CN109321611A - The production method of taxol - Google Patents
The production method of taxol Download PDFInfo
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- CN109321611A CN109321611A CN201811230779.0A CN201811230779A CN109321611A CN 109321611 A CN109321611 A CN 109321611A CN 201811230779 A CN201811230779 A CN 201811230779A CN 109321611 A CN109321611 A CN 109321611A
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Abstract
The present invention provides a kind of production method of taxol, comprising the following steps: 1) establishes stable Chinese yew forming layer stem cell cultivating system；2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is inoculated with yew cell, carries out ventilation culture under the conditions of artificial lighting, methyl jasmonate and sorrow careless extract of escaping are added into liquid suspension culture base and continues to cultivate；3) culture through step 2) suspension culture is collected, is washed using phosphate buffer solution, is centrifuged, obtain sediment, deionized water, ultrasonic disruption are added into the sediment, centrifugation obtains supernatant, ethyl acetate is added into the supernatant, it stands, water phase is collected after being layered, water phase repeats to be extracted with ethyl acetate, water phase is collected, addition ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；4) medicinal extract I is purified；5) it is recrystallized using tetrahydrofuran-acetone mixture.The present invention has the characteristics that extraction process is simple, with high purity.
Description
Technical field
The present invention relates to the technical field of cell culture of medicinal plant.It is more particularly related to a kind of taxol
Production method.
Background technique
Chinese yew is a kind of tree and shrubs of taxaceae (Taxaceae) Taxus (Taxus), the whole world totally 11
Kind.Chinese yew has wider distribution, from the subarctic region of Eurasia and North America to the subtropical zone of Sino-U.S. and Southeast Asia
Even torrid areas is distributed.Studies have shown that the taxol in Chinese yew is to treatment oophoroma, lung cancer, colon cancer, metastatic
The kinds cancers such as breast cancer, melanoma, leukaemia have special efficacy, cause taxol become current most popular anticancer drug it
One.However, in the prior art, taxol source is obtained with from the bark of Chinese yew, and content is very low, extraction process is complicated,
It is unable to satisfy people's demand growing to taxol at all.Therefore, cell culture is carried out with from cell tissue to Chinese yew
Middle extraction taxol has great importance.However, the content of taxol in yew cell is lower, culture medium medium used
Complicated component causes the problems such as extraction process is complicated and DNA purity is lower.
Summary of the invention
As various extensive and careful research and experiment as a result, it has been found by the inventor that in Chinese yew
Cell suspension cultures stage addition Chinese yew endogenetic fungus extracting solution can improve the content of taxol in unit cell.Based on this
It was found that completing the present invention.
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide a kind of production method of taxol, Japanese yew in unit cell can be improved
The content of alcohol, prevents yew cell from morphing during the cultivation process.
In order to realize these purposes and other advantages according to the present invention, a kind of production method of taxol is provided,
The following steps are included:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
After rinsing 10-30 minutes, it is placed in 75% alcohol and handles 10-30 seconds, then rushed after 0.1% mercuric chloride disinfection with sterile water again
It washes, cuts the part of explant discoloration, then carry out Fiber differentiation, choose continuous subculture 3~5 times and grow vigorous, quality and dredge
Loose, in stable condition uniform fresh embryo callus is inoculated into liquid suspension culture base, shake culture, every 7-8 days subcultures
Once, the Chinese yew forming layer stem cell cultivating system for stablizing growth is established through 2-3 months squamous subcultures；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, Chinese yew forming layer of the selection growth rate up to 9.0~12.0mg/gd
Stem cell cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, cultivates
Temperature is 24 DEG C -26 DEG C, and ventilation culture 6-8 days is carried out under the conditions of artificial lighting, jasmonic is added into liquid suspension culture base
Methyl esters and sorrow escape careless extract continue culture with induce Chinese yew cambial cell accumulation generate taxol；
3) culture through step 2) suspension culture is collected, carries out washing 3-5 times using phosphate buffer solution, centrifugation obtains
Sediment is obtained, deionized water is added into the sediment, ultrasonic disruption 5-10min, centrifugation obtains supernatant, toward on described
Ethyl acetate is added in clear liquid, is stood, water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 2-3 times, collects water
Phase, addition ethanol solution low-temperature reduced-pressure are concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
Preferably, germinating bed is set in culture dish, and first layer is that the filter paper second layer is to the germinating bed from top to bottom
Sponge, the height of the sponge are 2-4 millimeters, by the culture dish high pressure steam sterilization, adding liquid Fiber differentiation after cold cut
Base, the culture materials in the filter paper layer after addition disinfection, controls condition of culture, carries out Fiber differentiation, obtain callus.
Preferably, the condition of culture are as follows: 23-25 DEG C of temperature, replaced using blue LED lamp and red LED lamp
Irradiation, blue LED lamp irradiate 3-5 hours, intensity of illumination 1500-1800lux；Red LED lamp irradiates 1-2 hours, and illumination is strong
Degree is 2200-2500Lux.
Preferably, the induced medium is that α-naphthylacetic acid 0.8-1.2mg, benzyl amino are added in every liter of MS culture medium
Adenine 0.5-0.7mg, gibberellin 0.2-0.3mg and Quercetin 0.6-0.8mg.
Preferably, the Chinese yew endogenetic fungus extracting solution the preparation method comprises the following steps: Chinese yew endogenetic fungus is inoculated into
It is 27-29 DEG C in temperature in PDA liquid fermentation medium, revolving speed is vibration gear culture 4-6 days, training in the shaking table of 80-100r/min
Ultrasonic disruption 5-8min after supporting, addition carries out active constituent extraction with the isometric ethyl acetate of culture medium, to solution
Remove water layer after layering, obtain extract liquor, be dried under reduced pressure the extract liquor, adds the deionization of 0.2-0.4 times of extract liquor volume
Water obtains the Chinese yew endogenetic fungus extracting solution.
Preferably, isometric water is added toward the extract liquor, removes water layer after solution layering, carry out repeating extraction
2-3 times.
Preferably, the Chinese yew endogenetic fungus extracting solution additive amount is the 5- of the culture medium quality of suspension culture
10%.
Preferably, the sorrow escape careless extract the preparation method comprises the following steps: by sorrow escape grassland material dries pulverizing cross 100-200 mesh
Sieve obtains minus sieve powder, dehydrated alcohol is added into minus sieve powder, 24-28h is saved under conditions of 4-8 DEG C, filters, and obtains
Filtrate is evaporated under reduced pressure the filtrate and obtains ethyl alcohol extract coarse extract, adds deionization into the ethyl alcohol extract coarse extract
Water dissolution, then adds extracting n-butyl alcohol, medicinal extract is concentrated under reduced pressure to obtain in butanol extraction liquid, adds deionized water dissolving again,
Sorrow is obtained to escape careless extract.
The present invention is include at least the following beneficial effects: addition Chinese yew endogenetic fungus extracting solution mentions for the growth of Chinese yew
For necessary active material, the speed of growth of yew cell is improved；By the way that germinating bed is arranged, the metabolism of culture materials can be made
Product quickly by Adsorption of Filter Paper and then is diffused into fluid nutrient medium, avoids culture materials and growth inhibition effect occurs, greatly
The time for brown stain occur is extended greatly；By using blue light and feux rouges alternate culture, when can be shortened the culture of evoked callus
Between and improve induction success rate；It is avoided that aging and variation occurs in yew cell by adding sorrow careless extract of escaping, and promotes
Yew cell taxol biosynthesis；It can promote yew cell taxol biosynthesis by adding methyl jasmonate, improve unit fine
The content of taxol in born of the same parents.There is the present invention unit cell content of taxol height, cell line to stablize, and be not easy the spies such as aging degeneration
Point.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence
To implement.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
Embodiment 1
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, chooses the Chinese yew forming layer stem cell that growth rate reaches 9.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
26 DEG C, ventilation culture 8 days is carried out under the conditions of artificial lighting, and methyl jasmonate and sorrow grass of escaping are added into liquid suspension culture base and mentions
Object is taken to continue culture to induce the accumulation of Chinese yew cambial cell to generate taxol；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.6%.
Embodiment 2
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection, control
Condition of culture carries out Fiber differentiation, obtains callus；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, chooses the Chinese yew forming layer stem cell that growth rate reaches 9.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
26 DEG C, ventilation culture 8 days is carried out under the conditions of artificial lighting, and methyl jasmonate and sorrow grass of escaping are added into liquid suspension culture base and mentions
Object is taken to continue culture to induce the accumulation of Chinese yew cambial cell to generate taxol；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.5%.
Embodiment 3
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection carry out
Culture, cultivation temperature are 23 DEG C, carry out alternately irradiation using blue LED lamp and red LED lamp, and blue LED lamp irradiates 3 hours,
Intensity of illumination is 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, carries out induction training
It supports, obtains callus；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, chooses the Chinese yew forming layer stem cell that growth rate reaches 9.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
26 DEG C, ventilation culture 8 days is carried out under the conditions of artificial lighting, and methyl jasmonate and sorrow grass of escaping are added into liquid suspension culture base and mentions
Object is taken to continue culture to induce the accumulation of Chinese yew cambial cell to generate taxol；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.6%.
Embodiment 4
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection carry out
Culture, cultivation temperature are 23 DEG C, carry out alternately irradiation using blue LED lamp and red LED lamp, and blue LED lamp irradiates 3 hours,
Intensity of illumination is 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, carries out induction training
It supports, obtains callus；The induced medium is that α-naphthylacetic acid 0.8mg is added in every liter of MS culture medium, and benzyl amino gland is fast
Purine 0.5mg, gibberellin 0.2mg and Quercetin 0.6mg, obtain the induced medium；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, chooses the Chinese yew forming layer stem cell that growth rate reaches 9.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
26 DEG C, ventilation culture 8 days is carried out under the conditions of artificial lighting, and methyl jasmonate and sorrow grass of escaping are added into liquid suspension culture base and mentions
Object is taken to continue culture to induce the accumulation of Chinese yew cambial cell to generate taxol；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.1%.
Embodiment 5
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection carry out
Culture, cultivation temperature are 23 DEG C, carry out alternately irradiation using blue LED lamp and red LED lamp, and blue LED lamp irradiates 3 hours,
Intensity of illumination is 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, carries out induction training
It supports, obtains callus；The induced medium is that α-naphthylacetic acid 0.8mg is added in every liter of MS culture medium, and benzyl amino gland is fast
Purine 0.5mg, gibberellin 0.2mg and Quercetin 0.6mg, obtain the induced medium；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, is uniformly mixed, is mixed
Mixed-culture medium is placed in bioreactor by liquid, chooses the Chinese yew forming layer stem cell that growth rate reaches 9.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
26 DEG C, ventilation culture 8 days is carried out under the conditions of artificial lighting, and methyl jasmonate and sorrow grass of escaping are added into liquid suspension culture base and mentions
Object is taken to continue culture to induce the accumulation of Chinese yew cambial cell to generate taxol；Wherein, the Chinese yew endogenetic fungus extracts
Liquid the preparation method comprises the following steps: Chinese yew endogenetic fungus is inoculated into PDA liquid fermentation medium, be 27 DEG C in temperature, revolving speed is
Vibration gear culture 6 days, ultrasonic disruption 8min after culture, addition and the isometric acetic acid of culture medium in the shaking table of 80r/min
Ethyl ester carries out active constituent extraction, removes water layer after solution layering, obtains extract liquor, be dried under reduced pressure the extract liquor, adds
The deionized water of 0.2 times of extract liquor volume obtains the Chinese yew endogenetic fungus extracting solution；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.3%.
Embodiment 6
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection carry out
Culture, cultivation temperature are 23 DEG C, carry out alternately irradiation using blue LED lamp and red LED lamp, and blue LED lamp irradiates 3 hours,
Intensity of illumination is 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, carries out induction training
It supports, obtains callus；The induced medium is that α-naphthylacetic acid 0.8mg is added in every liter of MS culture medium, and benzyl amino gland is fast
Purine 0.5mg, gibberellin 0.2mg and Quercetin 0.6mg, obtain the induced medium；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, the Chinese yew endogenetic fungus extracts
Liquid additive amount is the 5% of the culture medium quality of suspension culture, is uniformly mixed, and obtains mixed-culture medium, mixed-culture medium is placed in
In bioreactor, the Chinese yew forming layer stem cell cultivating system that growth rate reaches 9.0mg/gd is chosen, not by rate of vaccination
Inoculum concentration less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is 26 DEG C, under the conditions of artificial lighting
Ventilation culture 8 days is carried out, methyl jasmonate and sorrow careless extract of escaping are added into liquid suspension culture base and continues culture to induce
The accumulation of Chinese yew cambial cell generates taxol；Wherein, the Chinese yew endogenetic fungus extracting solution the preparation method comprises the following steps: by red
Beans China fir endogenetic fungus is inoculated into PDA liquid fermentation medium, is 27 DEG C in temperature, revolving speed is the gear that shakes in the shaking table of 80r/min
Culture 6 days, ultrasonic disruption 8min after culture, addition carry out active constituent extraction with the isometric ethyl acetate of culture medium
It takes, removes water layer after solution layering, obtain extract liquor, add isometric water toward the extract liquor, gone after solution layering
Fall water layer, carries out repeating to extract 2 times, be dried under reduced pressure the extract liquor, add the deionized water of 0.2 times of extract liquor volume, obtain
The Chinese yew endogenetic fungus extracting solution；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10h, filtering add active carbon particle in filtrate, carry out decolorization, mistake under conditions of being placed in 46 DEG C
Filter, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges low temperature after eluent
Medicinal extract II is concentrated under reduced pressure to obtain；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2-3 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.1%.
Embodiment 7
A kind of production method of taxol comprising following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, cleans, set under flowing water through dish washing liquid and tap water
It rinses after ten minutes, is placed in 75% alcohol and handles 10 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again, cut
The part for going explant to change colour, then carries out Fiber differentiation, chooses continuous subculture 3 times and growth is vigorous, quality is loose, state is steady
Fixed uniform fresh embryo callus is inoculated into liquid suspension culture base, and shake culture, every 7 days subcultures were primary, through 2 months
Squamous subculture establish stablize growth Chinese yew forming layer stem cell cultivating system；Wherein, germinating bed is set in culture dish,
First layer is the filter paper second layer to the germinating bed from top to bottom is sponge, and the height of the sponge is 4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection carry out
Culture, cultivation temperature are 23 DEG C, carry out alternately irradiation using blue LED lamp and red LED lamp, and blue LED lamp irradiates 3 hours,
Intensity of illumination is 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, carries out induction training
It supports, obtains callus；The induced medium is that α-naphthylacetic acid 0.8mg is added in every liter of MS culture medium, and benzyl amino gland is fast
Purine 0.5mg, gibberellin 0.2mg and Quercetin 0.6mg, obtain the induced medium；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, the Chinese yew endogenetic fungus extracts
Liquid additive amount is the 5% of the culture medium quality of suspension culture, is uniformly mixed, and obtains mixed-culture medium, mixed-culture medium is placed in
In bioreactor, the Chinese yew forming layer stem cell cultivating system that growth rate reaches 9.0mg/gd is chosen, not by rate of vaccination
Inoculum concentration less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is 26 DEG C, under the conditions of artificial lighting
Ventilation culture 8 days is carried out, methyl jasmonate and sorrow careless extract of escaping are added into liquid suspension culture base and continues culture to induce
The accumulation of Chinese yew cambial cell generates taxol；Wherein, the Chinese yew endogenetic fungus extracting solution the preparation method comprises the following steps: by red
Beans China fir endogenetic fungus is inoculated into PDA liquid fermentation medium, is 27 DEG C in temperature, revolving speed is the gear that shakes in the shaking table of 80r/min
Culture 6 days, ultrasonic disruption 8min after culture, addition carry out active constituent extraction with the isometric ethyl acetate of culture medium
It takes, removes water layer after solution layering, obtain extract liquor, add isometric water toward the extract liquor, gone after solution layering
Fall water layer, carries out repeating to extract 2 times, be dried under reduced pressure the extract liquor, add the deionized water of 0.2 times of extract liquor volume, obtain
The Chinese yew endogenetic fungus extracting solution；The sorrow escape careless extract the preparation method comprises the following steps: by sorrow escape grassland material dries pulverizing mistake
100 meshes obtain minus sieve powder, dehydrated alcohol are added into minus sieve powder, save for 24 hours, filter under conditions of 4 DEG C, obtain
Filtrate is evaporated under reduced pressure the filtrate and obtains ethyl alcohol extract coarse extract, adds deionization into the ethyl alcohol extract coarse extract
Water dissolution, then adds extracting n-butyl alcohol, medicinal extract is concentrated under reduced pressure to obtain in butanol extraction liquid, adds deionized water dissolving again,
Sorrow is obtained to escape careless extract；
3) culture through step 2) suspension culture is collected, washing 5 times is carried out using phosphate buffer solution, is centrifuged, obtain
Sediment adds deionized water, ultrasonic disruption 5min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 3 times, collects water phase, addition
Ethanol solution low-temperature reduced-pressure is concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, is added and dehydrated alcohol same volume
Acetone, standing sedimentation 10h, filtering add active carbon particle in filtrate, carry out decolorization, mistake under conditions of being placed in 46 DEG C
Filter, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges low temperature after eluent
Medicinal extract II is concentrated under reduced pressure to obtain；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation is brilliant
Body is dissolved into tetrahydrofuran-acetone mixture again and carries out recrystallization 2 times, obtains the taxol of high-purity.
The purity of taxol manufactured in the present embodiment is 99.5%.
Comparative example 1
Group 1-1: it is not added with Chinese yew endogenetic fungus extracting solution in the culture medium for the culture that suspends, yew cell is trained
It supports, detects the speed of growth of yew cell, the results are shown in Table 1.
Group 1-2: it is not added with Chinese yew endogenetic fungus extracting solution in the culture medium for the culture that suspends, yew cell is trained
It supports, detects the speed of growth of yew cell, the results are shown in Table 1.
Table 1
| Vitro growth rates (g (dw)/g.d) | |
| Group 1-1 | 0.53 |
| Group 1-2 | 0.86 |
As can be known from Table 1, addition Chinese yew endogenetic fungus extracting solution can promote the speed of growth of yew cell.
Comparative example 2
Group 2-1: not adding methyl jasmonate in culture solution and sorrow is escaped careless extract, other incubations and embodiment 7 are complete
It is exactly the same, the content of the content of taxol and taxol in yew cell in culture solution is detected, the results are shown in Table 2.
Group 2-2: not adding methyl jasmonate in culture solution, other incubations are identical with embodiment 7, detection culture
In liquid in the content of taxol and yew cell taxol content, the results are shown in Table 2.
Group 2-3: not adding sorrow and escape careless extract in culture solution, other incubations are identical with embodiment 7, and detection is cultivated
In liquid in the content of taxol and yew cell taxol content, the results are shown in Table 2.
Table 2
| Extracellular content of taxol (mg/L) | Content of taxol (%) intracellular | |
| Group 2-1 | 18.3 | 0.031 |
| Group 2-2 | 20.7 | 0.046 |
| Group 2-3 | 19.4 | 0.041 |
| Embodiment 7 | 27.4 | 0.075 |
As can be known from Table 2, red bean can be improved in certain degree by adding methyl jasmonate and sorrow careless extract of escaping
The synthesis of China fir cell, and add methyl jasmonate jointly and sorrow careless extract of escaping is then more aobvious for the content for improving yew cell
It writes.
Comparative example 3
Group 3-1: the red LED lamp of the application is substituted using the light source of white during the cultivation process and blue LED lamp carries out
Illumination and dark alternate culture illumination cultivation 3 hours, dark culturing 3 hours, intensity of illumination 1500lux, count callus
Average induction time and inductivity, the results are shown in Table 3.
Group 3-2: alternately giving red LED lamp and blue LED lamp irradiation during the cultivation process, and blue LED lamp irradiation 3 is small
When, intensity of illumination 1500lux；Red LED lamp irradiates 2 hours, and intensity of illumination is that 2500Lux controls condition of culture, is lured
Culture is led, counts the average induction time and inductivity of callus, the results are shown in Table 3.
Group 3-3: whole process gives white light during the cultivation process, counts average induction time and the induction of callus
Rate, the results are shown in Table 3.
Table 3
| Induction time (d) | Inductivity (%) | |
| Group 3-1 | 30 | 91 |
| Group 3-2 | 19 | 100 |
| Group 3-3 | 35 | 80 |
As can be known from Table 3, using red LED lamp and blue LED lamp alternate culture, it can be shortened induction time and raising lure
Lead success rate.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (8)
1. a kind of production method of taxol, which comprises the following steps:
1) it selects the sprout in the tender stem of Chinese yew as culture materials, is cleaned through dish washing liquid and tap water, set and rinsed under flowing water
After 10-30 minutes, it is placed in 75% alcohol and handles 10-30 seconds, then use aseptic water washing after 0.1% mercuric chloride disinfection again,
Cut explant discoloration part, then carry out Fiber differentiation, choose continuous subculture 3~5 times and grow it is vigorous, quality is loose,
In stable condition uniform fresh embryo callus is inoculated into liquid suspension culture base, shake culture, every 7-8 days subcultures one
It is secondary, the Chinese yew forming layer stem cell cultivating system for stablizing growth is established through 2-3 months squamous subcultures；
2) Chinese yew endogenetic fungus extracting solution is added into the culture medium for the culture that suspends, be uniformly mixed, obtain mixed-culture medium, it will
Mixed-culture medium is placed in bioreactor, Chinese yew forming layer stem cell of the selection growth rate up to 9.0~12.0mg/gd
Cultivating system, the inoculum concentration by rate of vaccination not less than 10%, which is inoculated into bioreactor, carries out suspension culture, and cultivation temperature is
24 DEG C -26 DEG C, carried out under the conditions of artificial lighting ventilation culture 6-8 days, into liquid suspension culture base be added methyl jasmonate with
Sorrow escape careless extract continue culture with induce Chinese yew cambial cell accumulation generate taxol；
3) culture through step 2) suspension culture is collected, carries out washing 3-5 times using phosphate buffer solution, centrifugation is sunk
Starch adds deionized water, ultrasonic disruption 5-10min into the sediment, and centrifugation obtains supernatant, toward the supernatant
Middle addition ethyl acetate is stood, and water phase is collected after being layered, and water phase repeats to be extracted with ethyl acetate 2-3 times, collects water phase, add
Ethanol solution low-temperature reduced-pressure is added to be concentrated to give medicinal extract I；
4) after dissolving the medicinal extract I with appropriate dehydrated alcohol, crude extract is formed, third with dehydrated alcohol same volume is added
Ketone, standing sedimentation 10-11h, filtering are added active carbon particle in filtrate, are carried out at decoloration under conditions of being placed in 43-46 DEG C
Reason, filtering, macroporous resin adsorption is added into filtrate, then carries out gradient elution with the ethyl alcohol of various concentration, merges eluent
Low-temperature reduced-pressure is concentrated to give medicinal extract II afterwards；
5) it is dissolved by heating toward medicinal extract II addition tetrahydrofuran-acetone mixture, cold cut to room temperature, spontaneous nucleation, crystal is again
Secondary tetrahydrofuran-the acetone mixture that is dissolved into carries out recrystallization 2-3 times, obtains the taxol of high-purity.
2. the production method of taxol according to claim 1, which is characterized in that germinating bed is arranged in culture dish, institute
Stating germinating bed first layer is the filter paper second layer from top to bottom is sponge, and the height of the sponge is 2-4 millimeters, by the culture dish
High pressure steam sterilization, adding liquid induced medium after cold cut, the culture materials in the filter paper layer after addition disinfection, control
Condition of culture carries out Fiber differentiation, obtains callus.
3. the production method of taxol according to claim 2, which is characterized in that the condition of culture are as follows: temperature 23-25
DEG C, alternately irradiation is carried out using blue LED lamp and red LED lamp, blue LED lamp irradiates 3-5 hours, intensity of illumination 1500-
1800lux；Red LED lamp irradiates 1-2 hours, intensity of illumination 2200-2500Lux.
4. the production method of taxol according to claim 2, which is characterized in that the induced medium is in every liter of MS
α-naphthylacetic acid 0.8-1.2mg, benzyl aminoadenine 0.5-0.7mg, gibberellin 0.2-0.3mg and Quercetin are added in culture medium
0.6-0.8mg。
5. the production method of taxol according to claim 1, which is characterized in that the Chinese yew endogenetic fungus extracting solution
The preparation method comprises the following steps: Chinese yew endogenetic fungus is inoculated into PDA liquid fermentation medium, be 27-29 DEG C in temperature, revolving speed is
Vibration gear culture 4-6 days, ultrasonic disruption 5-8min after culture in the shaking table of 80-100r/min, the bodies such as addition and culture medium
Long-pending ethyl acetate carries out active constituent extraction, removes water layer after solution layering, obtains extract liquor, be dried under reduced pressure the extraction
Liquid adds the deionized water of 0.2-0.4 times of extract liquor volume, obtains the Chinese yew endogenetic fungus extracting solution.
6. the production method of taxol according to claim 5, which is characterized in that isometric toward extract liquor addition
Water removes water layer after solution layering, carries out repeating to extract 2-3 times.
7. the production method of taxol according to claim 1, which is characterized in that the Chinese yew endogenetic fungus extracting solution
Additive amount is the 5-10% of the culture medium quality of suspension culture.
8. the production method of taxol according to claim 1, which is characterized in that the sorrow is escaped the preparation side of careless extract
Method are as follows: sorrow grassland material dries pulverizing of escaping is crossed into 100-200 mesh, minus sieve powder is obtained, dehydrated alcohol is added into minus sieve powder,
24-28h is saved under conditions of 4-8 DEG C, is filtered, filtrate is obtained, and is evaporated under reduced pressure the filtrate acquisition acquisition ethyl alcohol extract and is slightly soaked
Cream adds deionized water dissolving into the ethyl alcohol extract coarse extract, then adds extracting n-butyl alcohol, butanol extraction liquid is subtracted
Pressure is concentrated to give medicinal extract, adds deionized water dissolving again, obtains sorrow and escapes careless extract.
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