CN1204246C - Method for producing indole alkaloid by large-scale culture of complete adaptive cell of catharanthus roseus - Google Patents

Method for producing indole alkaloid by large-scale culture of complete adaptive cell of catharanthus roseus Download PDF

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CN1204246C
CN1204246C CN 02136919 CN02136919A CN1204246C CN 1204246 C CN1204246 C CN 1204246C CN 02136919 CN02136919 CN 02136919 CN 02136919 A CN02136919 A CN 02136919A CN 1204246 C CN1204246 C CN 1204246C
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culture
cell
substratum
vinca
temperature
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CN1401781A (en
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郑珍贵
刘涤
胡之壁
周煜
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Shanghai University of Traditional Chinese Medicine
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Shanghai University of Traditional Chinese Medicine
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Abstract

The present invention belongs to the field of bioengineering technology. In particular to a novel method for generating indole alkaloid by culturing hormone-completely-adaptive type longshouxin cells. The invention takes the wild catharanthus roseus seeds as the original material to obtain the aseptic seedling of the catharanthus roseus, further induces the callus, establishes a hormone completely adaptive cell culture system suitable for large-scale culture through multi-generation directional induction and domestication, the total alkaloid content in the culture is equivalent to that of the wild catharanthus roseus, but the culture does not contain vinblastine and vincristine with cytotoxicity.

Description

The complete adaptation type cell large scale of Vinca is cultivated the method that generates indoles alkaloid
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of Vinca cell large scale and cultivate the method that generates indoles alkaloid.
Background technology
Human utilization to the Secondary Metabolism of Plant material has long history, and is current in the ascendant especially to the research of plant secondary substance.But the output of Secondary Metabolism of Plant material is subjected to the restriction of plant biomass and secondary substance content, and some secondary substance is very little at natural content, thereby has limited to the widespread use of plant secondary substance.Along with in the rapid progress that obtains aspect the biotechnology, people have accumulated in sight and how to utilize biotechnology with in the research that obtains plant secondary substance.Relevant with it for this reason scientists has begun unremitting research and effort, can realize that through the exploration discoveries of the nearly decades large scale culturing by vegetable cell the Secondary Metabolism of Plant material effectively produces.
Large scale culturing production Secondary Metabolism of Plant material by vegetable cell is from extracted form natural plant and artificial bionic is synthetic has its original advantage: (one) does not destroy natural resources; (2) not limited by natural causes such as region season, soil, disease and pest; (3) compare with chemical industry is synthetic, Processes and apparatus is simple, save energy, industrial investment are few, production process does not almost have environmental pollution, to the worker safety height: (four) with from natural plants extract phase ratio, with short production cycle, cell individual difference is little, growth Artificial Control, metabolic process can rationally be adjusted, and helps reducing cost, improving productive rate; (5) can produce the better material of drug effect for some crude substance after modified, also can obtain baroque modifier precursor by this technology; (6) for the active substance that is secreted in the outer nutrient solution of born of the same parents, produce then more convenient.In this regard, study and use many.China Luo Shiwei adopts the fluid suspension culture ginseng-cell, and productive rate can increase day by day one times, and the total content of ginsenoside of culture surpasses the content (9.6%) of cultivated ginseng up to 10.5%, and every cubic metre of nutrient solution can obtain 13.98 kilograms of trepangs.This technology has entered the pilot scale stage at present.Japan uitsui company by paniculatum cell is cultivated produce still can't synthetic Shikonin, the productive rate of culturing cell is 1000 times of n cell productive rates, present Japanese Asian puccoon source mainly is the cell tissue cultivation.Many Secondary Metabolism of Plant products such as trypterygine have all been obtained bigger achievement on producing in addition, and the cultivation scale that has can reach tons up to ten thousand.These experiments and facts have proved, stock plant can produce by the cultivation of vegetable cell by the synthetic secondary metabolites, thereby is considered to a new effective way of utilizing biotechnology to produce secondary metabolites (as medicine, natural pigment, spices, natural condiment containing etc.).
Vinca (Cgtharanthus rosouJ) is an Apocynaceae Vinca plant, and tcm clinical practice has tranquilizing and allaying excitement with all herbal medicine, the effect of flat liver step-down and anti-malignant tumor.So far, in Vinca cell and tissue culture, found more than 60 kind of secondary metabolite altogether, vinealeucoblastine(VLB) (Vinblastine) wherein, vincristine(VCR) (Vincristine), Ah agate's alkali (Ajmalicine), Serpophite (Serpentine) and vincaleucoblastine (Catharanthine) etc. all is important medicine.Vinealeucoblastine(VLB) and vincristine(VCR) have significant curative effect to malignant tumours such as leukemia, have hypoglycemic activity simultaneously: Ah agate's alkali is mainly used in and brings high blood pressure down; Serpophite has antibiotic and antitumor action (effective to mammary cancer), and central nervous system is had persistent stable effect, can be used for treating manic property psychosis.Vincaleucoblastine has antitumor action (ehrlich carcinoma, ascitic type liver cancer and leukemia are had restraining effect), its mechanism of action is similar to colchicine, can make cancer cells mitotic division stop at mid-term, it also has hypoglycemic activity (effect is slow, but effect is lasting).
At present, most of effective constituents all are to produce by extracting from the Vinca plant, and Vinca originates in Madagascar, the marshy terrain, output is limited, mainly cultivates in Hainan Island in China.The method of extract producing biological nicotine from natural phant has its significant disadvantages and limitation: (one) because raw material all comes from agricultural supplies with, thereby in the restriction that is subjected to multiple factors such as season, weather, water and soil, region, upgrowth situation in varying degrees: obtaining of (two) raw material must be to use a large amount of cultivated areas as cost; (3) extraction process complexity: the indole alkaloid in (four) Vinca plant, existing monovalent Ah agate's alkali, Serpophite, vincaleucoblastine etc., vinealeucoblastine(VLB) and vincristine(VCR) that divalence is also arranged, these divalence indole alkaloid have very strong cell toxicant, numerous and diverse their separate process, increased the ratio of input and output more.Break away from dependence if can make the production of unit price indole alkaloid to agriculture production, avoid from natural Vinca, extracting the limitation of this process, the natural product characteristic that keeps simultaneously its product again, take than field planting minimum soil and space, realization is to the industrialized biological production of this Alkaloid intensive style, to promoting of exploitation and the application of this Alkaloid, will be of great immediate significance in fields such as medicine, health cares.
Summary of the invention
Technical problem to be solved by this invention is at the problems referred to above, utilizes the large scale culturing technology of vegetable cell, produces the monocycle indoles alkaloid by the large scale culturing method of the complete adaptation type cell of Vinca.
(1) adaptation type Vinca clone preparation fully
The present invention is achieved by following technical proposals by the method that the complete adaptation type cell cultures of Vinca generates indoles alkaloid:
1. be material with the Vinca seed, the medical ethanol with 70% soaked 30 seconds, was transferred to subsequently and carried out surface sterilization in 10% aqueous sodium hypochlorite solution, was seeded in the B that high-temperature sterilization is crossed 5On the minimum medium, be 25+1 ℃ of following illumination cultivation, get the Vinca aseptic seedling after 10 days in temperature.
2. be explant with organs such as the stem of obtained aseptic seedling, leaves, at B 5The solid-based basal culture medium adds 2, and O.2mg/L 4-D 2mg/L and KT cultivate, and temperature 25+1 ℃, through dark cultivation of 4 weeks, can form callus at the cutting part of explant.
With resulting callus with cultivate on the same medium stable after, average three all subcultures once after 10 generations, form soft, stable callus, culture temperature 25+1 ℃, unglazed photograph.The callus of this moment can be set up suspension cell culture.
4. the callus with above gained continues succeeding transfer culture, in substratum, reduce hormone dosage gradually, cultivate 8-10 after generation, hormone is reduced to zero gradually, as find poor growth, can add 0.1-0.2mg/L 2,4-D, growth is removed hormone again after taking a turn for the better, and simultaneously the medium pH value is reduced to 5.4-5.6; After too much generation domestication, can form the complete adaptation type clone of stable hormone, in solid and liquid B 5All can well grow on the minimum medium, the culture cycle on the solid medium was 4 weeks, and the culture cycle on the liquid nutrient medium is 12-15 days.
(2) growth medium and production medium preparation
Growth medium used in the present invention and production culture medium prescription are as shown in table 1:
Table 1
Growth medium (mg/L) is produced substratum (mg/L)
(NH 4) 2SO 4 134 30
KNO 3 2000 400
CaCl 22H 2O. 150 210
MgSO 47H 2O 250 290
NaH 2PO 4H 2O 150 30
KI 0.75
H 3BO 3 3.0
MnSO 4.4H 2O 10
ZnSO 4.7H 2O 2.0
Na 2MoO 4.2H 2O 0.25
CuSO 4.5H 2O 0.025
CoCl 2.6H 2O 0.025
FeSO 4.7H 2O 27.8 27.8
Na 2.EDTA.2H 2O 37.3 37.3
Inositol 100 100
Nicotinic acid 1.0 0.5
Pyridoxine hydrochloride 1.0 0.5
Vitamin 10 3
Sucrose 2%-3% (w/v) white sugar 3% (w/v)
pH 5.6-5.8
(3) generation of indoles alkaloid
(1) set by step produce culture medium prescription preparation growth medium in (two), be adjusted to pH 5.4-5.6, be sub-packed in (every bottle of 50-60ml) in the 250ml triangular flask, seal and high-temperature sterilization;
(2) be provenance with the cell that makes in the step (), be inoculated in and cultivate amplification in the above-mentioned growth medium that culture condition is 25 ± 1 ℃ of temperature, the dark cultivation, shaking speed 110-120 rev/min, 10 days.
(3) will prepare to such an extent that substratum is sub-packed in 1 liter of culturing bottle in (1), every bottle of 300-400ml, seal and high-temperature sterilization, the culturing cell that makes with (2) is a seed, inoculate, inoculum size is 15% (W/V), continues can extract the acquisition alkaloid after 10-12 days with above-mentioned culture condition cultivation from nutrient solution.
When the final large scale culturing reactor of the present invention surpasses 10 liters, must possess feed supplement irritates, amplification culture needs to increase progressively according to 5-8 times of volume continuously, promptly 10 liters → 50 liters → 250 liters → 1000 liters, as 1000 liters, the need of producing substratum in order to adding are irritated in the feed supplement that be equipped with at least 800 liters at the final step of cultivating.Produce to cultivate generally and only carry out in final one-level of cultivating.
The present invention is a starting materials with the Vinca seed, obtain the aseptic seedling explant of Vinca, explant is carried out inducing culture, set up and filtered out and be fit to the hormone autotrophic type cell by two steps cell culture method production indole alkaloid and set up suspension culture system.Design corresponding growth, produced culture medium prescription.This method has characteristics such as culture cycle is short, productive rate is high, environmentally safe.
The present invention compares than prior art has following advantage:
(1) production process is controlled fully, is not subjected to the influence of physical environment;
(2) no hormone, no agricultural chemicals and heavy-metal residual;
(3) extraction process is simple;
(4) low cost is saved in a large number and is ploughed.
Embodiment
Be described in detail the technical process (being not limited to present embodiment) that the complete adaptation type cell large scale of Vinca of the present invention is cultivated the production indole alkaloid with the embodiment form below:
The culture plant cell reactor that embodiment is 1 10 liters is cultivated the method for the complete adaptation type cells produce of Vinca hormone unit price indole alkaloid
1. the preparation of triangular flask cell seed on the shaking table
(1) forms (seeing Table 1) by grown cultures based formulas of the present invention, the preparation growth medium.Measure the pH value with pH meter, and regulate the medium pH value in the 5.4-5.6 scope with the HCl of 1N and the NaOH of 1N.Be sub-packed in the triangular flask of 250ml every bottle of 50-60ml then.After sealing with cotton and paper, carry out autoclaving, pressure is 1.0-1.2Kg/cm 2, the time is 15-25 minute.
(2) be provenance with the complete adaptation type cell of Vinca hormone, in Bechtop, be inoculated in the above ready liquid growth medium and cultivate amplification that inoculum size (in the cell fresh weight) is 6%-8% (w/v).Postvaccinal culturing bottle is put on the shaking table of culturing room, and culture condition is: 25 ± 1 ℃ of temperature, and dark the cultivation, shaking speed 110-120 rev/min, but subculture is once after 10 days.
(3) growth medium for preparing is sub-packed in 1 liter culturing bottle, every bottle of 300-400ml.After sealing with cotton and paper, carry out autoclaving, pressure is 1.0-1.2Kg/cm 2, the time is 15-25 minute.
(4) be seed with 250mL shake-flask culture cell, be inoculated in youngster and shake in the bottle that inoculum size is 8% (w/v), culture condition is with (2).Cultivate and to insert in 10 liters of reactors after 10-12 days.
2.10 rising reactor cultivates
Dispose 10 liters of production substratum, place reactor, by specification requires autosterilization on the throne, after sterilization finishes, regulates the tank body air outlet valve, to guarantee the gas malleation in jar and the pipeline.Insert the cell of logarithmic phase in inoculation tank, inoculum size is 15% (W/V), cultivates 25 ± 1 ℃ of temperature, Ventilation Rate 0.5vvm, 100 rev/mins of stirring velocitys (helical runner).Dark.Cultivated 10-12 days, when PCV reaches more than 75%, the cell growth reaches index latter stage, takes a sample when Ah agate's alkali content reaches 3.5mg/L, extracts alkaloid.
Complete two steps of adaptation type cell of 2 70 liters of culture plant cell reactors of embodiment Vinca hormone are cultivated the method for production indole alkaloid
1. the same example of preparation of triangular flask cell seed on the shaking table.
2.70 rise the preparation of reactor assembly sterilization and substratum
(1) pipeline and the air filter of cultivating reactor carried out steam sterilizing: sterilisation vapour pressure is 1.2-1.4Kg/cm 2, time 20-30 minute.Steam sterilizing finishes, and uses 1.5-2.0Kg/cm immediately 2The air blow drying pipeline and the water of condensation in the air filter, need 20-30 minute usually, the terminal valve of blinding off a line is to guarantee the gas malleation in the pipeline.
(2) sky disappears: culture tank and the feed supplement jar of cultivating reactor carried out steam sterilizing, and sterilisation vapour pressure is 1.2-1.4Kg/cm 2, time 30-40 minute.Steam sterilizing finishes, and treats that tank body pressure reduces to 0.5Kg/cm 2The time open the intake valve of culture tank and feed supplement jar, regulate the tank body air outlet valve, to guarantee that gaseous tension maintains 0.3-0.5Kg/cm in the tank body 2Scope in.
(3) real disappearing: after the temperature for the treatment of culture tank and feed supplement jar is reduced to below 70 ℃, close the tank body air outlet valve, open the charging opening of culture tank and feed supplement jar, in culture tank, add growth medium mother liquor and distilled water to the 25-30 liter, in the feed supplement jar, add production substratum (seeing Table 1) mother liquor and distilled water to the 45-50 liter, regulate the medium pH value in the 5.0-6.0 scope, close the charging opening and the intake valve of culture tank and feed supplement jar.Pass to 0.2Kg/cm 2Water vapour, treat that tank body pressure rises to 0.5Kg/cm 2The time open the air outlet valve of culture tank and feed supplement jar, exitted crack subsequently the two air outlet valve 5-10 minute.Treat that a jar internal pressure rises to 1.2-1.3Kg/cm 2The time, the air outlet valve of regulating water vapour intake valve and culture tank and feed supplement jar is to guarantee that vapor pressure maintains 1.2-1.3Kg/cm in the tank body 2Scope in, carry out that high temperature is real to disappear.Close the water vapour intake valve after 25-30 minute, open water of condensation manual valve cooling tank body, treat that tank body pressure reduces to 0.5Kg/cm 2The time open the air outlet valve of culture tank and feed supplement jar, regulate the tank body air outlet valve, to guarantee that gaseous tension maintains 0.3-0.5Kg/cm in the tank body 2Scope in.
(4) inoculation: the culture tank tank deck rear (the inoculation mouth is positioned at tank deck) that aseptic level air-supply platform is installed on cell culture reactor, aseptic level air-supply is after 20-30 minute, close the culture tank air outlet valve, the culture tank intake valve open is extremely maximum, light inoculation mouthful pyrosphere, open the inoculation flap, with the ready triangular flask bottleneck flame sterilization that fills the cell provenance, pour into the cell provenance in the culture tank fast by the inoculation mouth of the pyrosphere that burning afterwards, cover inoculation flap and screwing rapidly, regulate culture tank intake valve and air outlet valve at last, guarantee that gaseous tension maintains 0.3-0.5Kg/cm in the tank body 2Scope in.
(5) grown cultures and monitoring: by culture condition of the present invention, 25 ± 1 ℃ of temperature, unglazed photograph, system's control automatically cultivates, and grown cultures 10 days reaches biomass index latter stage, and the sampling monitoring pcv value reaches promptly to be finished one step growth and cultivates more than 75%.
(6) produce cultivation and monitoring: drain growth medium with the marker pipe that has the cell filtration device, subsequently with the bacterium of having gone out in the feed supplement jar the production substratum mend in the cell culture reactor through aseptic pipeline pressure reduction, produce cultivation, by culture condition of the present invention, 28 ± 1 ℃ of temperature, unglazed photograph, system's control automatically cultivates, produce and cultivated 10-12 days, sampling monitoring culture Ah agate alkali content reaches can finish the production cultivation of two steps more than the 3.5mg/L.
(7) culture is discharged with the marker pipe that does not have the cell filtration device, carry out alkaloid extraction.
Embodiment 3
In the laboratory lab scale is cultivated, form by grown cultures based formulas of the present invention, the preparation growth medium, sucrose concentration 20g/L regulates the medium pH value 5.6.Be sub-packed in the triangular flask of 250ml every bottle of 50-70ml then.After sealing with cotton and paper, carry out autoclave sterilization, pressure is 1.0-1.2Kg/cm 2, the time is 15-25 minute.With Vinca hormone autotrophic type cell is provenance, is inoculated in above being ready to and cultivates in the liquid growth medium in Bechtop, and inoculum size is 5%-10% (w/v).Postvaccinal culturing bottle is put on the shaking table of culturing room, culture condition is: 25 ± 1 ℃ of temperature, unglazed photograph, 120 rev/mins of shaking speed, cultivate that every bottle of cell fresh weight can reach 35g after 10 days, it is gone to (the same growth medium of process for preparation) in the production substratum of the present invention, culture condition, 25 ± 1 ℃ of temperature, unglazed photograph cultivates that every bottle of cell fresh weight can reach (the P/V value reaches 85%) about 50g after 8-10 days, and the culture total alkaloid content reaches 0.48%, be more or less the same with total alkaloid content in the plant, do not have cytotoxic divalence indole alkaloid but do not contain in this culture.

Claims (1)

1. use the method that the Vinca cell large scale is cultivated the generation indoles alkaloid for one kind, it is characterized in that this method comprises the following steps:
(1) by formulated growth and production substratum:
Growth medium mg/L produces substratum mg/L
(NH 4) 2SO 4 134 30
KNO 3 2000 400
CaCl 22H 2O. 150 210
MgSO 47H 2O 250 290
NaH 2PO 4H 2O 150 30
KI 0.75
H 3BO 3 3.0
MnSO 4·4H 2O 10
ZnSO 4·7H 2O 2.0
Na 2MoO 4·2H 2O 0.25
CuSO 4·5H 2O 0.025
CoCl 2·6H 2O 0.025
FeSO 4·7H 2O 27.8 27.8
Na 2·EDTA·2H 2O 37.3 37.3
Inositol 100 100
Nicotinic acid 1.0 0.5
Pyridoxine hydrochloride 1.0 0.5
Vitamin 10 3
Sucrose 2%-3%w/v white sugar 3%w/v
pH 5.6-5.8
(2) adaptation type Vinca clone preparation fully
1) is material with the Vinca seed, obtains aseptic seedling through sterilization and cultivation;
2) be explant with organs such as the stem of obtained aseptic seedling, leaves, at B 5The solid-based basal culture medium adds 2, and O.2mg/L 4-D 2mg/L and KT cultivate, temperature 25+1 ℃, through dark cultivation of 4 weeks, at the cutting part formation callus of explant;
3) with resulting callus with above-mentioned 2) identical 10 generations of culture medium culturing, average three all subcultures once, culture temperature 25+1 ℃, unglazed photograph;
4) callus of gained is continued succeeding transfer culture, above-mentioned 2) reduce 2 gradually in the described substratum, the 4-D consumption is cultivated 8-10 after generation, and with 2,4-D reduces to zero, solid B 5Culture cycle on the substratum was 4 weeks, or liquid B 5Culture cycle on the substratum is 12-15 days, forms stable complete adaptation type Vinca clone;
(3) press growth medium formulation growth medium, be adjusted to pH5.4-5.6, be sub-packed in the 250ml triangular flask, seal and high-temperature sterilization, the cell that makes with step (2) method is a provenance, be inoculated in and cultivate amplification in the above-mentioned growth medium, culture condition is 25 ± 1 ℃ of temperature, dark cultivation, shaking speed 110-120 rev/min, 10 days subcultures once, as seed cell;
(4) produce substratum by producing the culture medium prescription preparation, be adjusted to pH5.4-5.6, be sub-packed in the 1000ml triangular flask every bottle of 300-400ml, insert seed cell by 15%W/V and cultivate, 25 ± 1 ℃ of temperature, shaking speed are 120 rev/mins, dark was cultivated after 10-12 days, extracted alkaloid.
CN 02136919 2002-09-10 2002-09-10 Method for producing indole alkaloid by large-scale culture of complete adaptive cell of catharanthus roseus Expired - Fee Related CN1204246C (en)

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CN100357296C (en) * 2005-04-27 2007-12-26 东北林业大学 Method for processing production raw materials of vinca alkaloid
CN100340163C (en) * 2005-12-08 2007-10-03 上海交通大学 Plant regeneration method of vinca from hairly root
CN100429215C (en) * 2005-12-19 2008-10-29 东北林业大学 Method for increasing vinblastine content in catharanthus roseus
CN101168732B (en) * 2007-08-24 2010-06-02 清华大学 Method for producing Vinca rosea alkaloid
CN102613088A (en) * 2012-04-23 2012-08-01 上海安体康生植物化学有限公司 Fast propagation method for cultivating salt-tolerant and low temperature-tolerant periwinkle strain
CN106754628A (en) * 2016-11-15 2017-05-31 天津市博爱生物药业有限公司 A kind of synthetic media for inducing Catharanthus Roseus Cell to secrete catharanthine
CN109122323A (en) * 2018-10-22 2019-01-04 覃家日 The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum

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