CN104630304B - A kind of method that utilization microorganism prepares sophoridine oxide - Google Patents
A kind of method that utilization microorganism prepares sophoridine oxide Download PDFInfo
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- CN104630304B CN104630304B CN201510057554.XA CN201510057554A CN104630304B CN 104630304 B CN104630304 B CN 104630304B CN 201510057554 A CN201510057554 A CN 201510057554A CN 104630304 B CN104630304 B CN 104630304B
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- sophoridine
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Abstract
A kind of method that utilization microorganism fungus kind is prepared sophoridine oxide by Sophoridine, the microorganism fungus kind 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.The microbial strains catalytic cpd Sophoridine only needs a step to obtain its nitrogen oxides Sophoridine, with the simple advantage of low, selective high, the environment-friendly, step of cost.
Description
Technical field
It is specially that one kind prepares anticancer using microorganism catalysis the invention belongs to microbial technique and new medical technology field
Oxidation of drug Sophoridine（Oxysophoridine, CAS No. 54809-74-4) method.
Background technology
Sophoridine Sophoridine (SR) is the alkaloid of the type containing kuh-seng, be legume Sophora alopecuroide (Sophoraalope caroidesL. content is compared with one of high alkaloid present in).Discovery is studied it according to scholar, during Sophoridine may be present in
Pivot nervous system, angiocarpy, immune system, and with the effect such as anti-tumor microorganism and antiendotoxin.Sophoridine (and its system
Agent hydrochloric acid Sophoridine parenteral solution (Sophoridine Hydroehloride Injection) is in the states of in 8Yue22Huo, 2005
New Drug Certificate (traditional Chinese medicines demonstrate,prove word H20O51131,30) and the approval number of the drug that food and medicine Surveillance Authority of family (SFDA) issues
(Chinese medicines quasi-word H20051682,81), is that China develops, possesses the national class PTS of independent intellectual property right.Primary Calculation machine
Computer Aided Design and structure activity study show that the oxidized rear bioactivity of SR changes, while its toxicity substantially weakens.Liu Ping
Deng research sophoridine oxide（Oxysophoridine）It is the freeze-dried powder of sophoridine oxide, soft during to, the effect of lung cancer, intestinal cancer
The active anticancer of a variety of formulations such as capsule and pill, dripping pill, film bag piece and controlled release capsule is better than Sophoridine, and toxicity is fixed less than Chinese scholartree
Alkali.
The traditional preparation technology of sophoridine oxide is to extract to separate from Sophora alopecuroide, but its yield is very low, and only 0.015% is left
The right side, excessively excavation may cause the species endangered, can so cause vegetation and environment for human survival, and bio-diversity
Irretrievable loss.In addition, the extraction process is numerous and diverse, cost is high, it is difficult to realize large-scale industrial production.State's outer chemical is closed
Metachloroperbenzoic acid and dichloromethane are used into method, process costs are higher, reaction stereoselectivity is poor, is unfavorable for industry
Production.And dichloromethane belongs to two class solvents, it should which limitation is used.Separately have been reported that, route that SR reacts with hydrogen peroxide is made
Standby sophoridine oxide, but the oxidation product of synthesis has two kinds of configurations.So letter treats that a kind of efficient, economic prepare of demand aoxidizes Chinese scholartree
Determine the new method of alkali.
The content of the invention
It is an object of the invention to provide a kind of method that microorganism catalysis efficiently, economic prepares sophoridine oxide.This is micro-
Biological bacterial strain is entitledXylariales.sp, depositary institution：China typical culture collection center, address：Wuhan University, protects
Hide day：On December 24th, 2014, deposit number：CCTCC NO：M 2014660.
Based on object above, microorganism fungus kind of the present invention be separated from the medicinal plant Huperzia serrata in Chongqing it is pure
Change is obtained.It is prepared by the PDA culture medium used：The g of peeled potatoes stripping and slicing 200, boils about 20 min, and 8 layers of filtered through gauze must be filtered
Liquid, adds the g of glucose 20,1 L is supplied with running water, if adding 20 g agar with solid medium 1L, is put into high pressure and goes out
121 DEG C of 20 min of sterilizing in bacterium pot.Similar alkaloid-the Sophoridine of cheap and easy to get, structure in nature is used for substrate, to utilize
The method of mentioned microorganism catalysis prepares sophoridine oxide.
The concentration of its substrate Sophoridine can be 0.01-10mg/mL.
The solvent of its substrate Sophoridine is：Absolute ethyl alcohol, acetone, methanol, DMSO or their mixed solvent.
It prepared the reaction time for 3-9 days.
It is 22-30 DEG C that it, which prepares reaction temperature,.
Compared with prior art, the present invention prepares oxidation Chinese scholartree using the method for microorganism catalysis, passes through oxidized precursor substrate
Sophoridine obtains the form of its nitrogen oxides, only needs a step to prepare target compound sophoridine oxide, advantage is：（1）
Cost is low：It is raw material from nature natural products Sophoridine cheap and easy to get, single step reaction, which can catalyze and synthesize oxidation Chinese scholartree, to be determined
Alkali, substantial amounts of organic solvent is used in addition, overcoming from plant in extracting and developing, purge process（2）Selectivity is high：Selection
Microbial enzyme is catalyst, with strong regioselectivity and stereoselectivity；（3）It is environment-friendly：Because course of reaction is gentle,
Without harsh conditions such as HTHPs, it is also desirable to the catalysis of metal reagent, so meeting the production requirement of current Green Chemistry；（4）
Step is simple：Several steps are chemically needed to react from Sophoridine to sophoridine oxide, and microorganism catalysis only needs a step.Summary
Described, raw material and catalyst that this method is used are cheap, be easy to get, and reaction condition is gentle, easily-controllable, environment-friendly, can expire well
The development of sufficient Green Chemistry and sustainable development chemistry, and its to modern environment protection and the demand of low-carbon economy.
Brief description of the drawings
Fig. 1 is the electron micrograph of microbial strains in the present invention；
Fig. 2 generates the situation of sophoridine oxide for microbial strains catalysis in the present invention；
Fig. 3 is the Phylogenetic Analysis of microbial strains in the present invention；
Fig. 4 is the separation process of sophoridine oxide；
Fig. 5 is target compound in the present invention13C-NMR；
Fig. 6 is target compound in the present invention1H-NMR。
Embodiment
Embodiments below facilitates a better understanding of the present invention.Test material used in following embodiments, such as without special
Illustrate, be to be commercially available from routine biochemistry Reagent Company.
The strain of the present invention is isolated and purified from the medicinal plant Huperzia serrata of Chongqing to be obtained, strain name：Xylariales.sp, depositary institution：China typical culture collection center, address：Wuhan University, preservation day：In December, 2014
24 days, preservation registration number：CCTCC NO：M 2014660.Reaction substrate Sophoridine is Nanjing ingot peak Pharmaceutical Technology Co., Ltd
Production.
1st, the screening of active bacterial strain.
Cultivate matrix manufacturing：The g of peeled potatoes stripping and slicing 200, boils about 20 min, and 8 layers of filtered through gauze obtain filtrate, add Portugal
Grape 20 g of sugar, 1 L is supplied with running water, and natural ph is put into 121 DEG C of 20 min of sterilizing in high-pressure sterilizing pot.100 mL triangles
The bottled mL of culture medium 40,121 DEG C of high-temp steam sterilizing 25min, every liter of culture medium of solid medium adds 20g agar.
The strain inclined plane of preservation is inoculated into the 100 mL triangular flasks equipped with PDA culture medium（Built-in 40 mL culture mediums）In
28 DEG C, concussion and cultivate 2-3 days under conditions of 140rpm add the mg/mL of substrate Sophoridine about 0.5（Ethanol dissolves）, similarity condition
It is lower to co-culture 5-7 days, with the extraction of isometric ethyl acetate once, through thin-layer chromatography after being concentrated under reduced pressure（TLC）Detect conversion results.
TLC conditions：Solvent：Chloroform-methanol=5：1, developer：Improve bismuth potassium iodide.
Course of reaction is repeated for positive reaction, to determine the stability of conversion reaction.
2nd, 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, in electric Microscopic observation.
It is the micrograph results (10*60) of positive strain such as Fig. 1.
One aspect of the present invention carries out kind by using Protocols in Molecular Biology by ITS rDNA sequence pairs active bacterial strain
Identification and analysis.RDNA (rDNA) is extracted with CTAB methods, selection universal primer ITS1 and ITS4 enters to ITS areas
Performing PCR is expanded, and program is as follows：94 DEG C of min of pre-degeneration 3；94 DEG C of 30 s of denaturation；55 DEG C of s of renaturation 30；72 DEG C of extensions 1
Min, after 30 circulations, extends 10 min after 72 DEG C, sequence arranged through splicing after with GenBank comparings, determine bacterium
The kind of strain.The ITS rRNA sequences of the higher typical strain of similitude are obtained from GenBank databases as reference pair
As carrying out clustering using the softwares of MEGA 5. 0 and building NJ phylogenetic trees.
It is the Phylogenetic Analysis of bacterial strain of the present invention such as Fig. 3.
3rd, active bacterial strain catalyzes and synthesizes the application of anticancer compound sophoridine oxide.
Precursor substrate Sophoridine Nanjing ingot peak Pharmaceutical Technology Co., Ltd.Microbial catalystXylariales.spCCTCC NO：M 2014660.The strain be separated from the medicinal plant Huperzia serrata for picking up from Chongqing it is pure
Change is obtained, and is now stored in China typical culture collection center.
Bacterial strainXylariales.spCCTCC NO：The culture that M 2014660 catalyzes and synthesizes sophoridine oxide is sent out using two steps
Ferment method, first by seed culture fluid（250 mL culture mediums/500mL triangular flasks）In 28 DEG C, 140 rpm constant-temperature shaking cultures 2-3 days,
With the 5% inoculum concentration access amplification mL of culture medium 1000/2000, cultivated 3-4 days under similarity condition, add the g of substrate 1
（Ethanol dissolves）, nutrient solution is extracted with ethyl acetate after 7 days, evaporated under reduced pressure obtains the g of crude extract 9.1.
The separation of converted product using silica gel column chromatography, gel column chromatography, prepare the methods such as thin-layer chromatography separated, it is pure
Change, separation process is shown in Fig. 4.Section is learned to do by physicochemical property and Modern spectroscopy（1H-NMR、13C-NM））Identify converted product
Structure.
If Fig. 2 is the situation that strain of the present invention is catalyzed generation sophoridine oxide（H：Sophoridine, YH：Sophoridine oxide）.
If Fig. 5 is for target compound in the present invention13C-NMR is composed.
If Fig. 6 is for target compound in the present invention1H-NMR is composed.
Compared with current published technology, novel processing step of the invention is obtained by oxidized precursor substrate Sophoridine
To the form of its nitrogen oxides, only a step is needed to prepare target compound sophoridine oxide, advantage is：（1）Cost is low：Choosing
It is raw material with nature natural products Sophoridine cheap and easy to get, single step reaction can catalyze and synthesize sophoridine oxide, in addition, gram
Take and used substantial amounts of organic solvent in extracting and developing, purge process from plant（2）Selectivity is high：Selection microbial enzyme be
Catalyst, with strong regioselectivity and stereoselectivity；（3）It is environment-friendly：Because course of reaction is gentle, no HTHP
Etc. harsh conditions, it is also desirable to the catalysis of metal reagent, so meeting the production requirement of current Green Chemistry；（4）Step is simple：From
Sophoridine chemically needs several steps to react to sophoridine oxide, and microorganism catalysis only needs a step.Summary is described, this method
The raw material and catalyst of use are cheap, be easy to get, and reaction condition is gentle, easily-controllable, environment-friendly, can meet well Green Chemistry and
The development of sustainable development chemistry, and its to modern environment protection and the demand of low-carbon economy.
Above example is only that the present invention is furture elucidated, has no the meaning for limiting the invention to the specific embodiment
Figure.One skilled in the art would recognize that present invention encompasses all possible alternative in Claims scope,
Improvement project and equivalents.
Claims (6)
1. a kind of method that utilization microorganism prepares sophoridine oxide, it is characterized in that：The microorganism fungus kind title：Xylariales.sp, depositary institution：China typical culture collection center, address：In Wuhan University, preservation day：2014 12
The moon 24, deposit number：CCTCC NO：M 2014660；Oxidation Chinese scholartree is prepared using microorganism fungus kind catalysis Sophoridine to determine
Alkali.
2. according to the method described in claim 1, it is characterized in that：The concentration of the substrate Sophoridine is 0.01-10mg/mL.
3. according to the method described in claim 1, it is characterized in that：The solvent of the substrate Sophoridine is：Absolute ethyl alcohol, acetone,
Methanol, DMSO or their mixed solvent.
4. according to the method described in claim 1, it is characterized in that：The preparation reaction time is 3-9 days.
5. according to the method described in claim 1, it is characterized in that：The preparation reaction temperature is 22-30 DEG C.
6. according to the method described in claim 1, it is characterized in that：The preparation reaction 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 |
-
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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