CN104630304A - Method for preparing sophoridine oxide by virtue of microorganism - Google Patents
Method for preparing sophoridine oxide by virtue of microorganism Download PDFInfo
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- CN104630304A CN104630304A CN201510057554.XA CN201510057554A CN104630304A CN 104630304 A CN104630304 A CN 104630304A CN 201510057554 A CN201510057554 A CN 201510057554A CN 104630304 A CN104630304 A CN 104630304A
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- sophorine
- oxysophoridine
- sophoridine
- microorganism
- oxide
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Abstract
The invention relates to a method for preparing sophoridine oxide from sophoridine by virtue of a microbial strain. The microbial strain is named as Xylariales. sp and is collected in China Center for Type Culture Collection (CCTCC) in Wuhan University on December 24, 2014 and has a collection number of CCTCC M2014660. The microbial strain is used for catalyzing a compound sophoridine to obtain sophoridine nitric oxide, namely, sophoridine oxide in one step and the method has the advantages of low cost, high selectivity, environmental friendliness and simple steps.
Description
Technical field
The invention belongs to microbial technique and new medical technology field, be specially one and utilize microorganism catalysis to prepare the method for cancer therapy drug Oxysophoridine (Oxysophoridine, CAS No. 54809-74-4).
Background technology
Sophorine Sophoridine (SR) is the alkaloid containing kuh-seng type, for leguminous plants Herba Sophorae alopecuroidis (
sophoraalope caroidesl.) content comparatively one of the high alkaloid existed in.Find its research according to scholar, sophorine can be present in central nervous system, cardiovascular, immunity system, and has the effects such as anti-tumor microorganism and antiendotoxin.Sophorine (and preparation hydrochloric acid sophorine injection liquid (Sophoridine Hydroehloride Injection) is in New Drug Certificate (the traditional Chinese medicines card word H20O51131 that Huo State Food and Drug Administration August 22 in 2005 (SFDA) issues, 30) and the approval number of the drug (the accurate word H20051682 of traditional Chinese medicines, 81) be, that China develops, has independent intellectual property right country one class PTS.Predesigne machine aided design and structure activity study display SR change through oxidation artifact activity, and its toxicity obviously weakens simultaneously.When Liu's equality research Oxysophoridine (Oxysophoridine) is to, the effect of lung cancer, intestinal cancer, the antitumour activity of the freeze-dried powder of Oxysophoridine, soft capsule, dripping pill, the multiple formulation such as film bag sheet and controlled release capsule is better than sophorine, and toxic side effects is less than sophorine.
The traditional preparation technology of Oxysophoridine is from Herba Sophorae alopecuroidis extraction and isolation, but its yield is very low, is only about 0.015%, excessively excavates and these species may be caused endangered, vegetation and environment for human survival can be caused like this, and species diversity irretrievable loss.In addition, this extraction process is numerous and diverse, cost is high, is difficult to realize large-scale industrial production.External chemical synthesis process employs metachloroperbenzoic acid and methylene dichloride, and process costs is higher, and reaction stereoselectivity is poor, is unfavorable for industrial production.And methylene dichloride belongs to two kind solvents, use should be limited.Separately have report, the route that SR and hydrogen peroxide react prepares Oxysophoridine, but the oxidation products of synthesis has two kinds of configurations.So the novel method that demand is a kind of efficiently, economy prepares Oxysophoridine treated by letter.
Summary of the invention
The object of this invention is to provide a kind of method that efficient, economic microorganism catalysis prepares Oxysophoridine.This microorganism strains name is called
xylariales.sp, depositary institution: China typical culture collection center, address: Wuhan University, preservation day: on December 24th, 2014, deposit number: CCTCC M 2014660.
Based on above object, the microbial strains that the present invention relates to is that separation and purification obtains from the medicinal plant Herba Lycopodii serrati in Chongqing.The PDA medium preparing used: peeled potatoes stripping and slicing 200 g, boil about 20 min, 8 layers of filtered through gauze obtain filtrate, add glucose 20 g, supply 1 L with tap water, if join solid medium 1L to add 20 g agar, put into high-pressure sterilizing pot 121 DEG C of sterilizing 20 min.Adopt occurring in nature alkaloid-sophorine cheap and easy to get, similar to be substrate, utilize the method for mentioned microorganism catalysis to prepare Oxysophoridine.
The concentration of its substrate sophorine can be 0.01-10mg/mL.
The solvent of its substrate sophorine is: dehydrated alcohol, acetone, methyl alcohol, DMSO or their mixed solvent.
Its preparation feedback time is 3-9 days.
Its preparation feedback temperature is 22-30 DEG C.
Compared with prior art, the present invention adopts the method preparation oxidation Chinese scholartree of microorganism catalysis, the form of its oxynitride is obtained by oxidized precursor substrate sophorine, only need a step can prepare target compound Oxysophoridine, advantage is: (1) cost is low: the natural product sophorine selecting nature cheap and easy to get is raw material, single step reaction can catalyze and synthesize Oxysophoridine, in addition, overcome and extract from plant, be separated, use a large amount of organic solvent (2) selectivity high in purge process: selection microbial enzyme is catalyzer, there is strong regioselectivity and stereoselectivity, (3) environmental friendliness: because reaction process is gentle, without severe condition such as High Temperature High Pressure, also need the catalysis of metal reagent, so meet the Production requirement of current Green Chemistry, (4) step is simple: from sophorine to Oxysophoridine, chemically need a few step to react, and microorganism catalysis only needs a step.Described in summary, the raw material that the method adopts and catalyzer cheap, be easy to get, reaction conditions is gentle, easily control, environmental friendliness, can meet the development of Green Chemistry and Sustainable development chemistry well, and the demand to modern environment protection and low-carbon economy.
Accompanying drawing explanation
Fig. 1 is the electron micrograph of microorganism strains in the present invention;
Fig. 2 is the situation that in the present invention, microorganism strains catalysis generates Oxysophoridine;
Fig. 3 is the Phylogenetic Analysis of microorganism strains in the present invention;
Fig. 4 is the separation process of Oxysophoridine;
Fig. 5 is target compound in the present invention
13c-NMR;
Fig. 6 is target compound in the present invention
1h-NMR.
Embodiment
Following embodiment is convenient to understand the present invention better.Test materials used in following embodiment, if no special instructions, is and obtains from the purchase of routine biochemistry Reagent Company.
Bacterial classification of the present invention separation and purification from the medicinal plant Herba Lycopodii serrati of Chongqing obtains, strain name:
xylariales.sp, depositary institution: China typical culture collection center, address: Wuhan University, preservation day: on December 24th, 2014, preservation registration number: CCTCC M 2014660.Reaction substrate sophorine is that ingot peak, Nanjing Pharmaceutical Technology Co., Ltd produces.
1, the screening of active bacterial strain.
Substratum makes: peeled potatoes stripping and slicing 200 g, boil about 20 min, 8 layers of filtered through gauze obtain filtrate, add glucose 20 g, supply 1 L, natural ph with tap water, put into high-pressure sterilizing pot 121 DEG C of sterilizing 20 min.100 mL triangle bottled substratum 40 mL, 121 DEG C of high-temp steam sterilizing 25min, solid medium often rises substratum and adds 20g agar.
The strain inclined plane of preservation to be inoculated in the 100 mL triangular flasks (in-built 40 mL substratum) that PDA substratum is housed 28 DEG C, under the condition of 140rpm, 2-3 days is cultivated in concussion, add substrate sophorine about 0.5 mg/mL(dissolve with ethanol), Dual culture 5-7 days under similarity condition, with equal-volume extraction into ethyl acetate once, detect conversion results through thin-layer chromatography (TLC) after concentrating under reduced pressure.
TLC condition: developping agent: chloroform-methanol=5:1, developer: improvement bismuth potassium iodide.
For positive reaction reaction repeated process, to determine the stability of conversion reaction.
2, the Species estimation analysis of active bacterial strain.
One aspect of the present invention carries out Species estimation analysis by microstructure to active bacterial strain, at electric Microscopic observation.
As Fig. 1, it is the micrograph results (10*60) of positive strain.
One aspect of the present invention carries out Species estimation analysis by adopting Protocols in Molecular Biology by ITS rDNA sequence pair active bacterial strain.Extract rDNA (rDNA) by CTAB method, select universal primer ITS1 and ITS4 to carry out PCR amplification to ITS district, program is as follows: 94 DEG C of denaturation 3 min; 94 DEG C of sex change 30 s; 55 DEG C of renaturation 30 s; 72 DEG C extend 1 min, 30 circulation after, extend 10 min after 72 DEG C, sequence through splicing arrange after with GenBank comparing, determine the kind of bacterial strain.From GenBank database, obtain the ITS rRNA sequence of the higher typical strain of similarity as reference subject, adopt MEGA 5. 0 software carry out cluster analysis and build NJ phylogenetic tree.
As Fig. 3, it is the Phylogenetic Analysis of bacterial strain of the present invention.
3, active bacterial strain catalyzes and synthesizes the application of anticancer compound Oxysophoridine.
Precursor substrate sophorine Nanjing ingot peak Pharmaceutical Technology Co., Ltd.Microbial catalyst
xylariales.spcCTCC M 2014660.This bacterial classification obtains from separation and purification the medicinal plant Herba Lycopodii serrati picking up from Chongqing, is now stored in China typical culture collection center.
Bacterial strain
xylariales.spthe cultivation that CCTCC M 2014660 catalyzes and synthesizes Oxysophoridine adopts two-step fermentation, first by seed culture fluid (250 mL substratum/500mL triangular flask) in 28 DEG C, 140 rpm constant-temperature shaking culture 2-3 days, with inoculum size access amplification culture base 1000/2000 mL of 5%, 3-4 days is cultivated under similarity condition, add substrate 1 g(ethanol to dissolve), be extracted with ethyl acetate nutrient solution after 7 days, evaporated under reduced pressure obtains crude extract 9.1 g.
The separation of converted product adopts the methods such as silica gel column chromatography, gel column chromatography, Preparative TLC chromatogram to carry out being separated, purifying, and separation process is shown in Fig. 4.By physico-chemical property and the Modern spectroscopy section of learning to do (
1h-NMR,
13c-NM) structure of converted product) is identified.
If Fig. 2 is the situation (H: sophorine, YH: Oxysophoridine) that bacterial classification catalysis of the present invention generates Oxysophoridine.
If Fig. 5 is target compound in the present invention
13c-NMR composes.
If Fig. 6 is target compound in the present invention
1h-NMR composes.
Compared with current published technology, novel processing step of the present invention is the form being obtained its oxynitride by oxidized precursor substrate sophorine, only need a step can prepare target compound Oxysophoridine, advantage is: (1) cost is low: the natural product sophorine selecting nature cheap and easy to get is raw material, single step reaction can catalyze and synthesize Oxysophoridine, in addition, overcome and extract from plant, be separated, use a large amount of organic solvent (2) selectivity high in purge process: selection microbial enzyme is catalyzer, there is strong regioselectivity and stereoselectivity, (3) environmental friendliness: because reaction process is gentle, without severe condition such as High Temperature High Pressure, also need the catalysis of metal reagent, so meet the Production requirement of current Green Chemistry, (4) step is simple: from sophorine to Oxysophoridine, chemically need a few step to react, and microorganism catalysis only needs a step.Described in summary, the raw material that the method adopts and catalyzer cheap, be easy to get, reaction conditions is gentle, easily control, environmental friendliness, can meet the development of Green Chemistry and Sustainable development chemistry well, and the demand to modern environment protection and low-carbon economy.
Above embodiment is only illustrate the present invention further, there is no the intention limiting the invention to this specific embodiment.One skilled in the art would recognize that all possible alternatives, improvement project and the equivalents that present invention encompasses in Claims scope.
Claims (6)
1. utilize microorganism to prepare a method for Oxysophoridine, it is characterized in that: described microbial strains title:
xylariales.sp, depositary institution: China typical culture collection center, address: in Wuhan University, preservation day: on December 24th, 2014, deposit number: CCTCC M 2014660; Described microbial strains is utilized to prepare the method for Oxysophoridine by sophorine.
2. method according to claim 1, is characterized in that: the concentration of described substrate sophorine is 0.01-10mg/mL.
3. method according to claim 1, is characterized in that: the solvent of described substrate sophorine is: dehydrated alcohol, acetone, methyl alcohol, DMSO or their mixed solvent.
4. method according to claim 1, is characterized in that: the described preparation feedback time is 3-9 days.
5. method according to claim 1, is characterized in that: described preparation feedback temperature is 22-30 DEG C.
6. method according to claim 1, is characterized in that: described preparation feedback temperature is 28 DEG C.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453276A (en) * | 2003-05-22 | 2003-11-05 | 王答祺 | Prepn of matrine, oxymatrine and sophoxidine from flavescent sophora root |
| CN1739510A (en) * | 2005-04-20 | 2006-03-01 | 刘平 | Use of sophoridine oxide in preparing anticancer medicine |
| CN101591618A (en) * | 2008-05-28 | 2009-12-02 | 中国科学院微生物研究所 | A kind of Xylaria gracillima strain and liquid fermentation culturing method thereof and application |
| CN101942393A (en) * | 2009-12-30 | 2011-01-12 | 朱笃 | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A |
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2015
- 2015-02-04 CN CN201510057554.XA patent/CN104630304B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453276A (en) * | 2003-05-22 | 2003-11-05 | 王答祺 | Prepn of matrine, oxymatrine and sophoxidine from flavescent sophora root |
| CN1739510A (en) * | 2005-04-20 | 2006-03-01 | 刘平 | Use of sophoridine oxide in preparing anticancer medicine |
| CN101591618A (en) * | 2008-05-28 | 2009-12-02 | 中国科学院微生物研究所 | A kind of Xylaria gracillima strain and liquid fermentation culturing method thereof and application |
| CN101942393A (en) * | 2009-12-30 | 2011-01-12 | 朱笃 | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A |
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Granted publication date: 20171017 Termination date: 20220204 |