CN101319199B - Method for cell abduction generation of taxone with Chinese yew - Google Patents
Method for cell abduction generation of taxone with Chinese yew Download PDFInfo
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Abstract
The invention relates to a plant cell culture technology, in particular to a method for inducible production of taxane by utilization of Chinese yew cells. The method comprises the following steps that: the Chinese yew cells are placed into a commonly used culture medium for culture, and the cell inoculum size is between 50 and 200 grams per liter green weight; in the initial stage of the exponential growth of the cells, a revulsant with a concentration of between 1 and 2,000 micrometers, 50 to 200 grams of sterile absorbent and 10 to 50 grams of sugar are added into per liter of the culture medium; and in the intermediate stage of the exponential growth of the cells, the revulsant with a concentration of between 1 and 2,000 micrometers is added for the second time; and the revulsant is an abiological revulsant or a fungal elicitor. Because a large part of taxane compounds are externally secreted to the outsides of the cells and absorbed on the absorbent, the operation of separation and purification is greatly simplified, thereby the cost is reduced and the industrialized production is easy to realize. Moreover, because the method can produce novel taxane compounds by means of induction, the taxane is taken as a precursor compound of a first-line anticarcinogenic drug, namely paclitaxel, and the technology has important commercial application prospect.
Description
Technical field
The present invention relates to plant cell culture technology, specifically a kind of taxus chinensis cell that increases substantially is cultivated the output of producing Taxan and is impelled its outer secreting, the method for inducing new taxane compounds to produce simultaneously.
Background technology
Taxol is as a line medication of treatment advanced ovarian cancer, mammary cancer, it is a few one of natural compounds the most with anticancer activity up to now, the sales volume that multi-million dollar is arranged every year, but the medicine source problem remains the matter of utmost importance that the taxol investigators will solve.It is few that the whole world can produce the floristics of taxol, poor growth, and the content of taxol is extremely low, even on average also have only 5/10000ths in the highest bark of content.And obtain the taxol havoc eubiosis and species diversity by the felling Chinese yew genus plants, cause resource exhaustion the most at last.Though the complete synthesis success already of taxol chemistry, synthetic route is long, and productive rate is low, and the cost height can not be commercially produced.Semi-synthetic is the main source of present clinical application taxol; baccatinIII (baccatin III) or the 10-deacetylbaccatin III (10-deacetylation baccatin III) of semisynthetic precursor for obtaining by separation in the Ramulus et folium taxi cuspidatae, but still do not satisfy needs clinical and experiment far away.Utilize the culture plant cell method produce taxol because of its environmental friendliness, with short production cycle, controllability is strong etc., and advantage is subject to people's attention day by day.But not paclitaxel produced or taxol yield hangs down and remains the difficult problem that this technology generally faces.
When making great efforts to expand the medicine source of taxol and improving taxol output, scientist also joins in the research of bearing taxanes, and expectation is found the better bearing taxanes of antitumour activity or found suitable lead compound.There has been hundreds of kind Taxan from former plant of Japanese yew or cell culture fluid, to separate.TAXUYUNNANINE C Tc (Taxuyunnanine C) is a kind of active compound of class nerve growth factor (NGF) that has, and can strengthen the effect of NGF, helps the treatment of senile dementia (Alzheimer ' s disease).Tc and taxol belong to the tricyclic diterpene compounds.Its structural formula is as follows:
Tc is presumably the metabolic intermediate in the taxol biosynthetic pathway, can be used as the synthetic precursor of taxol or other useful compound, has the potential commercial value.Since in Chinese Ramulus et folium taxi cuspidatae suspension cell culture system, finding the higher Tc of content, the bio-reactor National Key Laboratory at the clock Kien Giang professor place of East China University of Science uses various control techniques to improve the output of Tc, makes Tc output reach 612mg/L in 1 liter of airlift reactor.They awarded cooperation with the money rising sun Red Sect of Lamaism again in the laboratory afterwards, used the new elicitor of latter's synthetic, made to shake that the production peak of Tc reaches 827mg/L in the bottle, and the production peak of Tc reaches 738mg/L in the reactor.It is 885.9mg/L that Choi et al.Tc cultivates the production peak that obtained Tc on the 35th day at taxus chinensis cell.Take a broad view of and improve the employed various control techniques of Tc output, all be conceived to the regulation and control to the Tc biosynthetic pathway, less relevance is stored Tc artifact route of synthesis-the be synthetic back of Tc and the regulation and control of transhipment aspect.This is the inventive point place of our this technology just: by add suitable sorbent material in Chinese Ramulus et folium taxi cuspidatae suspension cell culture system, make Tc by being adsorbed onto efficiently outside the born of the same parents in the born of the same parents, the injury of cell growth when thereby solution high-content Tc is accumulated in the born of the same parents is to checking that Tc further produces.This both also regulates and control the technology that biosynthetic process is thereafter regulated and control to the Tc biosynthetic process has increased space and potentiality that Tc output further improves greatly.
In Chinese Ramulus et folium taxi cuspidatae suspension culture system, induce, mend that sugar adds and the sorbent material combined regulating improves TAXUYUNNANINE C output by inductor, yet there are no report.
Summary of the invention
The purpose of this invention is to provide a kind of Chinese Ramulus et folium taxi cuspidatae (Taxus chinensis) suspension culture system that utilizes, add the combined regulating means that combine with inductor superinduce, benefit sugar and sorbent material and improve the method that TAXUYUNNANINE C output induces new taxane compounds to produce simultaneously.
For achieving the above object, the technical solution used in the present invention is: place its substratum commonly used to cultivate taxus chinensis cell, the cell inoculation amount is 50～200g/L weight in wet base, at the cell index early growth period, adding concentration in every liter of substratum is the inductor of 1～2000 μ M, the no bacteria adsorbent of 50-200g and the sugar of 10～50g; Grow mid-term at cell index, add the inductor that concentration is 1～2000 μ M once more; Described inductor is abiotic inductor or fungal elicitor.
Described abiotic inductor is Silver Nitrate, Whitfield's ointment or jasmonic and derivative thereof, and described sugar is sucrose or glucose; Described derivative is methyl jasmonic ketone ester (MJA), 2-hydroxyethyl jasmonic (HEJA), 3-fluoro ethyl jasmonic (TFEJA) or 2,3-dihydroxypropyl jasmonic (DHPJA).
The culture condition of taxus chinensis cell is: rotary shaking table, and rotating speed 60～200rpm, culture temperature is 20～30 ℃, the dark cultivation; Culture cycle is 15～30 days, goes down to posterity once every 14-20 days; Described cell index early growth period is meant cell inoculation the 4th～10 day behind substratum, promptly adds the time first time of inductor; The cell index growth is meant cell inoculation the 10th～15 day behind substratum mid-term, promptly adds the time second time of inductor;
Add for the first time inductor and add for the second time inductor during also can add Taxan synthetic precursor 0.25～2.0 gram/every liter of substratum; Synthetic precursor can be sodium-acetate or phenylformic acid.
Described sorbent material is treated bearing taxanes to be had stronger adsorptive power and the nontoxic porous material of pair cell, as various macroporous adsorbent resins.
The substratum commonly used of taxus chinensis cell consists of, and adds 0.1～1.0mg/L 6-benzyladenine in the MS basic medium, and 0.1～0.5mg/L 2, the 4-dichlorphenoxyacetic acid, 0.1～1.0mg/L naphthylacetic acid, 20～200mg/L xitix and 10～60g/L sucrose, the pH value is 5.5～6.5.
The inventive method principle: can significantly improve Tc output at interpolation inductor and the sugared sorbent material that adds particular types and concentration simultaneously of benefit, its major cause has following three aspects: 1, inductor energy effective stimulus vegetable cell secondary metabolism ability promotes the biosynthesizing production of Taxan; 2, can remedy because of the quick carbon source deficiency that causes of growth and metabolism of cell by mending sugar; 3, make the Taxan on the cell walls secrete outside the born of the same parents outward by the particular adsorbent adding, thereby removed the feedback inhibition of product, the injury of cell growth reaches the obstruction to further production when having avoided Taxan to be present in the born of the same parents, has also avoided the degraded of Taxan in born of the same parents simultaneously.
The present invention has following advantage:
1, combined utilization superinduce, interpolation feed supplement and sorbent material promote the production of taxus chinensis cell TAXUYUNNANINE C Tc, and the output of acquisition is well beyond the maximum of present bibliographical information.At present bibliographical information in shaking bottle, cultivate taxus chinensis cell, the production peak of its TAXUYUNNANINE C Tc is 827 ± 29mg/L, and these control measures can make the Tc production peak reach 1830.42 ± 62.69mg/L, is 2.2 times of bibliographical information maximum.
2, the invention enables TAXUYUNNANINE C Tc to secrete more than 90% outward, be adsorbed in the sorbent material.The operation that this has just simplified the extraction separation purifying has greatly reduced cost, for further suitability for industrialized production provides very big possibility.
3, the present invention has also obtained two new Taxans simultaneously, for the research of Taxan and taxol biosynthetic pathway provides new clue.In the combined induction culture system, two kinds of new Taxans that the extraction separation purifying obtains are: 2-deacetylation TAXUYUNNANINE C C and 2,14-deacetylation TAXUYUNNANINE C C.2,14-deacetylation TAXUYUNNANINE C C is the new taxane compounds of finding first.
4, the present invention also can combine with other technology such as precursor interpolations, further improves the output of Taxan.
5, cell culture condition of the present invention can guarantee cell growth and assurance favorable uniformity fast.
Description of drawings
Fig. 1 is the inside and outside distribution condition figure of the 3rd group of experiment of embodiment 3 Tc born of the same parents.
Embodiment
Below will be described further related content of the present invention by embodiment.
A kind of taxus chinensis cell that utilizes is induced the method that produces Taxan, may further comprise the steps:
(1) cultivation of taxus chinensis cell:
The strain of this experimental cell is at first by the young stem inductive of Chinese Ramulus et folium taxi cuspidatae (T.Chinesis) callus suspension culture, adopt the disclosed Murashige-Skoog of document (MS) substratum, add 0.1～1.0mg/L 6-benzyladenine, 0.1～0.5mg/L 2, the 4-dichlorphenoxyacetic acid, 0.1～1.0mg/L naphthylacetic acid, 20～200mg/L xitix and 10～60g/L sucrose, the pH value is 5.5～6.5.Went down to posterity once every 14 days, use rotary shaking table, rotating speed 60～200rpm, culture temperature is 20～30 ℃, the dark cultivation.
(2) the combined induction production of Tc:
The taxus chinensis cell that step (1) is obtained places the bottle that shakes that contains above-mentioned substratum to cultivate, and the cell inoculation amount is 50～200g/L weight in wet base.Culture condition is: rotary shaking table, and rotating speed 60～200rpm, culture temperature is 20～30 ℃, the dark cultivation.Culture cycle is 15～30 days.
Utilize inductor to induce in the shake-flask culture process, in cell index growth early stage with once induce respectively mid-term, the ultimate density of inductor interpolation is 1～2000 μ M in the substratum.
Mend sugar when adding inductor for the first time, additional amount is 10～50g/L, and added sugar is sucrose.
Add sorbent material when adding inductor for the first time, add-on is more than the 50g/L.
Obtain cell part and sorbent material part by the culture separation that obtains, extract Tc respectively.Tc productivity reaches as high as 80.67mg/ (Ld), and production peak can reach 1830.42mg/L.
(3) extraction of Tc and mensuration:
Get 100mg stem cell powder (or 250mg sorbent material), the add 4mL Jia Chun ﹠amp of 1: 1 (v/v); Methylene dichloride, the centrifugal 10min of room temperature supersound extraction 3h and 4000rpm, the supernatant liquor rotary evaporation is dissolved in the 4mL methylene dichloride behind the evaporate to dryness, and with 1mL pure water extraction 4 times, 1 centrifugal 5min of 4000rpm of every extraction discards water layer.The dichloromethane layer rotary evaporation is dissolved in 1mL chromatographically pure methyl alcohol, and 0.22 μ m membrane filtration is HPLC fully.HPLC adopts Agilent 1100 high performance liquid chromatographs, and the detection wavelength is 227nm.102 groups-real-time analysis of chromatographic column adopting Dalian Inst of Chemicophysics, Chinese Academy of Sciences and the Alkyl Phenyl post (4.6mm * 250mm, 5 μ m) that detects seminar's filling, moving phase is acetonitrile: water=58: 42, flow velocity 1.0mL/min, sample size 10 μ L.
(4) preparation of new taxane compounds, purifying and structure are identified:
Collect the sorbent material that has adsorbed Taxan in several the experiments, with 8 times of volumes methanol: 1: 1 (v/v) room temperature of methylene dichloride supersound extraction 3h, the extracting solution rotary evaporation is dissolved in chromatographically pure methyl alcohol, filters, and goes to preparation.Preparation work is finished in 1804 groups-pharmaceutical chemistry seminar of Dalian Inst of Chemicophysics, Chinese Academy of Sciences.Preparative chromatograph is watersDP4000, and the preparative chromatography post is CAPCELL PAK C18 post (Type:UG80, SIZE:20 * 250mm, 5 μ m), and moving phase is acetonitrile: water=60: 40, flow velocity: 15ml/min.
Through preparing two than pure compound, not single point but detect discovery through TLC, continue to do purification by silica gel column chromatography.Two samples are respectively with sherwood oil: acetone=90: 10 and 95: 5 wash-outs, collect the pure product that obtain.Two pure product Shanghai Pharmaceutical Inst., Chinese Academy of Sciences have carried out the structure evaluation, and structural formula is as follows:
2,14-deacetylation TAXUYUNNANINE C C, molecular formula: C
24H
36O
5Molecular weight: 404.26;
2-deacetylation TAXUYUNNANINE C C, molecular formula: C
26H
38O
7Molecular weight: 462.26.
Embodiment 1
Adopt Chinese Ramulus et folium taxi cuspidatae suspension cell;
Culture condition: 100mL shakes a bottle suspension culture, adopts the MS substratum, and other adds the 0.2mg/L 2,4 dichlorophenoxyacetic acid, 0.5mg/L naphthylacetic acid, 0.5mg/L 6-benzyladenine, 100mg/L xitix and 30g/L sucrose.The 2g wet cell that filtration is obtained be inoculated into contain the above-mentioned substratum of 20mL shake in the bottle rotary shaking speed 100rpm, 25 ℃ of cultivations.
Added in the 7th day MJA to ultimate density be 100 μ M, add 100g/L XAD-7 simultaneously, continue to be cultured to 21 days, harvested cell and 50 ℃ of dryings.Cell density reached the highest (dry weight 12.7g/L) at the 15th day.Tc assay determination result such as following table (* represents maximum value):
| Incubation time (my god) | Tc content (mg/gDW) | Tc adsorption rate (mg/gXAD-7) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 7.0±0.64 * | 54.3±0.35 | 4.76±0.03 | |
| 12 | 2.24±0.23 | 2.08±0.36 | 222.34±25.28 | 16.78±1.11 |
| 15 | 2.43±0.12 | 3.67±0.12 | 409.79±29.79 | 25.92±1.05 * |
| 21 | 2.2±0.04 | 4.46±0.56 * | 477.37±29.4 * | 21.73±1.14 |
Obtain the derivative of two kinds of TAXUYUNNANINE C Tc simultaneously: 2,14-deacetylation TAXUYUNNANINE C C and 2-deacetylation TAXUYUNNANINE C C.
Embodiment 2
Culture condition is with embodiment 1, added at the 7th day DHPJA to ultimate density be 100 μ M, add 100g/L XAD-7HP simultaneously, continue to be cultured to 21 days.Cell density reached the highest (dry weight 13.2g/L) at the 12nd day.The throughput of Tc sees the following form.
| Incubation time (my god) | Tc content (mg/gDW) | Tc adsorption rate (mg/gXAD) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 7.49±0.88 * | 56.83±2.58 | 5.12±0.21 | |
| 12 | 2.72±0.26 | 0.36±0.04 | 35.85±6.3 | 1.24±0.16 |
| 15 | 2.91±0.18 | 5.53±0.47 | 597.8±16.94 | 38.45±0.71 |
| 18 | 6.52±0.28 | 7.85±0.62 * | 855.05±28.49 * | 46.33±0.97 * |
| 21 | 1.51±0.11 | 3.42±0.37 | 373.52±7.49 | 16.79±0.46 |
Obtain the derivative of two kinds of TAXUYUNNANINE C Tc simultaneously: 2,14-deacetylation TAXUYUNNANINE C C and 2-deacetylation TAXUYUNNANINE C C.
Embodiment 3
Culture condition added DHPJA to 100 μ M with 1, the first group of embodiment at the 7th day, add sucrose 20g/L simultaneously; Second group was added DHPJA to 100 μ M respectively at the 7th day and the 12nd day, added sucrose 20g/L at the 7th day; The 3rd group was added DHPJA to 100 μ M respectively at the 7th day and the 12nd day, added sucrose 20g/L at the 7th day, added XAD-7 100g/L simultaneously, continued to be cultured to 21 days, and cell density reached the highest (dry weight 18.4g/L) at the 21st day.The throughput of Tc sees the following form.
First group (inducing and mend sugar simultaneously)
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 6.27±0.34 | 44.13±2.9 | 3.30±0.23 |
| 12 | 53.6±3.07 | 504.37±19.76 | 40.28±0.65 |
| 15 | 64.43±2.25 * | 730.07±32.97 * | 47.27±1.12 * |
| 18 | 55.92±2.69 | 702.85±4.7 | 37.88±0.09 |
| 21 | 23.76±0.56 | 464.35±36.58 | 21.11±0.88 |
Second group (mending sugar and superinduce)
| Incubation time (my god) | Tc content (mg/gDW) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 6.27±0.34 | 44.13±2.9 | 3.30±0.23 |
| 12 | 56.87±2.58 | 520.27±30.4 | 41.61±1.53 |
| 15 | 68.47±4.34 * | 725.77±28.34 * | 46.98±0.87 * |
| 18 | 26.73±0.58 | 370.12±25.4 | 19.4±0.38 |
| 21 | 25.89±0.56 | 522.82±29.04 | 23.9±0.42 |
The 3rd group (mend sugar, add sorbent material and superinduce)
| Incubation time (my god) | Tc content (mg/gDW) | Tc adsorption rate (mg/gXAD) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 6.27±0.34 | 44.13±2.9 | 3.30±0.23 | |
| 12 | 8.39±0.99 | 3.54±0.23 | 454.47±39.7 | 36.12±2.31 |
| 15 | 9.23±1.81 * | 8.5±0.54 | 982.43±43.14 | 64.1±1.87 |
| 18 | 7.56±0.76 | 11.91±0.38 | 1343.26±108.87 | 73.46±4.56 |
| 21 | 5.15±0.62 | 15.45±0.63 * | 1715.13±124.12 * | 80.67±4.90 * |
Obtain the derivative of two kinds of TAXUYUNNANINE C Tc simultaneously: 2,14-deacetylation TAXUYUNNANINE C C and 2-deacetylation TAXUYUNNANINE C C.
Embodiment 4
Culture condition added DHPJA to 100 μ M with embodiment 1 respectively at the 7th day and the 12nd day, added sucrose 20g/L at the 7th day, added XAD-7HP 100g/L simultaneously, continued to be cultured to 30 days.Cell density reached the highest (dry weight 17.1g/L) at the 18th day.The throughput of Tc sees the following form.
| Incubation time (my god) | Tc content (mg/gDW) | Tc adsorption rate (mg/gXAD-7HP) | Tc output (mg/L) | Tc productive rate (mg/ (Ld)) |
| 7 | 9.08±1.35 * | 62.25±13.89 | 5.89±0.31 | |
| 12 | 2.28±0.14 | 2.13±0.33 | 230.02±15.1 | 17.42±0.41 |
| 15 | 2.1±0.21 | 3.95±0.47 | 411.02±7.35 | 26.0±0.27 |
| 18 | 4.79±1.13 | 7.29±1.52 | 700.14±32.52 | 37.73±1.30 |
| 21 | 3.39±0.9 | 8.83±0.46 | 837.92±7.99 | 38.90±0.16 |
| 24 | 1.48±0.26 | 16.93±0.68 | 1602.9±166.25 | 65.91±2.33 |
| 27 | 1.52±0.03 | 18.38±0.42 * | 1830.42±62.69 * | 67.02±1.94 * |
| 30 | 2.34±0.39 | 12.44±0.72 | 1252.55±17.25 | 41.05±0.27 |
Obtain the derivative of two kinds of TAXUYUNNANINE C Tc simultaneously: 2,14-deacetylation TAXUYUNNANINE C C and 2-deacetylation TAXUYUNNANINE C C.
Claims (2)
1. one kind is utilized taxus chinensis cell to induce the method that produces Taxan, it is characterized in that: place its substratum commonly used to cultivate taxus chinensis cell, the cell inoculation amount is 50～200g/L weight in wet base, at the cell index early growth period, adding concentration in every liter of substratum is the inductor of 100 μ M, the no bacteria adsorbent macroporous adsorbent resin of 50-200g and the sucrose of 10～50g; Grow mid-term at cell index, add the inductor that concentration is 100 μ M once more; Described inductor is 2,3-dihydroxypropyl jasmonic;
The substratum commonly used of described taxus chinensis cell consists of, in the MS basic medium, add 0.1～1.0mg/L 6-benzyladenine, 0.1～0.5mg/L 2, the 4-dichlorphenoxyacetic acid, 0.1～1.0mg/L naphthylacetic acid, 20～200mg/L xitix and 10～60g/L sucrose, pH value are 5.5～6.5;
The interpolation time first time of inductor is cell inoculation the 7th day behind substratum, and the interpolation time second time of inductor is cell inoculation the 12nd day behind substratum.
2. according to the described method of claim 1, it is characterized in that: the culture condition of described taxus chinensis cell is: rotary shaking table, and rotating speed 60～200rpm, culture temperature is 20～30 ℃, the dark cultivation; Culture cycle is 15～30 days, goes down to posterity once every 14-20 days.
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| CN2007100116277A CN101319199B (en) | 2007-06-08 | 2007-06-08 | Method for cell abduction generation of taxone with Chinese yew |
| PCT/CN2008/070709 WO2008151526A1 (en) | 2007-06-08 | 2008-04-14 | A method of inducing taxane production |
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| CN2007100116277A CN101319199B (en) | 2007-06-08 | 2007-06-08 | Method for cell abduction generation of taxone with Chinese yew |
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| CN102071233A (en) * | 2010-12-06 | 2011-05-25 | 张苑金 | Technical method for synthesizing camptothecin through artificial induction of nothapodytes nimmoniana (graham) mablerley endophytes |
| CN105400731A (en) * | 2015-12-18 | 2016-03-16 | 天津市博爱生物药业有限公司 | Taxus chinensis cell culture medium promoting paclitaxel excretion |
| CN105420178A (en) * | 2015-12-18 | 2016-03-23 | 天津市博爱生物药业有限公司 | Composition influencing plant cell metabolic regulation |
| CN108841778B (en) * | 2018-04-28 | 2022-05-17 | 大连普瑞康生物技术有限公司 | Taxus chinensis cell tissue culture |
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