CN101492703B - Process for the separation and preparation of 4'-demethyl podophyllic acid - Google Patents
Process for the separation and preparation of 4'-demethyl podophyllic acid Download PDFInfo
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Abstract
The invention discloses a method for converting 4'- demethylpodophyllotoxin to 4'- demethylation epipodophyllic acid, comprising the following steps: the 4'- demethylpodophyllotoxin solution is added during the fermentation process of microorganism clostridium, pseudomonas aeruginosa, rhodococcus erythropolis, corynebacterium pekinense, hay bacillus, erwinia uredovora, bacillus or bending pseudomonas for carrying out biotransformation reaction, thus obtaining biotransformation matrix with the 4'-demethylation epipodophyllic acid. The invention also discloses a method for separating the 4'-demethylation epipodophyllic acid from the biotransformation matrix, comprising the following steps: the macroporous absorbent resin column is adopted to carry out initial separation on the biotransformation matrix and gel column chromatography is adopted to carry out fine separation, thus obtaining 4'-demethylation epipodophyllic acid. In the invention, modification is carried out on the 4'- demethylpodophyllotoxin structure by biotransformation, thus obtaining 4'-demethylation epipodophyllic acid, in addition, the reaction selectivity is high, the operation is easy, the reaction conditions are moderate, the generated waste is little, the separation process is simple and the yield is high.
Description
Technical field
The present invention relates to the preparation method of Podophyllum emodi var chinense lignans, relate in particular to a kind of with 4 '-the demethyl epipodophyllotoxin is converted into 4 '-method of '-demethyl podophyllic acid, the invention still further relates to from prepared biotransformation matrix, separate 4 '-method of '-demethyl podophyllic acid, belong to field of biological pharmacy.
Background technology
4 '-the demethyl epipodophyllotoxin is a podophyllotoxin E-4 ' position demethyl gained derivative, assemble different with podophyllotoxin by tubulin in the destruction cell mitogen process with the formation of microtubule, 4 '-the demethyl epipodophyllotoxin is brought into play antitumor action by the activity that suppresses the DNA topoisomerase II, and activity significantly strengthens than podophyllotoxin.Because of closely similar with conventional chemotherapy medicine Etoposide, teniposide structure, its derivative more may become the medicine with remarkable anti-tumor activity, thereby receives much concern.
4 '-chemically modified of demethyl epipodophyllotoxin research originates in the fifties in last century, and people such as Wang of Tongji University generation dragon modify its structure by chemical process and have obtained 4 '-'-demethyl podophyllic acid.But chemical conversion reaction exist product yield low, easily cause shortcoming such as environmental pollution.Compare with chemical conversion, bio-transformation has highly selective, high efficiency, advantages of environment protection, has reduced the complicacy of process on the one hand, improves product yield simultaneously, reduces the pollution to environment.At present both at home and abroad still useless bioconversion method to 4 '-the demethyl epipodophyllotoxin carries out the relevant report of structural modification.
Summary of the invention
Technical problem to be solved by this invention be overcome prior art preparation 4 '-the '-demethyl podophyllic acid process in problem such as low, the easy contaminate environment of existing poor selectivity, reaction yield, provide a kind of new 4 '-preparation and the separation method of '-demethyl podophyllic acid, this method with 4 '-the demethyl epipodophyllotoxin is as reaction substrate, by bioconversion method obtain 4 '-'-demethyl podophyllic acid, the inventive method is easy and simple to handle, reaction preference is strong, environmental friendliness, production cost are low.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of preparation 4 '-method of '-demethyl podophyllic acid, comprising:
At microorganism clostridium (Bacillus fusiformis), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Rhodococcus (Rhodococcus erythropolis), Beijing rod bacillus (Corynebacteriumpekinenese), Bacillus subtilus (Bacillus subtilis), uredo erwinia phage (Erwinia uredovora), in the fermenting process of genus bacillus (Bacillus sp.) or crooked pseudomonas (Pseudomonas geniculata), with 4 '-demethyl Etoposide cellulose solution joins in any microbial fermentation system and carries out bioconversion reaction, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid.
Described clostridium, Pseudomonas aeruginosa, Rhodococcus, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage, genus bacillus or crooked pseudomonas all can be bought by various commercial sources and obtain, for example clostridium can be available from Chinese industrial microbial strains preservation center, and it is numbered CICC 20463.
The culturing process of described clostridium, Pseudomonas aeruginosa, Rhodococcus, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage, genus bacillus or crooked pseudomonas can be with reference to various conventional cultural methods, comprise that slant strains is cultivated, liquid seeds is cultivated, fermentation culture, wherein, the various substratum of using and culture condition all be the conventional substratum and the culture condition of this area, these are all understood thoroughly by those skilled in the art.
Preferably, described clostridial culturing process is as follows:
(1) slant strains is cultivated: substratum: glucose 2%, extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, agar 2%, the pH value is 7.0 (each components contents is percent weight in volume, and promptly gram is/100 milliliters, down together), sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, bevel, inoculation clostridium bacterial classification was cultivated 6 hours for 37 degrees centigrade;
(2) liquid seeds is cultivated: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, pH 7.0, and liquid amount is 50 milliliters/250 ml shake flasks, sterilize 30 minutes for 115 degrees centigrade, the sterilization postcooling, the inoculation slant strains was cultivated 4 hours for 37 degrees centigrade, 180 rev/mins, as liquid seeds;
(3) fermentation culture: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, sucrose 1%, pH value are 7.0, liquid amount 50 milliliters/250 ml shake flasks, sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size are 10%, 37 degree centigrade, 180 rev/mins and cultivated 3 hours.
Preferably, described Pseudomonas aeruginosa culturing process is as follows:
(1) slant strains is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, agar 2%, pH value are 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Pseudomonas aeruginosa bacterial classification was cultivated 4 hours for 37 degrees centigrade;
(2) liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 3 hours for 37 degrees centigrade, 200 rev/mins, as liquid seeds;
(3) fermentation culture: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, and liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 3 hours for 10%, 37 degree centigrade, 200 rev/mins.
Described 4 '-demethyl Etoposide cellulose solution is mixed with dehydrated alcohol, its concentration be preferably every milliliter of dehydrated alcohol contain 4 of 0.25-20 milligram '-the demethyl epipodophyllotoxin, more preferably every milliliter of dehydrated alcohol contain 2.5 milligrams 4 '-the demethyl epipodophyllotoxin.
In order to reach better bio-transformation effect, preferred, with substrate 4 '-demethyl Etoposide cellulose solution add in the microbial fermentation system to its final concentration be the 5-2000 mcg/ml; Preferred, with 4 '-demethyl Etoposide cellulose solution add in the microbial fermentation system to its final concentration be 50 mcg/ml.
Described bioconversion reaction preferably carries out according to following condition: the bioconversion reaction temperature is 20-40 degree centigrade, and the bioconversion reaction time is 3-96 hour, and rotating speed is 50-350 rev/min;
Preferred, described bioconversion reaction preferably carries out according to following condition: the bioconversion reaction temperature is 37 degrees centigrade, and the bio-transformation time is 20 hours, and rotating speed is 180 rev/mins.
The present invention utilize first clostridium, Pseudomonas aeruginosa, Rhodococcus, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage, genus bacillus or crooked pseudomonas biological transform 4 '-the demethyl epipodophyllotoxin, prepare 4 '-'-demethyl podophyllic acid, product yield height, reaction conditions gentleness, simple to operate.
The biotransformation matrix of above-mentioned gained carried out high performance liquid chromatography (HPLC) is analyzed and thin-layer chromatography (TLC) analysis, the result shows, resulting bioconversion product is 4 '-'-demethyl podophyllic acid.
Another technical problem to be solved by this invention provide a kind of with 4 '-method that '-demethyl podophyllic acid is separated from above-mentioned biotransformation matrix.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind ofly from above-mentioned biotransformation matrix, separate 4 '-method of '-demethyl podophyllic acid, comprising:
(1) the supernatant liquor absorption with macroporous adsorbent resin after centrifugal with biotransformation matrix carries out gradient elution with aqueous ethanolic solution, the Fractional Collections elutriant;
(2) collect the 20%-50% ethanol eluate, with gel filtration chromatography segment from, 4 '-'-demethyl podophyllic acid.
Preferably, centrifugal described in the step (1), preferable methods is under 3000-7000 rev/min rotating speed centrifugal 20-40 minute; Preferred, under 5500 rev/mins rotating speed centrifugal 30 minutes; Described filling macroporous adsorbent resin model is preferably D312, AB-8, YWD04c, YWDBC, MCA or 860021, more preferably D312 is (available from resin subsidiary factory of Lukang Medical Co., Ltd., Shandong, nonpolar, particle diameter 0.315-1.25 millimeter, specific surface area 500-650 meters squared per gram, mean pore size 10-20 nanometer);
Gel column described in the step (2) is preferably Sephadex LH-20 gel column.
The present invention has set up a kind of novel method that is prepared the Podophyllum emodi var chinense lignans compound by microbial transformation, bioconversion method of the present invention can be directly add 4 in the fermenting process of clostridium, Pseudomonas aeruginosa, Rhodococcus, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage, genus bacillus or crooked pseudomonas '-demethyl Etoposide cellulose solution carries out bio-transformation, the product yield height, to substrate 4 '-molar yield of demethyl epipodophyllotoxin can reach 63.3%.
Present method utilize bio-transformation to 4 '-the demethyl epipodophyllotoxin carries out structural modification, obtain 4 '-'-demethyl podophyllic acid, the substrate conversion efficiency height, simple to operate; The bioconversion reaction mild condition, the waste of generation is less; The bioconversion product sepn process is simple, and the yield height.
In a word, biotransformation method of the present invention has the following advantages with respect to traditional chemical synthesis: 1, easy and simple to handle, efficiency of pcr product is high; 2, reaction conditions gentleness, environmental friendliness; 3, separation method is simple, agents useful for same toxicity is low, expense is low, save cost.
Description of drawings
Fig. 1 by 4 '-demethyl epipodophyllotoxin to 4 '-the bio-transformation route map of '-demethyl podophyllic acid;
Fig. 2 the present invention 4 '-preparation and the separation process figure of '-demethyl podophyllic acid.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
One, test materials:
Clostridium, available from Chinese industrial microbial strains preservation center, it is numbered CICC 20463; Pseudomonas aeruginosa, available from China typical culture collection center (Wuhan University), it is numbered CCTCC AB93066; Rhodococcus, available from Chinese common micro-organisms culture presevation administrative center, it is numbered CGMCC 1.2362; Beijing rod bacillus, available from China typical culture collection center (Wuhan University), it is numbered CCTCC AB97005; Bacillus subtilus, available from China typical culture collection center (Wuhan University), it is numbered CCTCC AB93174; Uredo erwinia phage, available from Chinese common micro-organisms culture presevation administrative center, it is numbered CGMCC 1.1215; Genus bacillus, available from China typical culture collection center (Wuhan University), it is numbered CCTCC AB95018; Crooked pseudomonas, available from China typical culture collection center (Wuhan University), it is numbered CCTCCAB93074.
Two, detection method
(1) 4 '-demethyl epipodophyllotoxin and 4 '-HPLC of '-demethyl podophyllic acid measures: the post model is Agela C
18Post (5 microns, 4.6 millimeters * 250 millimeters), moving phase is methanol-water (40: 60), and flow velocity is 0.8 ml/min, and the ultraviolet detection wavelength is 230 nanometers, and column temperature is 45 degrees centigrade.
(2) 4 '-demethyl epipodophyllotoxin and 4 '-TLC of '-demethyl podophyllic acid measures: silica gel G chromatoplate (Qingdao Marine Chemical Co., Ltd.), with biotransformation matrix with ethyl acetate extraction after standing demix, ethyl acetate layer concentrates and volatilizes, dehydrated alcohol redissolves, with kapillary point sample on the exsiccant thin plate, thin plate is put into the chromatography cylinder chromatography.Developping agent is chloroform-ethyl acetate-glacial acetic acid (7: 4: 2), and iodo steam displaing color presents the tawny spot.
Embodiment 1
1, clostridial cultivation:
Slant strains is cultivated: substratum: glucose 2%, extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, agar 2%, pH value are 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation clostridium bacterial classification was cultivated 6 hours for 37 degrees centigrade;
Liquid seeds is cultivated: substratum: yeast powder 0.5%, and peptone 0.5%, sodium-chlor 0.5%, the pH value is 7.0, liquid amount is 50 milliliters/250 ml shake flasks, sterilizes 30 minutes cooling for 115 degrees centigrade, the inoculation slant strains was cultivated 4 hours for 37 degrees centigrade, 180 rev/mins, as liquid seeds;
Preparation of fermentation liquid: substratum is a sucrose 1%, yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7.0, liquid amount is 50 milliliters/250 ml shake flasks, sterilized 30 minutes cooling, inoculation liquid seeds for 115 degrees centigrade, inoculum size was cultivated 3 hours for 10%, 37 degree centigrade, 180 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 2.5 milligrams 4 '-demethyl Etoposide cellulose solution; With 4 '-to join clostridia fermentation system to its final concentration be 50 mcg/ml to demethyl Etoposide cellulose solution, carry out bio-transformation under the following conditions: 37 degrees centigrade of bioconversion reaction temperature, shaking speed is 180 rev/mins, the bioconversion reaction time is 20 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 66.3%.
3, the separation and purification of bioconversion product and structure are identified
5500 rev/mins of above-mentioned biotransformation matrix is centrifugal 30 minutes, discard precipitation, directly (the D312 macroporous adsorbent resin is available from resin subsidiary factory of Lukang Medical Co., Ltd., Shandong with the D312 macroporous adsorptive resins for supernatant liquor, non-polar resin, particle diameter 0.315-1.25 millimeter, specific surface area 500-650 meters squared per gram, mean pore size 10-20 nanometer) absorption, with the ethanol-water solution gradient elution, 20-50% ethanol elution component concentrated volatilize, methyl alcohol redissolves, and removes by filter insoluble impurities, and Sephadex LH-20 gel is (available from the easy science and technology limited Company of intelligent moral in Beijing, particle diameter: the column chromatography for separation 20-150 micron), methanol-eluted fractions, white unsetting powder, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid.(
1H?NMR(D
2O)δ1.70(1H，H-3)，2.12(2H，4-OH，11-OH)，2.70(1H，H-2)，3.44(6H，MeO-3′，MeO-5′)，3.47(1H，H-11)，3.72(1H，H-11)，4.15(1H，H-1)，4.58(1H，H-4)，5.71(1H，H-13)，5.73(1H，H-13)，6.08(2H，H-2′，H-6′)，6.30(1H，H-8)，6.80(1H，H-5)；
13C?NMR(D
2O)δ38.65(C-1)，48.24(C-2)，51.89(C-3)，56.88(MeO-3′，MeO-5′)，61.63(C-11)，67.15(C-4)，101.20(C-13)，108.10(C-2′，C-6′)，108.75(C-8)，109.53(C-5)，1229.43(C-4′，C-10)，130.15(C-9)，132.65(C-1′)，146.31(C-6)，147.12(C-7)，150.73(C-3′，C-5′)，182.97(C-12)；ESI-MS?m/z：nanal.calcd.for?C
21H
22O
9：417.3924(M
++H)；found，417.0.)
Embodiment 2
1, the cultivation of pseudomonas aeruginosa
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2%, the pH value is 7.0,115 degrees centigrade of sterilizations 30 minutes, cooling, bevel, inoculation Pseudomonas aeruginosa bacterial classification was cultivated 12 hours for 37 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 10 hours for 37 degrees centigrade, 200 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, and liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 10 hours for 10%, 37 degree centigrade, 200 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 2.5 milligrams 4 '-demethyl Etoposide cellulose solution, with 4 '-demethyl Etoposide cellulose solution join in the Pseudomonas aeruginosa fermentation system to its final concentration be 50 mcg/ml, carry out bio-transformation under the following conditions: 37 degrees centigrade of temperature, rotating speed is 200 rev/mins, the bio-transformation time is 20 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 52%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 3
1, the cultivation of Rhodococcus:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 1.0%, sodium-chlor 0.5%, agar 1.5%, the pH value is 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Rhodococcus bacterial classification was cultivated 12 hours for 30 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, the pH value is 7.0, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation inclined-plane seed, cultivated 10 hours for 30 degrees centigrade, 180 rev/mins, as liquid seeds;
Fermentation culture: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, the pH value is 7.0, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, 2%, 30 degree centigrade of inoculum size was cultivated 10 hours for 180 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 20 milligrams 4 '-demethyl Etoposide cellulose solution, with 4 '-demethyl Etoposide cellulose solution join in the Rhodococcus fermentation system to its final concentration be 500 mcg/ml, carry out bio-transformation under the following conditions: 30 degrees centigrade of temperature, rotating speed is 200 rev/mins, the conversion reaction time is 30 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 50.0%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 4
1, the cultivation of Beijing rod bacillus:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2.0%, the pH value is 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Beijing rod bacillus species was cultivated 12 hours for 28 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7.0, liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 12 hours for 28 degrees centigrade, 140 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7.0, and liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 12 hours for 2%, 28 degree centigrade, 140 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 10 milligrams 4 '-demethyl Etoposide cellulose solution, with 4 '-demethyl Etoposide cellulose solution join in the rod bacillus fermentation system of Beijing to its final concentration be 100 mcg/ml, carry out bio-transformation under the following conditions: 28 degrees centigrade of temperature, rotating speed is 140 rev/mins, the conversion reaction time is 10 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 36%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 5
1, the cultivation of Bacillus subtilus:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2.0%, the pH value is 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Bacillus subtilus bacterial classification was cultivated 12 hours for 30 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 6 hours for 30 degrees centigrade, 180 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, and liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 6 hours for 2%, 30 degree centigrade, 180 rev/mins.
2, bio-transformation
With 4 '-to be mixed with concentration with dehydrated alcohol be that every milliliter of ethanol contains 5.0 milligrams podophyllotoxin solution to the demethyl epipodophyllotoxin, with 4 '-demethyl Etoposide cellulose solution join in the fermenting bacillus subtilis system to its final concentration be 127 mcg/ml, carry out bio-transformation under the following conditions: 30 degrees centigrade of temperature, rotating speed is 180 rev/mins, the bio-transformation time is 12 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, from this biotransformation matrix, take a sample, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 37%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 6
1, the cultivation of uredo erwinia phage:
Slant strains is cultivated: substratum: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, agar 1.5%, pH value are 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation uredo erwinia phage bacterial classification was cultivated 12 hours for 30 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 10 hours for 30 degrees centigrade, 180 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, the pH value is 7, and liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 10 hours for 2%, 30 degree centigrade, 180 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 2.5 milligrams 4 '-demethyl Etoposide cellulose solution, with 4 '-demethyl Etoposide cellulose solution join in the uredo erwinia phage fermentation system to its final concentration be 5 mcg/ml, carry out bio-transformation under the following conditions: 30 degrees centigrade of temperature, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 12%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 7
1, the cultivation of genus bacillus:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and yeast extract paste 0.5%, peptone 0.5%, agar 1.5%, the pH value is 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation genus bacillus bacterial classification was cultivated 12 hours for 28 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, yeast extract paste 0.5%, peptone 0.5%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 10 hours for 28 degrees centigrade, 180 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, yeast extract paste 0.5%, peptone 0.5%, the pH value is 7, and liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 10 hours for 5%, 28 degree centigrade, 180 rev/mins.
2, bio-transformation
With 4 '-the demethyl epipodophyllotoxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 20 milligrams 4 '-demethyl Etoposide cellulose solution; With 4 '-demethyl Etoposide cellulose solution join in the fermentation of bacillus system to its final concentration be 1800 mcg/ml, carry out bio-transformation under the following conditions: 28 degrees centigrade of temperature, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 16%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Embodiment 8
1, the cultivation of crooked pseudomonas:
Slant strains is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, agar 2.0%, pH value are 7.0,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel are inoculated crooked pseudomonas bacterial classification, cultivate 12 hours for 25 degrees centigrade;
Liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, cultivated 10 hours for 25 degrees centigrade, 180 rev/mins, as liquid seeds;
Fermentation culture: substratum: extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, the pH value is 7, and liquid amount 200 milliliters/500 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 10 hours for 5%, 25 degree centigrade, 180 rev/mins.
2, bio-transformation
With 4 '-demethyl table mortar ghost toxin with dehydrated alcohol be mixed with concentration be every milliliter of ethanol contain 20 milligrams 4 '-demethyl Etoposide cellulose solution, with 4 '-demethyl Etoposide cellulose solution join in the crooked pseudomonas fermentation system to its final concentration be 50 mcg/ml, carry out bio-transformation under the following conditions: 25 degrees centigrade of temperature of reaction, rotating speed is 2000 rev/mins, the bio-transformation time is 20 hours, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid, measure through HPLC, 4 '-demethyl epipodophyllotoxin transformation efficiency is 28%.
3, the separation and purification of bioconversion product and structure are identified
Adopt the separation purifying technique identical, obtain white unsetting powder with embodiment 1, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS) be defined as 4 '-'-demethyl podophyllic acid (structure evaluation parameter is with embodiment 1).
Claims (9)
1. one kind 4 '-preparation method of '-demethyl podophyllic acid, comprising:
At microorganism clostridium (Bacillus fusiformis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Rhodococcus (Rhodococcus erythropolis), Beijing rod bacillus (Corynebacterium pekinenese), Bacillus subtilus (Bacillus subtilis), uredo erwinia phage (Erwinia uredovora), in the fermenting process of genus bacillus (Bacillus sp.) or crooked pseudomonas (Pseudomonas geniculata), with substrate 4 '-demethyl Etoposide cellulose solution joins in any microbial fermentation system and carries out bioconversion reaction, obtain containing 4 '-biotransformation matrix of '-demethyl podophyllic acid;
Wherein, described clostridial microbial preservation is numbered CICC 20463; The microbial preservation of Pseudomonas aeruginosa is numbered CCTCC AB93066; The microbial preservation of Rhodococcus is numbered CGMCC 1.2362; The microbial preservation of Beijing rod bacillus is numbered CCTCC AB97005; The microbial preservation of Bacillus subtilus is numbered CCTCCAB93174; The microbial preservation of uredo erwinia phage is numbered CGMCC 1.1215; The microbial preservation of genus bacillus is numbered CCTCC AB95018; The microbial preservation of crooked pseudomonas is numbered CCTCC AB93074.
2. according to the described preparation method of claim 1, it is characterized in that: described substrate 4 '-demethyl Etoposide cellulose solution prepares with dehydrated alcohol, its concentration be every milliliter of dehydrated alcohol contain the 2.5-20 milligram 4 '-the demethyl epipodophyllotoxin.
3. according to the described preparation method of claim 1, it is characterized in that: with 4 '-demethyl Etoposide cellulose solution add in the microbial fermentation system to its final concentration be the 5-2000 mcg/ml.
4. according to the described preparation method of claim 3, it is characterized in that: with 4 '-demethyl Etoposide cellulose solution join in the microbial fermentation system to its final concentration be 50 mcg/ml.
5. according to the described preparation method of claim 1, it is characterized in that described bioconversion reaction carries out according to following condition: the bioconversion reaction temperature is 20-40 degree centigrade, and the bioconversion reaction time is 3-96 hour, and rotating speed is 50-350 rev/min.
6. according to the described preparation method of claim 5, it is characterized in that described bioconversion reaction carries out according to following condition: the bioconversion reaction temperature is 37 degrees centigrade, and the bioconversion reaction time is 20 hours, and rotating speed is 200 rev/mins.
By any one method of claim 1-6 prepare contain 4 '-biotransformation matrix of '-demethyl podophyllic acid.
An Accessory Right require to isolate in the 7 described biotransformation matrix 4 '-method of '-demethyl podophyllic acid, comprising:
(1) supernatant liquor after centrifugal carries out just separating with macroporous adsorptive resins D312 with biotransformation matrix, carries out gradient elution with aqueous ethanolic solution, the Fractional Collections elutriant;
(2) with collected 20-50% ethanol eluate with gel filtration chromatography segment from, obtain 4 '-'-demethyl podophyllic acid.
9. it is characterized in that in accordance with the method for claim 8: described gel column is a Sephadex LH-20 gel column.
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Non-Patent Citations (3)
| Title |
|---|
| Guo HZ, Guo DA,Fei XY,Cui YJ,Zheng JH.Biotransformation of podophyllotoxin to picropodophyllin by microbes.《J Asian Nat Prod Res》.1998,第1卷(第2期),第99-102页. * |
| 张辅民,田暄.新的4".去甲表鬼臼衍生物的合成及其抗癌活性.《化学学报》.2002,第60卷(第4期),第720-724页. * |
| 曹长年，米琴，王彦广.硒代-4-脱氧-4′-去甲表鬼臼毒素衍生物的合成研究.《青海大学学报(自然科学版)》.2004,第22卷(第4期),第57-58,72页. * |
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