raw material, after 60～80 ℃ of oven dry, pulverizing, ethanol lixiviate, solvent distribute, get CYD, get CYDd behind the removal polysaccharide, reclaim through resin isolation, 50% ethanol again and promptly get CYDd-2.

8, the analgesic that utilizes claim 4 or 5 described analgesic compounds to be prepared from is characterized in that this medicine comprises capsule, tablet and pulvis.

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