CN101492704A - Method for converting podophyllinic acid lactone into podophyllic acid and picropodophyllin - Google Patents
Method for converting podophyllinic acid lactone into podophyllic acid and picropodophyllin Download PDFInfo
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Abstract
The invention discloses a method for converting podophyllotoxin to podophyllic acid and picropodophyllin, comprising the following steps: the substrate podophyllotoxin solution is added during the fermentation process of microorganism pseudomonas aeruginosa, bacillus, rhodococcus erythropolis, clostridium, corynebacterium pekinense, hay bacillus, erwinia uredovora or bending pseudomonas for carrying out biotransformation reaction, thus obtaining biotransformation matrix with the podophyllic acid and picropodophyllin. The invention also discloses a method for separating the podophyllic acid and picropodophyllin from the obtained 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 respectively obtaining the picropodophyllin and the podophyllic acid. In the invention, modification is carried out on the picropodophyllin structure by microbiological transformation, thus obtaining the picropodophyllin and the podophyllic acid, in addition, the conversion rate of the substrate 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 and a kind of podophyllotoxin is converted into the method for Podophyllinic acid and picropodophyllotoxin, the invention still further relates to the method for from biotransformation matrix, separating Podophyllinic acid and picropodophyllotoxin, belong to field of biological pharmacy.
Background technology
Podophyllotoxin (Podophyllotoxin) is to separate a kind of natural radioactivity lead compound with antitumor action that obtains from Podophyllum emodi var chinense class plant, the molecular structure uniqueness has the aryl substituent group that four chiral centres, trans lactonic ring and C-1 position axially join.Podophyllotoxin is by destroying the tubulin carrying out in the mitotic cell and assemble and the formation of microtubule, make cell mitogen stop at the M phase, suppress the tumour cell proper splitting, the performance antitumor action, but it has the intensive toxic side effect, low etc. as poorly water-soluble, bioavailability, limited its application clinically.
Chinese scholars since last century the '30s carried out the chemical conversion research of podophyllotoxin successively, part scholar utilizes chemical process to modify podophyllotoxin and obtains Podophyllinic acid and picropodophyllotoxin, its method is as follows:
1, Borsche was substrate in 1932 with the podophyllotoxin, synthetic respectively Podophyllinic acid and picropodophyllotoxin, and reaction scheme is seen Fig. 1.
2, the Auterhoff of Tuebingen, Germany university was substrate in 1958 with the podophyllotoxin, chemosynthesis Podophyllinic acid and picropodophyllotoxin, and reaction scheme is seen Fig. 2.
3, Sandoz company (NL 6405480) was substrate in 1964 with the podophyllotoxin, chemosynthesis Podophyllinic acid and picropodophyllotoxin, yield is respectively 3.93% and 3.87%, reaction scheme is seen Fig. 3.
Chemical conversion reaction exist product yield lower, 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.
Chinese scholars has been made desk study to the bio-transformation of podophyllotoxin.The discovery U.S. such as the Broomhead of Univ Nottingham UK gold clock capsule of weeping forsythia suspended culture cell has the ability that podophyllotoxin is oxidized to podophyllotoxone, produces a spot of Podophyllum emodi var chinense picrotone simultaneously.The Teng of Univ Melbourne Australia etc. transform podophyllotoxin with the barley suspended culture cell and obtain isopicropodophyllone.The fruit Dean professor of Peking University utilized Penicillium notatum and sorrel cell transformation podophyllotoxin respectively at 1998 and 2005, obtained picropodophyllotoxin, and reaction scheme is seen Fig. 4.Up to now, still do not utilize bioconversion method to prepare the relevant report of Podophyllinic acid both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention is to overcome in the existing preparation Podophyllum emodi var chinense lignans compound method problems such as existing product yield is low, easy contaminate environment, a kind of new preparation Podophyllinic acid and the bio-transformation and the separation method of picropodophyllotoxin are provided, this method with podophyllotoxin as reaction substrate, obtain Podophyllinic acid and picropodophyllotoxin by bio-transformation, the inventive method is easy and simple to handle, efficiency of pcr product is high, environmental friendliness, production cost are low.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of podophyllotoxin is converted into the method for Podophyllinic acid and picropodophyllotoxin, comprises:
At microorganism Pseudomonas aeruginosa (Pseudomonas aeruginosa), genus bacillus (Bacillus sp.), Rhodococcus (Rhodococcus erythropolis), clostridium (Bacillus fusiformis), Beijing rod bacillus (Corynebacterium pekinenese), Bacillus subtilus (Bacillus subtilis), in the fermenting process of uredo erwinia phage (Erwiniauredovora) or crooked pseudomonas (Pseudomonas geniculata), substrate podophyllotoxin solution joined in any microbial fermentation system carry out bioconversion reaction, obtain containing the biotransformation matrix of Podophyllinic acid and picropodophyllotoxin.
Described Pseudomonas aeruginosa, genus bacillus, Rhodococcus, clostridium, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage, crooked pseudomonas can be bought by various commercial sources and obtain, for example Pseudomonas aeruginosa can be available from China typical culture collection center, and it is numbered CCTCC AB93066;
The culturing process of described Pseudomonas aeruginosa, genus bacillus, Rhodococcus, clostridium, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage 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 Pseudomonas aeruginosa 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 (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 Pseudomonas aeruginosa bacterial classification was cultivated 4 hours for 37 degrees centigrade;
(2) liquid seeds is cultivated: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, sucrose 1%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks, sterilized 30 minutes sterilization postcooling, inoculation slant strains, 37 degrees centigrade for 115 degrees centigrade, cultivated 3 hours for 200 rev/mins, as liquid seeds;
(3) fermentation culture: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, sucrose 1%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, the sterilization postcooling, the inoculation liquid seeds, 10%, 37 degree centigrade of inoculum size was cultivated 3 hours for 200 rev/mins.
Preferably, described Rhodococcus culturing process is as follows:
(1) slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 1.0%, sodium-chlor 0.5%, agar 1.5%, the pH value is 7,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Rhodococcus bacterial classification was cultivated 12 hours for 30 degrees centigrade;
(2) liquid seeds is cultivated: substratum: extractum carnis 0.3%, peptone 1.0%, sodium-chlor 0.5%, pH value are 7, liquid amount 50 milliliters/250 ml shake flasks, sterilized 30 minutes for 115 degrees centigrade, the sterilization postcooling, inoculation slant strains, 30 degrees centigrade, cultivated 10 hours for 180 rev/mins, as liquid seeds;
(3) fermentation culture: 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 liquid seeds, 10%, 30 degree centigrade of inoculum size was cultivated 10 hours for 180 rev/mins.
Preferably, the excellent bacillus culturing process in described Beijing is as follows:
(1) slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2.0%, the pH value is 7,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Beijing rod bacillus species was cultivated 12 hours for 28 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 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;
(3) 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 12 hours for 10%, 28 degree centigrade, 140 rev/mins.
Described podophyllotoxin solution is prepared with dehydrated alcohol, and its concentration is preferably the podophyllotoxin that every milliliter of dehydrated alcohol contains the 2.5-20 milligram, and more preferably every milliliter of dehydrated alcohol contains 5.0 milligrams podophyllotoxin.
In order to reach better bio-transformation effect, described bioconversion reaction preferably carries out according to following condition: with substrate podophyllotoxin solution add in the microbial fermentation system to its final concentration be the 20-1000 mcg/ml, the bioconversion reaction temperature is 20-45 degree centigrade, the conversion reaction time is 3-200 hour, and rotating speed is 50-350 rev/min.
Particularly preferred technical scheme of the present invention: with podophyllotoxin solution add in the microbial fermentation system to its final concentration be 90 mcg/ml, the bioconversion reaction temperature is 37 degrees centigrade, rotating speed is 200 rev/mins, the bio-transformation time is 20 hours.
The present invention utilizes Pseudomonas aeruginosa, genus bacillus, Rhodococcus, clostridium, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage or crooked pseudomonas to transform podophyllotoxin first, the preparation separation obtains Podophyllinic acid and picropodophyllotoxin, product yield height, reaction conditions gentleness, simple to operate.
The biotransformation matrix of above-mentioned gained is carried out high performance liquid chromatography (HPLC) analysis and thin-layer chromatography (TLC) mensuration, and the result shows that resulting bioconversion product is the mixture of Podophyllinic acid and picropodophyllotoxin.
Another technical problem to be solved by this invention provides a kind of method that Podophyllinic acid and picropodophyllotoxin are separated from above-mentioned biotransformation matrix.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method of separating Podophyllinic acid and picropodophyllotoxin from above-mentioned biotransformation matrix comprises:
(1) supernatant liquor after centrifugal carries out just separating with macroporous adsorptive resins with biotransformation matrix, carries out gradient elution with aqueous ethanolic solution, the Fractional Collections elutriant;
(2) with collected 30%-40% ethanol eluate, with gel filtration chromatography segment from, obtain Podophyllinic acid;
(3) with collected 60%-80% ethanol eluate, concentrate, remove by filter insoluble impurities, crystallization gets picropodophyllotoxin.
Preferably: centrifugal described in the step (1), preferable methods are with biotransformation matrix under 5500 rev/mins rotating speed centrifugal 30 minutes; Described aqueous ethanolic solution is the aqueous ethanolic solution of 10%-90%; Described macroporous adsorbent resin model is preferably D312, AB-8, S-8, ADS-7, YWD04C, YWDBC, DM130, MCA, 860021, DM-18; More preferably D312 (available from resin subsidiary factory of anti-medical limited-liability company in the Shandong, Shandong, nonpolar, particle diameter: 0.315-1.25 millimeter, specific surface area: 500-650 meters squared per gram, mean pore size: the 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 directly add podophyllotoxin solution and carry out bio-transformation in the fermenting process of Pseudomonas aeruginosa, genus bacillus, Rhodococcus, clostridium, Beijing rod bacillus, Bacillus subtilus, uredo erwinia phage or crooked pseudomonas, the product yield height can reach 70.2% to the molar yield of substrate podophyllotoxin.
Present method utilizes microbial transformation that the podophyllotoxin structure is modified, and obtains Podophyllinic acid and picropodophyllotoxin, substrate conversion efficiency height, simple to operate; The waste of bioconversion reaction mild condition, generation is less; The bioconversion product sepn process is simple, and rate of recovery height.
In a word, microbe transformation 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, and agents useful for same toxicity is low, expense is low, save cost.
Description of drawings
The chemical conversion route map of Fig. 1 Podophyllinic acid and picropodophyllotoxin;
The chemical synthesis route figure of Fig. 2 Podophyllinic acid and picropodophyllotoxin;
The chemical reaction route map of Fig. 3 Podophyllinic acid and picropodophyllotoxin;
Fig. 4 Penicillium notatum, sorrel cell are to the bio-transformation of podophyllotoxin;
The preparation of Fig. 5 Podophyllinic acid of the present invention and picropodophyllotoxin and separation process figure.
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:
Pseudomonas aeruginosa, available from China typical culture collection center (Wuhan University), it is numbered CCTCCAB93066; Genus bacillus, available from China typical culture collection center (Wuhan University), it is numbered CCTCCAB95018; Rhodococcus, available from Chinese common micro-organisms culture presevation administrative center, it is numbered CGMCC1.2362; Clostridium, available from Chinese industrial microbial strains preservation center, it is numbered CICC 20463; Beijing rod bacillus, available from China typical culture collection center (Wuhan University), it is numbered CCTCCAB97005; Bacillus subtilus, available from China typical culture collection center (Wuhan University), it is numbered CCTCCAB93174; Uredo erwinia phage, available from Chinese common micro-organisms culture presevation administrative center, it is numbered CGMCC 1.1215; Crooked pseudomonas, available from China typical culture collection center (Wuhan University), it is numbered CCTCC AB93074.
Two, detection method
(1) HPLC of podophyllotoxin, Podophyllinic acid and picropodophyllotoxin analyzes: the post model is Akasil C18 column (5 microns, 4.6 millimeters * 250 millimeters); Moving phase is 0.05 mol potassium primary phosphate-methyl alcohol-acetonitrile (52: 32: 16), and the pH value is 3.00; Flow velocity is 0.8 ml/min, and the ultraviolet detection wavelength is 230 nanometers, and column temperature is 40 degrees centigrade.
(2) TLC of podophyllotoxin, Podophyllinic acid and picropodophyllotoxin analyzes: silica gel G chromatoplate (Qingdao Marine Chemical Co., Ltd.), with biotransformation matrix to be measured with ethyl acetate extraction after standing demix, ethyl acetate layer concentrates and volatilizes, dehydrated alcohol redissolves, with kapillary point sample on exsiccant silica gel G chromatoplate, chromatoplate is put into the chromatography cylinder chromatography.Developping agent is chloroform-ethyl acetate-methyl alcohol-glacial acetic acid (7: 3: 2: 0.5).Iodo steam displaing color presents the tawny spot.
Embodiment 1
1, the cultivation of Pseudomonas aeruginosa:
Slant strains is cultivated: substratum: glucose 2%, extractum carnis 0.3%, peptone 0.5%, sodium-chlor 0.5%, agar 2%, pH value are 7,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation Pseudomonas aeruginosa bacterial classification was cultivated 4 hours for 37 degrees centigrade;
Liquid seeds is cultivated: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, sucrose 1%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks, sterilized 30 minutes sterilization postcooling, inoculation slant strains, 37 degrees centigrade for 115 degrees centigrade, cultivated 3 hours for 200 rev/mins, as liquid seeds;
Fermentation culture: substratum: yeast powder 0.5%, peptone 0.5%, sodium-chlor 0.5%, sucrose 1%, the pH value is 7, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, the sterilization postcooling, the inoculation liquid seeds, 10%, 37 degree centigrade of inoculum size was cultivated 3 hours for 200 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 5.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin solution join in the Pseudomonas aeruginosa fermentation system to its final concentration be 90 mcg/ml, carry out bio-transformation under the following conditions: 37 degrees centigrade of temperature of reaction, rotating speed is 200 rev/mins, the bio-transformation time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 90 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 27 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 70%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
5500 rev/mins of above-mentioned biotransformation matrix is centrifugal 30 minutes, discard precipitation, supernatant liquor directly with the D312 macroporous adsorptive resins (available from resin subsidiary factory of anti-medical limited-liability company in the Shandong, Shandong, nonpolar, particle diameter: 0.315-1.25 millimeter, specific surface area: 500-650 meters squared per gram, mean pore size: the 10-20 nanometer) absorption, with alcohol-water (1: 9 to 9: 1) gradient elution, 60%-80% ethanol elution component is concentrated, filter, get coarse crystallization, with the dehydrated alcohol recrystallization, get white needle-like crystals, be defined as picropodophyllotoxin (C by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS)
22H
22O
8 1H NMR (300MHz, CDCl
3): δ 7.12 (1H, H-5), 6.57 (2H, H-2 ', H-6 '), 6.19 (1H, H-8), 5.91 (2H, H-13), 4.63 (1H, H-11), 4.49 (1H, H-4), 4.45 (1H, H-11), 3.98 (1H, H-1), (3.84 9H, MeO-3 ', MeO-4 ', MeO-5 '), 3.28 (1H, H-2), 2.65 (1H, H-3).
13C?NMR(300MHz，CDCl
3)：δ178.92(C-12)，153.07(C-3’，C-5’)，146.45(C-6，C-7)，139.28(C-1’)，136.37(C-10)，133.50(C-9)，130.45(C-4’)，107.68(C-8)，105.86(C-2’，C-6’)，104.16(C-5)，100.63(C-13)，69.62(C-4)，67.62(C-11)，59.89(MeO-4′)，55.30(MeO-3′，MeO-5′)，44.98(C-2)，43.74(C-1)，42.57(C-3)。ESI-MS(m/z)485.1(M
++H)，442.9，292.7。Ultimate analysis C
22H
22O
8H
2O: calculated value C, 61.11%, H, 5.59%, measured value C, 61.85; H, 5.747).
30%-40% ethanol elution component concentrated volatilize, methyl alcohol redissolves, remove by filter insoluble impurities, the SephadexLH-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 gets white unsetting powder, is defined as Podophyllinic acid (C by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS)
22H
24O
9,
1H NMR (300MHz, CD
3OD): δ 6.83 (1H, H-5), 6.54 (2H, H-2 ', H-6 '), 6.24 (1H, H-8), 5.85 (2H, H-13), 4.86 (1H, H-4), 4.20 (1H, H-1), 3.76 (6H, MeO-3 ', MeO-5 '), 3.73 (3H, MeO-4 '), 3.41 (1H, H-11), 3.38 (1H, H-11), 3.30 (1H, H-2), 2.45 (1H, H-3).
13C?NMR(300MHz，CD
3OD)：δ172.35(C-12)，153.03(C-3’，C-5’)，147.74(C-6)，146.60(C-7)，142.00(C-1’)，136.21(C-10)，132.20(C-9)，129.93(C-4’)，109.18(C-8)，108.82(C-5)，106.69(C-2’，C-6’)，101.08(C-13)，68.29(C-4)，60.21(C-11)，59.90(MeO-4′)，55.35(MeO-3′，MeO-5′)，46.98(C-2)，45.34(C-1)，45.06(C-3)。ESI-MS(m/z)431.1(M
++H)，421.1，387.1，324.9，283.0，255.0)。
Embodiment 2
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 7,115 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 10%, 28 degree centigrade, 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 5.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol; With podophyllotoxin solution join in the fermentation of bacillus system to its final concentration be 172 mcg/ml, carry out bio-transformation under the following conditions: 28 degrees centigrade of temperature of reaction, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 172 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 75 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 56%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purification method identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that 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 7,115 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, and liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation slant strains, 30 degrees centigrade, cultivated 10 hours for 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, liquid amount 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, 10%, 30 degree centigrade of inoculum size was cultivated 10 hours for 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 2.5 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin solution join in the Rhodococcus fermentation system to its final concentration be 50 mcg/ml, carry out bio-transformation under the following conditions: 30 degrees centigrade of temperature of reaction, rotating speed is 180 rev/mins, the conversion reaction time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 50 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 22 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 56.0%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purification method identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
Embodiment 4
1, clostridial cultivation:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2%, the pH value is 7,115 degrees centigrade of sterilizations 30 minutes, sterilization postcooling, bevel, inoculation clostridium 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 6 hours for 37 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 50 milliliters/250 ml shake flasks were sterilized 30 minutes for 115 degrees centigrade, sterilization postcooling, inoculation liquid seeds, inoculum size was cultivated 6 hours for 10%, 37 degree centigrade, 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 5.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol; With podophyllotoxin solution join in the clostridia fermentation system to its final concentration be 46 mcg/ml, carry out bio-transformation under the following conditions: 37 degrees centigrade of temperature of reaction, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 46 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 20 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 56%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purifying technique identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
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 7,115 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, 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: 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 12 hours for 10%, 28 degree centigrade, 140 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 10 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin solution join in Beijing rod bacillus fermentation system to its final concentration be 100 mcg/ml, carry out bio-transformation under the following conditions: 28 degrees centigrade of temperature of reaction, rotating speed is 140 rev/mins, the conversion reaction time is 10 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 100 mcg/ml, transform 10 hours artifacts and transform that podophyllotoxin concentration is 54 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 46%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purifying technique identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
Embodiment 6
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 7,115 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 10%, 30 degree centigrade, 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 10.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin 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 of reaction, rotating speed is 180 rev/mins, the bio-transformation time is 12 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 127 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 80 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 37%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purifying technique identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
Embodiment 7
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 7,115 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 10%, 30 degree centigrade, 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 5.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin solution join in the uredo erwinia phage fermentation system to its final concentration be 62 mcg/ml, carry out bio-transformation under the following conditions: 30 degrees centigrade of temperature of reaction, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 62 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 42 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 32%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purifying technique identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
Embodiment 8
1, the cultivation of crooked pseudomonas:
Slant strains is cultivated: substratum: extractum carnis 0.3%, and peptone 0.5%, sodium-chlor 0.5%, agar 2.0%, the pH value is 7,115 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 10%, 25 degree centigrade, 180 rev/mins.
2, bio-transformation
It is the podophyllotoxin solution that every milliliter of ethanol contains 10.0 milligrams of podophyllotoxins that podophyllotoxin is mixed with concentration with dehydrated alcohol, with podophyllotoxin solution join in the crooked pseudomonas fermentation system to its final concentration be 109 mcg/ml, carry out bio-transformation under the following conditions: 25 degrees centigrade of temperature of reaction, rotating speed is 180 rev/mins, the bio-transformation time is 20 hours, obtain containing the biotransformation matrix of picropodophyllotoxin and Podophyllinic acid, from this biotransformation matrix, take a sample, HPLC measures: the podophyllotoxin initial concentration is 109 mcg/ml, transform 20 hours artifacts and transform that podophyllotoxin concentration is 78 mcg/ml in the matrix, the podophyllotoxin transformation efficiency is 28%.
3, the separation and purification of product and structure are identified in the biotransformation matrix
Adopt the separation purifying technique identical with embodiment 1, obtain white needle-like crystals and white unsetting powder from biotransformation matrix, by nucleus magnetic resonance (NMR) and mass spectrum (ESI-MS), white needle-like crystals is confirmed as picropodophyllotoxin; The unsetting powder of white is confirmed as Podophyllinic acid (structure identifies that parameter is with embodiment 1).
Claims (10)
1, a kind of podophyllotoxin is converted into the method for Podophyllinic acid and picropodophyllotoxin, comprises:
At microorganism Pseudomonas aeruginosa (Pseudomonas aeruginosa), genus bacillus (Bacillus sp.), Rhodococcus (Rhodococcus erythropolis), clostridium (Bacillus fusiformis), Beijing rod bacillus (Corynebacterium pekinenese), Bacillus subtilus (Bacillus subtilis), in the fermenting process of uredo erwinia phage (Erwiniauredovora) or crooked pseudomonas (Pseudomonas geniculata), substrate podophyllotoxin solution joined in above-mentioned any microbial fermentation system carry out bioconversion reaction, obtain containing the biotransformation matrix of Podophyllinic acid and picropodophyllotoxin.
2, in accordance with the method for claim 1, it is characterized in that: described substrate podophyllotoxin solution is prepared with dehydrated alcohol, and its concentration is the podophyllotoxin that every milliliter of dehydrated alcohol contains the 2.5-20 milligram.
3, in accordance with the method for claim 1, it is characterized in that: with podophyllotoxin solution join in the microbial fermentation system to its final concentration be the 20-1000 mcg/ml.
4, in accordance with the method for claim 3, it is characterized in that, with podophyllotoxin solution join in the microbial fermentation system to its final concentration be 90 mcg/ml.
5, in accordance with the method for claim 1, it is characterized in that described bioconversion reaction carries out according to following condition: the bioconversion reaction temperature is 20-45 degree centigrade, and the conversion reaction time is 3-200 hour, and rotating speed is 50-350 rev/min.
6, in accordance with the method for 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 bio-transformation time is 20 hours, and rotating speed is 200 rev/mins.
7, prepare the biotransformation matrix that contains Podophyllinic acid and picropodophyllotoxin by any one method of claim 1-6.
8, a kind of Accessory Right requires to isolate in the 7 described biotransformation matrix method of Podophyllinic acid and picropodophyllotoxin, comprising:
(1) supernatant liquor after centrifugal carries out just separating with macroporous adsorptive resins with biotransformation matrix, carries out gradient elution with aqueous ethanolic solution, the Fractional Collections elutriant;
(2) with collected 30%-40% ethanol eluate with gel filtration chromatography segment from, obtain Podophyllinic acid;
(3) with collected 60%-80% ethanol eluate, concentrate, remove by filter insoluble impurities, crystallization gets picropodophyllotoxin.
9, in accordance with the method for claim 8, it is characterized in that: described macroporous adsorbent resin is selected from macroporous adsorbent resin D312, AB-8, S-8, ADS-7, YWD04C, YWDBC, DM130, MCA, 860021 or DM-18.
10, in accordance with the method for claim 8, it is characterized in that: described gel column is a Sephadex LH-20 gel column.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234669A (en) * | 2010-04-29 | 2011-11-09 | 湖北工业大学 | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin |
| CN103189381A (en) * | 2010-08-31 | 2013-07-03 | 阿克塞拉公司 | New process for preparing cyclolignans |
| US9314525B2 (en) | 2010-10-08 | 2016-04-19 | Axelar Ab | Picropodophyllin polymorph C and its use in cancer therapy |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234669A (en) * | 2010-04-29 | 2011-11-09 | 湖北工业大学 | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin |
| CN102234669B (en) * | 2010-04-29 | 2013-10-16 | 湖北工业大学 | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin |
| CN103189381A (en) * | 2010-08-31 | 2013-07-03 | 阿克塞拉公司 | New process for preparing cyclolignans |
| US8987475B2 (en) | 2010-08-31 | 2015-03-24 | Alexar AB | Process for preparing cyclolignans |
| CN103189381B (en) * | 2010-08-31 | 2016-06-08 | 阿克塞拉公司 | Prepare the new method of cyclolignan |
| US9314525B2 (en) | 2010-10-08 | 2016-04-19 | Axelar Ab | Picropodophyllin polymorph C and its use in cancer therapy |
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