CN101280326B - Preparation and application of compound for inhabiting aspergillus fumigatus activity - Google Patents
Preparation and application of compound for inhabiting aspergillus fumigatus activity Download PDFInfo
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- CN101280326B CN101280326B CN200810106546XA CN200810106546A CN101280326B CN 101280326 B CN101280326 B CN 101280326B CN 200810106546X A CN200810106546X A CN 200810106546XA CN 200810106546 A CN200810106546 A CN 200810106546A CN 101280326 B CN101280326 B CN 101280326B
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- siterui
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Abstract
The present invention relates to a methyl asterrate preparation method and the application thereof. The solid fermentation is performed to Fungi Geomyces sp, chemical compound methyl asterrate can be obtained by performing purification to fermentation products, and the chemical compound methyl asterrate prepared by the invention is confirmed methyl asterrate by applying technologies such as mass spectrum, nuclear magnetic resonance spectrum and infrared spectrum and documents. The present invention also provides usages of the chemical compound in preparing drugs which can restraint the activity of aspergillus fumigatus.
Description
Technical field
The present invention relates to medical technical field, in particular to the preparation method of compound methyl Ai Siterui (methyl asterrate), and the application of compound methyl Ai Siterui in the medicine of preparation inhibition activity of Aspergillus fumigatus.
Background technology
Aspergillus fumigatus is the deuteromycetes Eurotium, is the condition pathomycete of a kind of serious harm industrial and agricultural production and human health.In wet environment, Aspergillus fumigatus can infect cereal such as corn, dregs of beans, causes the decline of flat food output and quality; Aspergillus fumigatus also can infection animal, and the toxin that Aspergillus fumigatus produced can cause horse alba malacosis, causes that poultry loses weight, diarrhoea, feed efficiency is low and hepatic necrosis, causes pancreas necrosis, liver injury and the pulmonary edema etc. of pig.Visible smoke aspergillus brings very big harm can for human industrial and agricultural production.In addition, along with the widespread use of high-efficiency broad spectrum microbiotic, adrenocortical hormone, immunosuppressor clinically, and AIDS, organ-tissue transplant and the increasing gradually of immunocompromised host patients such as chemotherapy, and it is serious day by day to make that the condition pathomycete infects, and human lives's health has faced significant threat.Aspergillus fumigatus brings significant threat can for human lives's health.But the report that the Aspergillus fumigatus infected person was just arranged as far back as 1848, it remains important clinically condition pathomycete at present, and compared with 1848, its infection rate has risen about 14 times, surpassed candidiasis become modal in the world fungi infestation disease [referring to, Nature (nature) .2005,438,1105-1115].
If be widely used in the drug main polyenoid class medicine amphotericin B of treatment infection by Aspergillus fumigatus at present clinically; The wide spectrum triazole type medicine is as itraconazole, voriconazole; Echinocandin class medicine Caspofungin (caspofungin).Amphotericin B has very strong toxicity, can cause Gao Re and renal toxicity, so limited its clinical application.Though azole drug and echinocandin class medicine have security preferably, continue use and can bring out the appearance of Resistant strain.For example, be that the third generation antifungal drug of representative is the choice drug for the treatment of aspergillus fumigatus infection at present clinically with the fluconazole.Fluconazole belongs to imidazoles, mainly be to play a role by competitive inhibition fungi lanosterol 14 α demethylases, make that lanosterol is accumulated, the membrane structure function forms the synthetic shortage as ergosterol, cause many enzyme activity changes on fungi membrane permeability and the film, thereby suppress fungi growth.But the imidazoles antifungal drug can act on simultaneously 3A in a plurality of cytochrome P 450 albumen, particularly human cell's cytochrome p 450 enzyme of human body
4Enzyme, and the 3A in the Cytochrome P450
4And 2C
8～2C
10Enzyme is an enzyme main in the adult human liver, therefore, use fluconazole in a large number after, can cause than serious adverse effects, its adverse reaction rate is up to 10%～16%.Therefore, seek infection by Aspergillus fumigatus effectively preventing method and medicament, extremely urgent [referring to, Chinese prescription drugs .2004,3,69-71].
The methyl Ai Siterui that the present invention relates to (methyl asterrate) structural formula is as shown in the formula shown in (I):
Wherein Me is a methyl.
At home and abroad in the patent documentation report, all do not relate to compound methyl Ai Siterui and can suppress the active report of Aspergillus fumigatus (Aspergillis fumigatus).
In document " New Chlorinated Diphenyl Ethers from an Aspergillus Species (from a strain aspergillus tubigensis, separating the chloride diphenyl ether compound that obtains); Journal of NaturalProducts (natural product magazine) .2002; 65; 7-10 ", the author passes through nuclear magnetic resonance spectrum, the structure of the active compound methyl Ai Siterui in the patent of the present invention has been determined in the X-ray crystal analysis, and tested it to intestinal bacteria (E.coli.), the anti-microbial activity of phytopathogen gaeumannomyce bacterium (Gaeumannomycesgraminis vartritici) and dry thread Pyrenomycetes (Rhizoctonia solani), the result shows, all the bacterial strains all unrestraint activity of this compound to being tested.Wen Zhongwei carries out active testing with this compound to this class pathogenic fungi of Aspergillus fumigatus (Aspergillis fumigatus).
At document Fungal metabolites, asterric acid derivatives inhibit vascularendothelial growth factor (VEGF)-induced tube formation of HUVECs (what derive from fungi has inhibiting asterric acid derivative to vascular endothelial growth factor (a kind of factor that can cause the human umbilical vein blood cell proliferation)) .J.Antibiot. (microbiotic magazine) 2002,55, in 552, the author has reported that compound methyl Ai Siterui has the activity of inhibition to VEGF, does not equally also have compound to suppress the activity report of Aspergillus fumigatus (Aspergillis fumigatus).
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing microbial fermentation to prepare methyl Ai Siterui, and methyl Ai Siterui is provided the application in the medicine of preparation inhibition activity of Aspergillus fumigatus.
The inventor has found that the solid fermentation extract of producing bacterial strain Geomyces sp. has Aspergillus fumigatus inhibition activity on the high flux screening basis of microorganism secondary metabolite active compound; By the further separation and purification under the active tracking instruction, prepared this active compound methyl Ai Siterui.Compare with other class anti-aspergillus fumigatus compound of patent documentation report, compound methyl Ai Siterui has the activity of significant inhibition Aspergillus fumigatus.
Particularly, the present invention thes contents are as follows:
The invention provides the method for a kind of Geomyces of utilization sp. fermentative preparation as shown in the formula the methyl Ai Siterui shown in (I),
Wherein Me is a methyl, and described method is characterised in that and comprises the following steps:
A) Geomyces sp. is carried out solid fermentation;
B) after the solid fermentation of step a), in cultured products, add organic solvent extraction, obtain extracting solution, with described extracting solution underpressure distillation, obtain crude extract;
C) described crude extract is reduced pressure silica gel column chromatography and gel chromatography separation and obtain active component solution, described active component solution is separated the compound that obtains formula (I) with high performance liquid chromatography.Wherein the production bacterial strain of methyl Ai Siterui is Geomyces sp., this strains separation is from South Pole pedotheque, and, being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 05 07th, 2008, deposit number is CGMCC No.2481.
In aforesaid the inventive method, those skilled in the art be to be understood that described organic solvent comprise multiple water-fast at room temperature be liquid organic solvent, such as chloroform, ethyl acetate, benzene,toluene,xylene etc., preferably, described organic solvent is an ethyl acetate.
The present invention also provides the compound methyl Ai Siterui shown in the following formula (I) purposes in the active medicine of preparation inhibition Aspergillus fumigatus (Aspergillis fumigatus),
Wherein Me is a methyl.
The present invention also provides the purposes of compound methyl Ai Siterui in the active test kit of preparation inhibition Aspergillus fumigatus (Aspergillis fumigatus) of formula (I).Those skilled in the art are to be understood that, can utilize routine techniques in the medicament production field that the compound methyl Ai Siterui of formula (I) is used for producing and suppress the active test kit of Aspergillus fumigatus (Aspergillis fumigatus), the effective constituent in this test kit is the compound methyl Ai Siterui of formula (I).
The invention provides the method for utilizing Geomyces sp. fermentative preparation methyl Ai Siterui, and provide methyl Ai Siterui preparing the purposes that suppresses in the active medicine of Aspergillus fumigatus (Aspergillis fumigatus), compare with other anti-aspergillus fumigatus compound of bibliographical information, compound methyl Ai Siterui has the effect of significant inhibition activity of Aspergillus fumigatus.The present invention provides candidate compound for the new anti-aspergillus fumigatus medicine of research and development.
Culture presevation
The used bacterial classification Geomyces sp. of the present invention was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 05 07th, 2008, and deposit number is CGMCCNo.2481.
Embodiment
Embodiment 1: the preparation of compound methyl Ai Siterui
A. the solid fermentation of bacterial strain Geomyces sp.
The activation of bacterial strain Geomyces sp..The PDA substratum: potato 200g, glucose 20g, agar 15g, water 1000mL, 121 ℃ of high pressure steam sterilization 30min make the test tube slant, and the picking mycelium is inoculated on the test tube slant, cultivates 7 days for 25 ℃;
The solid fermentation of bacterial strain Geomyces sp..The preparation of rice medium (100g rice and 100mL water are added in the 500mL triangular flask, soaked overnight, 121 ℃ of high pressure steam sterilization 30min cool off stand-by); (spore concentration is 1 * 10 with the bacteria suspension for preparing
6Individual/mL) 5mL is inoculated on the rice medium, between 25 ℃ of sterile culture, cultivated 40 days.
B. the extraction of methyl Ai Siterui
Treat that fermentation finishes, ethyl acetate is added in the triangular flask extract three times, the organic solvent underpressure distillation to dry, is obtained the 5.0g crude extract;
C. the separation and purification of crude extract
With crude extract with normal hexane-methylene chloride-methanol system silica gel column chromatography that reduces pressure, the order ascending according to polarity is divided into 20 components with crude extract, follow the tracks of under (antibacterial method of testing is tested the inhibition activity of 20 components to Aspergillus fumigatus respectively with the scraps of paper) guidance in activity, discovery is at methylene dichloride: the component of methyl alcohol=as moving phase wash-out obtain at 200: 1 has and suppresses active, collects active eluant (400mg).
Active eluant is passed through gel chromatography separation, the gel column filler is Sephadex LH-20, moving phase is methylene dichloride: methyl alcohol=1: 1, the order descending according to molecular weight is divided into 10 components with component, then, test the inhibition activity of 10 components respectively by the antibacterial method of testing of the scraps of paper again, obtain the 150mg active ingredient Aspergillus fumigatus.
Active ingredient is obtained 50mg active compound methyl Ai Siterui by the HPLC purifying, and wherein the condition of HPLC is: adopt Agilent 1100 types partly to prepare high pressure liquid chromatograph, AgilentC
18(as moving phase, preparation condition: 47% acetonitrile/water isocratic elution 20min obtains the retention time t of methyl Ai Siterui to anti-phase half preparative chromatography post with acetonitrile and water for 10 μ m, 10 * 250mm), flow velocity 2.0mL/min
R=16.8min.
The methyl Ai Siterui of method for preparing is carried out infrared spectra and nmr analysis, and its middle infrared spectrum provides test by chemistry institute of Peking University, and nuclear magnetic resonance spectrum provides test by institute of Materia Medica,Chinese Academy of Medical Sciences.The physico-chemical constant of the methyl Ai Siterui of the present invention's preparation, hydrogen spectrum and carbon spectrum data see Table 1 and 2 respectively.
The physico-chemical constant of table 1. compound methyl Ai Siterui
The hydrogen spectrum of table 2. compound methyl Ai Siterui and carbon spectrum data (DMSO-d
6)
Compare with the data of being reported in the document " New Chlorinated Diphenyl Ethers from an AspergillusSpecies (from a strain aspergillus tubigensis, separating the chloride diphenyl ether compound that obtains); Journal ofNatural Products (natural product magazine) .2002; 65; 7-10 ", confirm that the product that obtains is methyl Ai Siterui.
To sum up, the 5.0g crude extract obtains 50mg pure compound methyl Ai Siterui through separation and purification, and its purity is more than 99%, and yield is 1%.
Embodiment 2: adopt Alamar Blue fluorescence detection to detect methyl Ai Siterui for the active inhibition of Aspergillus fumigatus (Aspergillis fumigatus)
1. test and use reagent:
The methyl Ai Siterui of the present invention preparation is prepared 4,2,1,0.5,0.2,0.1,0.05,0.02,0.01,0.005 and the solution to be measured of 0.002mg/mL with methyl-sulphoxide (DMSO) respectively as solvent;
Positive control fluconazole (purchasing the company in Sigma) is mixed with the solution of 4mg/mL with DMSO;
2. the cultivation of Aspergillus fumigatus (Aspergillis fumigatus)
With Aspergillus fumigatus (Aspergillis fumigatus 48238E, this bacterium is provided by DSMZ of Institute of Microorganism, Academia Sinica) be seeded in PDB substratum (starch 40g/L, glucose 20g/L) on, after 28 ℃ of shaking tables are cultivated 2 days and at microscopically meter spore count, adjust the spore number 1 * 10
5Individual/mL.
3. test method
Test conditions is as shown in table 3.
Table 3 active testing is volume required
| Negative control | Blank | Positive control | Soup to be measured | |
| The PDB substratum | 190μL | |||
| The Aspergillus fumigatus nutrient solution | 190μL | 190μL | 190μL | |
| Methyl Ai Siterui solution | 1μL | |||
| Fluconazole solution (4mg/mL) | 1μL | |||
| Alamar Blue dye liquor | 10μL | 10μL | 10μL | 10μL |
By method as shown in table 3, get 190 μ L PDB substratum and 10 μ L Alamar Blue dye liquors and join in 96 orifice plates and be made into negative control group; Getting 190 μ L Aspergillus fumigatus nutrient solutions and 10 μ L AlamarBlue dye liquors joins and is made into the blank group in 96 orifice plates; Get 190 μ L Aspergillus fumigatus nutrient solutions, the fluconazole of 10 μ L Alamar Blue dye liquors and 1 μ L 4mg/mL joins in 96 orifice plates and is made into positive controls; The methyl Ai Siterui solution (concentration be respectively 4,2,1,0.5,0.2,0.1,0.05,0.02,0.01,0.005 and 0.002mg/mL) of getting 1 μ L the present invention preparation joins respectively in 96 orifice plates, add 190 μ L Aspergillus fumigatus nutrient solutions more respectively, 10 μ L Alamar Blue dye liquors make the ultimate density for test agent be respectively 20,10,5,2.5,1.0,0.5,0.25,0.1,0.05,0.025 and 0.01 μ g/mL.Treat that soup and bacteria suspension are mixed evenly, under 28 ℃ and dark condition, cultivated 48 hours.
Under 590nm, test the fluorescent absorption value (OD value) of each test hole in 96 orifice plates with microplate reader (the GENios Plus of Tecan company).
Calculate inhibiting rate I%:I%=[1-(OD
The test soup-OD
Blank)/(OD
Negative control-OD
Blank)] * 100%
Calculate IC
50Value: IC
50=[C
L(I
H-50)+C
H(50-I
L)]/(I
H-I
L)
C
L: lower concentration values; C
H: high concentration value; I
H: the I% under the high density; I
L: the I% under the lower concentration.
4. test-results
The IC that the compound methyl Ai Siterui of the present invention's preparation suppresses Aspergillus fumigatus (Aspergillis fumigatus48238E)
50Value is 0.078 μ g/mL.The IC of positive control fluconazole
50Be 2.25 μ g/mL.
The result shows: compound methyl Ai Siterui of the present invention is better than fluconazole to active 29 times of the inhibition of Aspergillus fumigatus (Aspergillis fumigatus 48238E) to the activity of Aspergillus fumigatus (Aspergillisfumigatus 48238E).
Claims (4)
1. method of utilizing ground silk mould genus bacterial classification (Geomyces sp.) fermentative preparation as shown in the formula the compound methyl Ai Siterui shown in (I),
Wherein Me is a methyl, and described method comprises the following steps:
A) silk mould genus bacterial classification in ground is carried out solid fermentation;
B) after the solid fermentation of step a), in cultured products, add organic solvent extraction, obtain extracting solution, with described extracting solution underpressure distillation, obtain crude extract;
C) described crude extract is reduced pressure silica gel column chromatography and gel chromatography separation and obtains active component solution, described active component solution is separated the compound that obtains formula (I) with high performance liquid chromatography,
The wherein said ground mould genus bacterial classification of silk is that preserving number is the ground silk mould bacterial classification of CGMCC No.2481.
2. the described method of claim 1, wherein said organic solvent is an ethyl acetate.
3. the compound methyl Ai Siterui of following formula (I) is preparing the purposes that suppresses in the active medicine of Aspergillus fumigatus (Aspergillisfumigatua),
Wherein Me is a methyl.
4. the purposes of compound methyl Ai Siterui as claimed in claim 3 in the test kit of preparation inhibition activity of Aspergillus fumigatus.
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| CN102242067B (en) * | 2010-05-13 | 2013-07-31 | 上海康地恩生物科技有限公司 | Bacterial strain for producing low temperature neutral cellulase and method for preparing said cellulose |
| CN102531906B (en) * | 2010-09-29 | 2014-03-19 | 北京大学 | Application of natural compound P21 in inhibition of reproductive growth of tumor cell |
| CN103768075B (en) * | 2014-01-26 | 2016-06-22 | 宜春学院 | Ethyl asterrate application in preparing anti senile dementia drug |
| CN104804007B (en) * | 2015-03-14 | 2017-01-18 | 王能飞 | Geomyces sp.wnf-15A(phy) coloring compound |
| CN110218744B (en) * | 2019-05-10 | 2020-11-27 | 北京师范大学 | Method for preparing kojic acid by microbial fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1950104A (en) * | 2004-05-07 | 2007-04-18 | 希格马托制药工业公司 | Medicament for the treatment of fungal infections, particularly aspergillosis |
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| CN1950104A (en) * | 2004-05-07 | 2007-04-18 | 希格马托制药工业公司 | Medicament for the treatment of fungal infections, particularly aspergillosis |
Non-Patent Citations (4)
| Title |
|---|
| J Hargreaves et al.New chlorinated diphenyl ethers from an Aspergillus species.《J.Nat.Prod》.2002,第65卷(第1期),7-10. * |
| JP特开2001-322997A 2001.11.20 |
| JP特开平7-107989A 1995.04.25 |
| S Khera.A Novel Antibacterial Iridoid and Triterpene from Caiophora coronata.《J.Nat.Prod》.2003,第66卷(第12期),1628-1631. * |
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