CN1047795C - A method for producing taxol and taxanes from embryo cultures of taxus species - Google Patents

A method for producing taxol and taxanes from embryo cultures of taxus species Download PDF

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CN1047795C
CN1047795C CN94190464A CN94190464A CN1047795C CN 1047795 C CN1047795 C CN 1047795C CN 94190464 A CN94190464 A CN 94190464A CN 94190464 A CN94190464 A CN 94190464A CN 1047795 C CN1047795 C CN 1047795C
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李辅植
孙圣镐
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Abstract

Disclosed herein are methods of taxol and taxane production through extraction of tissue of Taxus species and cell culture thereof. The method of production of taxol and its derivatives by extracting tissues and/or culture medium of Taxus species is characterized in that said tissue is zygote embryo. Cell culture of zygote embryo of Taxus species can be carried out by inducing somatic embryo or embryogenic callus.

Description

From the embryo culture thing of Japanese yew, produce the method for taxol and taxanes
The present invention relates to from somatic embryo and/or substratum, produce the method for taxol and derivative thereof, particularly relate to cultivation zygotic embryo or somatic embryo to obtain the method for somatocyte polyembryony or embryo generation callus.
Taxol is a kind of isolating alkaloid from taxus, and the kinds of tumor cells system performance that comprises the B16 melanoma is had notable antitumor activity.Known taxol mainly is contained in short dried Japanese yew, and (Taxusb in bark brevifolia), is about 0.02% of its gram dry weight.
Though as if proved that the total synthetic method of taxol is successful, this method is unsuitable for commercial mass production because of cost is high.In addition, also use Baccatine III or 10-deacetylation baccatin III successfully semi-synthetic taxol sample compound (for example tax-olere).But there are the problems referred to above too in this method.
Therefore, can only be from this rare natural origin, for example the coarse raw materials of Shou Huo Japanese yew obtains taxol.Because the content of taxol is very low in the yew tree, thus must cut down 2000 to 4000 trees for producing the 1kg taxol, and the taxol that natural origin production enough needs will cause the havoc of forest economy system.Therefore, attempt from the cell of Japanese yew kind and tissue culture, to produce taxol and related compound thereof.
For example, United States Patent (USP) 5,019 discloses a kind of bark, form layers, needle and root tissue of cultivating the Japanese yew kind in nutritional medium for No. 504 to produce callus or suspended cell culture, therefrom reclaims taxol or the alkaloidal method of taxol sample then.But aforesaid method produces 1.0-3.0mg taxol from 1 liter of cell suspension culture base.Above-mentioned patent is addressed, and the yew tree bark can directly obtain the taxol of optimum yields, and can produce taxol to induce undifferentiated cell mass (callus) and/or to constitute cell suspension culture from the callus of breeding by cultivating these tissues.
So far, the not commercialization as yet of the method for the various Japanese yew tissues of above-mentioned use, some reasons are because the taxol productive rate of relevant cell is low, perhaps owing to fail fully to set up the technology of scale operation secondary purpose metabolite.
The tissue, particularly its female gamete parent cell of cultivating taxus disclosed for international monopoly WO92-No. 13961 and from callus or by reclaiming taxol it suspended culture cell that obtains.This method can obtain accounting for the taxol of suspended culture cell dry weight 0.05%.
These methods can not be with the taxol of industrial-scale production q.s, still remains to be improved taxol throughput so far.These true promptings, need provide a kind of increases in cell and/or the substratum taxol content and screens the modification method of high taxol production clone, to adapt to the final purpose of industrial-scale production.
In these cases, the inventor has carried out research in depth for the method for setting up a kind of industrial-scale production taxol.As a result, we find that zygotic embryo contains more substantial taxol (based on same dry weight) than its hetero-organization that contains taxol of reporting so far for the first time unpredictablely.
Because embryo its volume for the method for developing industry scale production taxol itself is too little, so the inventor has done further exploration for a kind of method that increases embryo quality is provided.As a result, we be surprisingly found out that when with embryo culture on nutritional medium during with inductor somatic embryo or embryo generation callus, the comparable original size of embryo quality increases hundreds of thousands of doubly.
Finished the present invention based on above-mentioned discovery.
Therefore, an object of the present invention is to produce the method for taxol, be characterised in that said tissue is a zygotic embryo by the tissue that extracts Japanese yew.
Another object of the present invention provides a kind ofly cultivates the tissue of Japanese yew and reclaims taxol to produce the method for taxol or derivatives thereof from callus or substratum, is characterised in that said tissue is a zygotic embryo.
A further object of the present invention provides a kind of method of producing the taxol or derivatives thereof, and this method comprises the steps:
(a) zygotic embryo alive is provided and sterilizes it from taxus;
(b) on substratum, cultivate the disinfectant embryo to produce callus from the embryo;
(c) callus that obtains in the culturing step (b) is to produce somatic embryo from said callus;
(d) somatic embryo that obtains in disinfectant embryo who obtains in the culturing step (a) or the step (c) is to produce embryo's generation callus;
(e) the embryo generation callus that obtains in somatic embryo that obtains in the liquid culture step (c) or the step (d); And
(f) from cell, reclaim taxol or derivatives of taxol from the substratum neutralization.
The present invention is different from above-named prior art and is that it is based on the discovery zygotic embryo and contains a large amount of taxols unpredictablely, and further increase embryo's quality by inductor somatic embryo or embryo generation callus, and, it may and be better than prior art because making liquid culture somatic embryo or embryo generation callus become with the industrial-scale production taxol.
By following description, those skilled in the art will readily understand other features of the present invention and application.
Fig. 1 (A) is the HPLC tomographic map of standard substance taxanes (taxane).
Fig. 1 (B) is the HPLC tomographic map of substratum that derives from the purifying of embodiment 7.
Fig. 2 (A) shows the photo that does not have the rat tumor cell of processing in the EXPERIMENTAL EXAMPLE 1.
Fig. 2 (B) is the photo that shows the rat tumor cell of the extract-treated of using embodiment 7 in the EXPERIMENTAL EXAMPLE.
Fig. 3 (A) is the typical curve of ELISA data.
Fig. 3 (B) is the curve of gained ELISA data in the EXPERIMENTAL EXAMPLE 2.
Fig. 4 shows to produce the diagram of substratum to the influence of taxol production among the embodiment 8.
The inventor has analyzed the taxol content of 24, the 000 strain Taxus cuspidatas (Taxus cuspidala) that are planted in Korea S country forest land. Although the content of every tree has obvious difference, find to collect and in the dried embryo of the 100g of good tree, contain the 5g taxol. Then obtain respectively 4.5mg and 2.0mg taxol from the dried bark of same amount and needle.
Therefore, first purpose of the present invention provides a kind of method of extracting the production taxol of taxol from the embryo of Cercocarpus. To the kinds of Japanese yew trees without limits, so long as belong to Cercocarpus, any trees all can utilize, but preferred what utilize is Taxus cuspidata.
To the extracting method of from the embryo, producing taxol without limits. Yet the method that generally can be used on the cryodesiccated embryo's powder of dipping in the alcohol, particularly methyl alcohol is extracted taxol from the embryo.
Unfortunately, contain water more than 90% among the very little and embryo of the volume of zygotic embryo. Must just only produce taxol in a small amount with very a large amount of embryos, and see that from economic angle it is disadvantageous directly extracting taxol from the embryo.
Therefore, the inventor has carried out in depth research, so that a kind of method of the problem of above not assert with economized form production taxol to be provided, and can be because of the using-system culture technique as its result, particularly make embryo quality increase hundreds of thousands doubly by inductor blast and embryo generation callus. Therefore, second purpose of the present invention provides a kind of by zygotic embryo inductor blast or embryo generation callus from Cercocarpus, use and cultivate said somatic embryo or embryo generation callus in shaking flask or the bioreactor liquid medium within, and from culture medium and cell, reclaim taxol to produce the method for taxol or derivatives thereof.
A feature of the present invention is that the present invention can be by inductor blast or embryo generation callus so that embryo's mass penalty be tens thousand of to hundreds thousand of times, organize from various except the embryo and to carry out simply callus induction and then be difficult to reach such expanding effect, this may be different by used merismatic Cell Differentiation ability. Specifically, inducing embryo generation callus might make embryo quality increase hundreds of thousands doubly, is very important therefore.
Hereinafter will describe from zygotic embryo inductor blast or the callus of embryo generation and the method for producing taxol.
For the sterile culture embryo, the seed disinfection that should be collected from yew tree for 8 to November or do surperficial degerming. Can use routine techniques to carry out surperficial degerming sterilization. For example, with seed 30-60 seconds kinds of submergence in 70% ethanol, with aseptic washing 2 or 3 times and with 1-3% (v/v) liquor natrii hypochloritis degerming 24 hours. With sterilized water with the seed drip washing 5 times of surperficial degerming and separate the embryo.
Can from the embryo, extract taxol by the said extracted technology.
Also the embryo can be placed on the solid medium to induce callus by it. Available PEDC (cell occurs to determine front embryo) or IEDC (cell occurs to determine the embryo of inducing) program are carried out inducing of somatic embryo.
Use in the steps below PEDC mode inductor blast:
With embryo culture on the solid medium that is added with 2,4-D that 1-naphthylacetic acid (hereinafter referred to as " NAA "), kinetin and callus induction use.In case induced callus, soon the small pieces of callus are cultivated and make it propagation on the substratum that contains proper concn master nutrient substance, micro-nutrients and VITAMIN.
In order to induce and to breed callus, can use the conventional plant tissue culture medium (TCM) and do not have what restriction.For example, can use mB 5(improved CamborgShi B 5Substratum), Durzan, MS ((Marashige and Skoog substratum), WPM (Lloyd ﹠amp; Mc-Cown), DKW (Driver-Kuniyuk-Walnut), GD (Gresshoff ﹠amp; Doy), SH (Schenk ﹠amp; The Hildebrandt substratum) or LP (Quoirin ﹠amp; Lepi-over).
Certainly these substratum are changed, removed different compositions as adding, or the change ratio.MB preferably wherein 5Or Durzan.As the intermediate steps that somatic embryo takes place, can use with the identical or different substratum of the substratum that is used for breeding rapidly callus and induce.From obtaining the angle of maximum number somatic embryo, should preferentially use mB 5Substratum.
Because above-mentioned most of substratum all are that can obtain from commercial channels or very sophisticated, so the public can obtain at an easy rate.Therefore, determine and/or optimal selection is used to induce and rapidly the substratum of propagation callus be that those skilled in the art are in power.For example, preferentially be used for mB of the present invention 5Substratum is the Gamberg ' s B that changes a little 5Substratum, and its composition is as shown in table 1.
Table 1
Compound mg/l compound mg/l
KNO 3 2500 CoCl 2·6H 2O 0.025 KCl 150 Na 2MoO 4·5H 2O 0.25 CaCl 2 150 CuSO 4·5H 2O 0.025 KI 1 H 3BO 3 3 NaH 2PO 4300 vitamins, 2 MgSO 4·7H 2O 250 pyridoxine hydrochlorides 1 (NH 4) 2SO 4150 nicotinic acid, 1 MnSO 4·4H 2O 10 inositols 100 ZnSO 4·7H 2O 2 sucrose 30000 Na 2-EDTA 37.2 FeSO 4·7H 2O 27.8 pH 5.6-5.7
In addition, can in being added with the substratum of above enumerating of different plant growth hormoness in addition, cultivate the embryo, to finish the IEDC method of producing somatic embryo.
For initial incubation, add 2,4-D of 2-4ppm.When generating callus and cell mass from embryo surface and increase by 10-20 times, use usually to add the same substratum of partly measuring the concentration plant hormone and cultivate again.
Because secrete phenolic compound in the cell that may never breed, and make most of callus lose its energy for growth, so will mix 0.5% (w/v) activated carbon or 1-2% (w/v) polyethylene polypyrrole alkane.Do not having on the substratum of growth regulator again cultured calli 2-4 months, generating somatic embryo with meristematic tissue from callus.
So the somatic embryo that generates contains a large amount of taxols or derivatives of taxol (hereinafter referred to as " taxanes " (" taxanes ")) and can be with it in order to extract taxanes.Can use ordinary method, particularly hereinafter described method is extracted taxanes from somatic embryo.But induce the callus that embryo takes place in order further to increase cell use somatic embryo best in quality.
According to the present invention, can reach the somatic embryo that as above produces and make a high proportion of relatively taxanes by the callus through extracting the embryo generation.Can produce the callus that embryo takes place through cultivating the somatic embryo or the zygotic embryo that obtain with above-mentioned PEDC or IEDC method.The method of producing the callus that embryo takes place is as described below: adding 1.0-4.0ppm NAA and 0.5-2.0ppm kinetin, better adding in the solid medium that is suitable for cultured calli of 2.0ppm NAA and 1.0ppm kinetin disinfectant embryo or somatic embryo were cultivated 2-4 months.Can in substratum, add 1-4ppm, 2,4-D and add kinetin to replace NAA.Then in the same substratum that adds 1-5ppm phytokinin such as benzylamine purine, N-isopentene group aminopurine, kinetin or zeatin with derivative callus culture 1-2 months, on the surface of callus, to form embryoid, and this embryoid is placed on adds 2-10ppm, 2, on the same substratum of 4-D, cultivated 2-3 months under (illumination/dark) condition in 26 ℃ and 16/8 hour.This cultivation can be with very a large amount of, and promptly the amount up to hundreds thousand of times of proembryo liver masses obtains the callus that embryo takes place.The callus that embryo takes place can show color widely, for example from yellow, brown to green.
In order to induce and to breed the callus that embryo takes place, can use any substratum that can be used for conventional plant cell and tissue culture ad lib.
The callus of the embryo generation that as above produces contains a large amount of taxols also can be directly in order to extract taxol and taxanes.
Available routine techniques, particularly method is hereinafter described extracted the callus that embryo takes place.
Quality for technical scale amplification embryo generation callus cuts into individual cells or little cell aggregation thing with embryo generation callus.For setting up cell suspension culture, with these cell inoculations of 10% (V/V) to containing in the 250ml Erlenmeyer flask of liquid nutrient medium that 50ml added 2ppm 2,4-D.Preferably use air lift type or impeller type bio-reactor for ease of carrying out cultured continuously.
Can use two kinds of different substratum to carry out bioreactor culture: a kind of is (growth medium) that is used for the cell growth, and another kind is (the production substratum) that is used to produce taxanes.Growth medium better is the mB that adds 2-4pppm, 2,4-D 5Substratum, producing substratum better is the MS substratum that adds 2-10ppm NAA.
Specifically, produce substratum and better contain inducer to increase taxol production.Inducer can be selected from fungi inducer, female gamete parent cell extract (1-5ml/l), phenylalanine (50-300mM) and GA3 (0.5-1.0ppm), the fungi inducer comprises by Cy-lospora abietis ATCC No.38688 and Penicillium minioluteum (Dierckx) NRRL18467 through pulverizing and a series of extraction and the extract that obtains.In addition, also can preferentially select and cultivate the fungi of Pestalotiopsis, and the amount of its extract with 0.5-1.0ml/L is added in the substratum.
Taxol is present in the cell and is secreted in the substratum.Therefore, cultivate recovery taxol and taxanes in available known technology culture in back or the substratum.Available centrifuging (for example 1000xg, 5-10 minutes) or decantation isolated cell from substratum.If utilize a kind of technology in back to separate, nutrient solution can be placed about 24 hours with the sedimentation cell and the substratum that inclines.Use suction pipe to remove substratum in the cell that still is retained in collection through vacuum take-off.
1: 1 mixture of available alcohol or methyl alcohol and methylene dichloride extracts the callus and the substratum of somatic embryo or embryo generation, therefrom to reclaim taxol.The throw out that forms during can using ultrasonic disintegrator (Branson 250) fragmentation centrifugal, and extract.
According to the present invention, reclaim taxol itself and various derivatives of taxol.Derivatives of taxol comprises 10-take off the red toxin III of acetyl berry, Baccatine III, 10-take off acetyl taxol, cephanolmanin and 7-table-10-take off acetyl taxol (referring to Fig. 1 (B)).Derivatives of taxol can so use, and perhaps changes into taxol with semi-synthesis method in case of necessity.
Available HPLC, cell toxicity test and use the ELISA that carries out as the taxol of standard substance and six kinds of (6) derivatives of taxol that provides by NCI to identify and derive from the taxanes that the embryo generation callus of the embryo of zygotic embryo produces by somatic embryo.
By non-limiting example the present invention is described in more detail below.
Embodiment 1
From the zygotic embryo of Japanese yew, reclaim taxol
Gather in the crops seed from good northeast yew tree.Dry embryonic tissue 3 days to water-content is less than 10% and weigh in about 40 ℃ vacuum drying oven.Then with the liquid nitrogen freezing embryo and pulverize it.The embryo of pulverizing was soaked about 7 days in containing about 28 ℃ of thermostat containers of 100 times of weight methyl alcohol approximately.Extract obtained by filter membrane (aperture 0.2 μ m) filtration, and the content of use HPLC methods analyst taxol.
Bark that processing is collected from same tree and needle and by above-mentioned same methods analyst taxol content.The results are shown in the table 2.
Table 2
Organize the taxol content in the stem organization (100g)
Embryo 5g
Bark 4.5mg
Needle 2.0mg
Embodiment 2
Use the PEDC mode to produce somatic embryo from the embryo.
The seed that will obtain from the genotype of the selection of northeast Japanese yew soaked respectively among 70% ethanol and 2% sodium perchlorates 50 seconds and 24 hours, and stopped the back with sterile distilled water drip washing 3 to 5 minutes in each step.
Use the mB that adds 2.0ppm NAA, 0.5ppm kinetin and 0.5ppm 2,4-D 5Substratum (having composition shown in the table 1 and composition as mentioned).8 weeks of vitro culture in adjusting to 26 ℃ growth room.
Between preceding 8 all incubation periods, the embryo increases and has induced callus from the surface of most of embryos.Cut away the green spot in the callus and make it at mB without any growth regulator 5Cultivate 3 months in the substratum to induce somatic embryo.
Embodiment 3
Use the IEDC mode to produce somatic embryo from zygotic embryo.
Seed by method sterilization northeast Japanese yew among the embodiment 2 also therefrom separates the embryo.The embryo is 23-28 ℃ of 2 weeks of cultivation in the Durzan substratum that adds 2ppm 2,4-D.After cultivating for 2 weeks, the callus that all embryos are therefrom induced wraps quilt fully.The quality of cultivating 4 all backs callus reaches tens times of original quality.
For fear of generating or the death of hanking because of its excretory phenol suppresses callus, the same substratum of additional about 1% polyvinylpolypyrrolidone of use is cultured calli again.8 weeks moved into the mB that does not contain plant-growth regulator with callus after cultivating when the mean diameter of callus reaches 1-1.5cm 5In the substratum, callus is grown to induce somatic embryo at it.Observe the differentiation of root in some embryo.
Embodiment 4
For the callus culture thing of inducing embryo to take place, the zygotic embryo of sterilization and the somatic embryo in embodiment 3 and 4 are as parent material among the usefulness embodiment 2.
With the mB of above-mentioned materials at additional 2.0ppm NAA and 1.0ppm kinetin 5Cultivate about 2.5 months with evoked callus for 23-28 ℃ in the substratum.After finishing callus induction, use the same substratum that is added with the 3ppm zeatin to carry out about 6 weeks of subculture, green to obtain to yellowy embryoid.
Under 16/8 (illumination/dark) hour condition, embryoid was cultivated 2 months containing in the same substratum of 3ppm 2,4-D 26 ℃, reach proembryo hundreds of thousands of embryo generation callus doubly to produce quality.Resulting embryo generation callus is similar with the normal callus that derives from all kinds Japanese yew tissue on morphology, and produces the similar outward appearance of zygotic embryo sometimes.
Yet the surface of embryo generation callus is made of the color of multiple different color and luster scopes, comprises red, yellow, dark green, shallow brown, dark brown and black.With blade each colored cell mass is cut into slices to obtain little cell aggregation, subclone cultivated for 2-4 weeks in liquid nutrient medium then.After from liquid culture, removing big cell mass, individual cells and/or little cell aggregation are inoculated on the plastics plate of 20ml substratum.The method of mixing is to carry out the liquid bed board and select different band color clone after cultivating for 4 weeks.Select to obtain different band color clone through conventional naked eyes.
Embodiment 5
The embryo generation callus that in 25 ℃ of dry embodiment 2 or 3 somatic embryos that obtain or embodiment 4, obtains, and be dissolved in the 1 μ l methylene dichloride.Add 1 μ l distilled water and this mixture was stirred for 10 seconds, centrifugal then (25,000xg).In 25 ℃ of dry sediments, be dissolved in the 50 μ l methyl alcohol and by 0.2 μ m membrane filtration to obtain extract.
Carry out HPLC with anti-phase microbore columns is housed, use the typical curve (correction coefficient 0.999) of external perimysium reference product to analyze extract.HPLC result shows that the peak of standard taxanes occurs in the same residence time (14.3 minutes) with the peak that contains the taxanes in the said extracted thing.
Embodiment 6
Extract the embryo generation callus that obtains among the somatic embryo that obtains in embodiment 2 and 3 and the embodiment 4 as follows, to produce taxanes:
Each 0.5g of embryo generation callus among somatic embryo in embodiment 2 and 3 and the embodiment 4 is placed in the centrifuge tube, and adds 2 μ l hexanes.Preserved 12 hours with this mixture of glass stick thorough mixing and at-20 ℃, then in 25 ℃, 25, centrifugal 20 minutes of 500xg.
In throw out, add methyl alcohol: the mixing solutions of ethylene chloride, with using ultrasonic disintegrator (Branson 250) to carry out supersound process.It is centrifugal that (25,000xg) gained solution and in 60 ℃ of dry gained throw outs obtains the exsiccant extract.
The taxol content of separation and Extraction and the extract that obtains the somatic embryo of following result: embodiment 2 contain the every gram of 0.21-0.27mg/ stem cell, and (total taxol content is 0.5-1.2mg); The extract of the somatic embryo of embodiment 3 contains 0.20-0.27mg, and (total taxol content is 0.55-1.1mg); And the extract of the embryo generation callus of embodiment 4 contains 0.23-0.28mg, and (total taxol content is 0.6-1.4mg).
Embodiment 7
Make mass production embryo generation callus, used the impeller type biomass generator to cultivate the embryo generation callus of embodiment 4.
The embryo generation callus of example 4 is inoculated in 10%PCV (accumulation cell volume) contains 50ml liquid mB 5Substratum (wherein is added with in 2,4-D) the 250ml culturing bottle of 2ppm.When under aseptic condition, when cultivating 18 days for 25-28 ℃, reaching stationary phase.
Culture is placed in 5 liters of impeller type bio-reactors that contain MS production substratum (being supplemented with 2ppm NAA).Under aeration condition, culture was kept 30 days in 25-28 ℃.Cultivate after 30 days, nutrient solution is placed 24 hours with sedimentation cell, and from substratum, isolate cell.From cell, thoroughly remove substratum with suction pipe.
Extract isolated cells like this and substratum to obtain taxol and derivative thereof by the same quadrat method described in the embodiment 5.
The HPLC peak (Fig. 1 (A)) of display standard product taxol and its derivative as a result appears at the same residence time with being contained in the taxol in the said extracted thing and the peak (Fig. 1 (B)) of its derivative, shows that these compounds are identical.
Calculated every gram stem cell and contained the 0.09mg taxol, and substratum contains the 8mg taxol for every liter approximately.
Embodiment 8
In order to detect the influence that dissimilar substratum are produced taxol, the culture of embodiment 7 is seeded in various substratum such as MS, mB 5, on WPM, DKW, Durzaan, White, LP, GD, B5, DCR or the SH substratum.All tested substratum are all added 2.0ppmNAA, and the balance adjustment comprises other factors of microenvironment condition.Culture cycle reduces to 20 days.
The results are shown among Fig. 4.As can be seen from the figure, the type of substratum has a significant impact the generation of taxol, and visible MS and mB 5Can provide the highest taxol output.
Embodiment 9
Can in producing substratum, add various inducers to increase taxol and taxanes turnout.Estimate the influence that inducer produces taxol as follows:
(1) is the object of the invention, in substratum, adds the extract that appears at the fungi Pestalotiopsis sp. in the Japanese yew as the inducer of taxol.
Plant tissue such as seed and internal layer bark are immersed in 70% ethanol, use 15%H 2O 2The expression sterilization is immersed in 70% ethanol after 15 minutes once more.With sterile distilled water will organize drip washing more than 4 times or 4 times to remove remaining reagent.The tissue of surface sterilization like this is placed on the substratum: malt extract nutrient agar (1 liter of malt extract 20.0g, peptone 5.0g, agar 15.0g and distilled water), growth nutrient agar (1 liter of glucose 40g, bactosoyton10g, sodium acetate 1g, Sodium Benzoate 50mg, agar 20.0g and distilled water) and water culture medium (1 liter of agar 20.0g and distilled water), and maintain in the thermostatically controlled growth room of 12/12 (illumination/dark) hour light irradiation apparatus.
When Pestalotiopsis sp. fungi when cultivation manifests in back 3 days, with fungi further at malt extract and growth nutrient agar (yeast extract 3g, bacto soyton5g, MgSO 40.5g, glucose 5g, sucrose 10g/L) cultivated 4 days.During results, centrifugal culture is to isolate cell from substratum.Behind the dried cellular, pulverize and with methanol extraction to obtain the carbohydrate part.
The carbohydrate extract of the Pestalotiopsis sp. that so obtains is added to produce in the substratum cultivates and analyze to 1ppm concentration and by method described in the embodiment 7.The results are shown in the table 3.
(2) adding repeats the method for embodiment 7 as female gamete parent cell extract 2ml/L, phenylalanine 100mM and the gibberic acid 1ppm of inducer in producing substratum.The results are shown in the table 3.
Table 3
Inducer The amount of taxol
Every liter of substratum Every 100g viable cell
Fungus extract female gamete parent cell extract phenylalanine gibberic acid 30mg 25mg 21mg 28mg 6.3mg 5.5mg 4.1mg 6.0mg
EXPERIMENTAL EXAMPLE 1
Use the rat tumor cell to carry out cell toxicity test, derive from the taxol of Japanese yew resin embryo culture thing from checking.
Usually can optionally kill based on taxol and be in this fact of cell fission intermediary tumour cell, and use the rat tumor cell to carry out cell toxicity test to confirm the activity of taxol.Will be by Central Research and Development Center of PacificCorporation, the rat tumor cell cultures that Kyounggi-do, Korea provide is in containing the plastic culture bottle of Zooblast culture medium.At the commitment of cultivating, when cell fission takes place, in a bottle, add the extract (processing) of 1 embodiment 7, and in another bottle, do not add extract (contrast).Check the cell fission and the viability of each tumour cell then.The results are shown among Fig. 2 (A) and Fig. 2 (B).
As can from Fig. 2 (A) and 2 (B), seeing the vigorous growth of control group tumor cells showed (Fig. 2 (A)), then tumor cells showed death of treatment group (Fig. 2 (B)).Manifesting in back 3 hours in processing has death of neoplastic cells, and dead fully at about 24 hours thereafter.Therefore, do not need to detect cell fission or viability.
EXPERIMENTAL EXAMPLE 2
Also use the ELISA method (enzyme-linked immunosorbent assay) of monoclonal antibody to identify the taxanes that derives from yew tree embryo culture thing as follows:
Use taxol is played the TA01 medicine box of specific reaction and taxanes played the TA03 medicine box of specific reaction in this experiment available from Hawaii Biotechnology Croup.With PBS (phosphate buffered saline (PBS)) taxol and taxanes are diluted 100 times, and each 100 μ l diluent branch is added in the elisa plate The Small Well.25 ℃ of insulations were washed flat board 4 times at least with TBS-7 (lavation buffer solution) solution after 1 hour, and to wherein adding 50 μ l PBST (salt solution-tween during phosphoric acid salt is slow).The extract of taxol standard substance, taxanes standard substance or embodiment 7 is assigned in the The Small Well in continuous three times of dilution modes.
With PBST taxol antibody and taxanes antibody are diluted 100 times and 1000 times respectively, and distribute each 50 μ l diluent.25 ℃ of insulations were washed dull and stereotyped 4 times with lavation buffer solution after 1 hour.To dilute 2000 times 100 μ lHRP (horseradish peroxidase) with PBST is assigned in the The Small Well and in 25 ℃ of insulations 1 hour, washes 4 times with the slow middle liquid of washing then.Add 200 μ l OPD (O-Phenylene Diamine) and be incubated 1 hour with colour developing in 25 ℃.Use the ELISA reader to measure the 490nm light absorption ratio.The detected result of the substratum of the embodiment 7 shown in the detected result of the taxol shown in Fig. 3 (A) and Fig. 3 (B) has proved the taxol activity of the embryo culture thing of Japanese yew.
Should be clear and definite be that detailed description above just provides in illustrational mode, obviously can be under the situation that does not deviate from spirit and scope of the invention make invention and change and change.

Claims (8)

1. method of producing the taxol or derivatives thereof, wherein this method may further comprise the steps:
(a) seed from Japanese yew provides zygotic embryo and sterilizes it;
(b) cultivate the said disinfectant zygote embryo be seeded in the solid medium with evoked callus, cultivate this callus again mending on the fair same medium that the compound that can stop phenolic compound to suppress callus growth arranged then, so that its propagation;
(c) in not containing the solid nutrition base of any plant-growth regulator, cultivate the callus of propagation with the inductor somatic embryo;
(d) in order to induce embryogenic callus, on the solid medium that is supplemented with 1～4ppm NAA and 0.5～2ppm kinetin, cultivate described somatic embryo with evoked callus therefrom, have in benefit and cultivate this callus on the same medium of 1～5ppm phytokinin on the callus surface, to form embryoid, then this embryoid is had in benefit on the same medium of 2-10ppm, 2,4-D and cultivate;
(e) or further the described embryogenic callus of liquid culture; And
(f) reclaim the taxol or derivatives thereof from this liquid nutrient medium and in the cell or in the embryogenic callus (d).
2. according to the process of claim 1 wherein that step (c) utilizes preceding embryo that the decision programmed cell takes place or inductive embryo generation decision programmed cell inductor somatic embryo is finished.
3. according to the process of claim 1 wherein that described step (e) is by the growth phase that uses growth medium with use the taxol production phase of producing substratum to form.
4. according to the method for claim 3, wherein growth medium is to contain 2-4ppm2 in addition, the mB of 4-D 5Substratum, and the production substratum is MS or the mB that contains 1-2ppm NAA in addition 5Substratum.
5. according to the method for claim 3, wherein produce extract, phenylalanine or gibberic acid 3 that substratum contains the female gamete parent cell of the carbohydrate extracting section thing that derives from Pcstalotiopsis sp. as inducer, Japanese yew.
6. according to the process of claim 1 wherein that the Japanese yew kind is the northeast Japanese yew.
7. according to the process of claim 1 wherein that the compound that replenishes in the callus proliferated culture medium is activated carbon or polyvinylpolypyrrolidone in (b) step.
8. according to the process of claim 1 wherein that phytokinin is benzylamine purine, N-isopentene group aminopurine, kinetin or zeatin.
CN94190464A 1993-07-06 1994-07-06 A method for producing taxol and taxanes from embryo cultures of taxus species Expired - Fee Related CN1047795C (en)

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