CN1077600C - Epherdrine producing method by using large-scale ephedra cell culture as material - Google Patents
Epherdrine producing method by using large-scale ephedra cell culture as material Download PDFInfo
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Abstract
The present invention relates to a method for producing ephedrine by using ephedra cell (Ephedra) ZHJ-25CGMCCNo. 0359 cultures as raw material on a large scale. The method comprises ephedra cells (Ephedra) ZHJ-25CGMCCNo. 0359 are inoculated in a saccharide culture medium by three ways, and cultured at the temperature of 25 (+/-) 2DEG C for 20 to 50 days; then, the cultures are taken out and dried at 60DEG C to obtain the raw material containing ephedrine. The method is simple, and can be used for producing ephedrine in an industrialized continuous mode and an industrialized semicontinuous mode. The obtained product is stable in ephedrine content and high in yield.
Description
The present invention relates to utilize biotechnology to produce the method for ephedrine.Be particularly related to the suspension culture by utilizing ephedra cell or utilize cell culture that the bio-reactor large scale culturing obtains, continuous or semi-continuous suitability for industrialized production ephedrine as raw material.
Mainly contain ephedrine, pseudoephedrine, L-N N-Methylephedrine, d-N methyl pseudoephedrine, L-demethyl ephedrine, d-demethyl pseudoephedrine etc. in the Chinese ephedra plant.Herba Ephedrae is a kind of herbal medicine commonly used, has sweating, relievings asthma, effect such as dispel the wind.Ephedrine is the same with suprarenin can exciting sympathetic nerves; But ephedrine can be oral, and long action time, and this point can not for suprarenin institute.Ephedrine can make bronchiectasis, and nasal mucosa shrinks, and makes elevation of blood pressure, clinical specific treatment cough, asthma, spring fever, the Whooping cough etc. of being used for; And can lower the pain of cramp disease, also can make mydriatic.
As far back as 1904, the existing people chemical synthesis that begins one's study prepared ephedrine.Yet the ephedrine that the method for chemosynthesis obtains is the mixture of racemization, wherein contains the left-handed and sudabid of equivalent, and sanedrine does not almost have medical functions, and sudabid is difficult to separate from mixture.Nineteen twenty-one, Hildebrandt and Klavehn utilize Nueberg to invent biological condensation method, can make phenylformic acid and acetaldehyde condensation make 1-acetylbenzene methyl alcohol, again with the methylamine condensation, and nationality active aluminum reduction again, blended rubber shape platinum is contact substance, obtains the 1-ephedrine at last.This method complex process, cost is also higher.
At present, the production of ephedrine is to utilize natural Chinese ephedra plant to extract, and as the Chifeng Pharmaceutics Factory of maximum ephedrine factory one Inner Mongol, the whole nation and domestic tens producers of size, all is to utilize the natural phant Chinese ephedra to produce ephedrine.Consume Chinese ephedra plants in these nearly about 60,000 tons, the Chinese ephedra plant is about to exhausted in several years in factory's years.This method need expend a large amount of natural resource, with carrying out smoothly of guaranteeing to produce.Through excavating for many years, the Chinese ephedra plant resources quite lacks.The Chifeng Pharmaceutics Factory of Inner Mongol is also planted large-area Chinese ephedra plant, but the amount of Herba Ephedrae still can not meet the needs of production.Reason is that the Chinese ephedra plant of plantation generally needed for 3 years just can gather in the crops.The soil of planting the Chinese ephedra plant has toxicity, need fall into disuse can plant other plant in 1-2 years.And artificial growth need take a large amount of arable lands.Address Chinese ephedra plant community poorness in " discussion of Xinjiang Chinese ephedra plant land occupation condition " Xinjiang phytology research collected works one literary composition of Liu Guojun, only put down in writing 5-10 groups on the ground at 10 * 5 square metres samples, what have has only 2-3 groups.In addition, the Xinjiang Chinese ephedra is through assay, and the amount usable with industrial value only is 1/3 of standing stock, the cost height, and productive rate is low.In view of above situation, be necessary the additive method of Development and Production ephedrine.
The objective of the invention is to overcome the method complicated and that from natural Herba Ephedrae, extract ephedrine of technological line in the existing chemosynthesis ephedrine method and need consume a large amount of Herba Ephedrae; and the Chinese ephedra plant is tending towards exhausted and plantation takies shortcomings such as a large amount of arable lands; for protecting national resource and ecotope; save the output of ploughing and improving ephedrine; carry out the production of Sustainable development; so a kind of ephedra cell large scale culturing of utilizing is provided, utilizes cell culture to produce the method for ephedrine.
The present invention is based on from the culture of Chinese ephedra vegetable cell (Ephedra) ZHJ-25 and produce ephedrine, the object of the present invention is achieved like this:
1. the title of microorganism: ephedra cell (Ephedra) ZHJ-25:
2. the preservation of microorganism: ephedra cell (Ephedra) ZHJ-25CGMCCNo.0359 ephedra cell (Ephedra) ZHJ-25, be deposited in Chinese microbial preservation center on September 23rd, 1998, it abbreviates CGMCC as, preservation be numbered CGMCC0359:
3. the feature of microorganism: utilize the Chinese ephedra plant induction to produce the ephedra cell ZHJ-25 of raw ephedra alkali, this ephedra cell has following feature:
(1) morphological specificity of ephedra cell:
Containing 2, observe after 2 days in 23-26 ℃ of cultivations on the nutrient agar of 4-D and indolylacetic acid, can find out that cell is increasing, on original cell mass, breed tiny granular cell, cultivate the cell mass that just can be observed 0.5-5mm size after 3-5 days again.Ephedra cell is spheroid and spheroid, and the diameter of cell is approximately about 10-150 microns.
(2) the Chinese ephedra callus feature of carefully knitting
The cell of Chinese ephedra forms callus through propagation, and the Chinese ephedra callus has presents aggregate, and what have presents the state that disperses shape.It is wavy that the callus edge presents the class particulate, little sticking.Present the tiny particulate state about 1mm when suspension culture, be suspended in the nutrient solution, by the time the later stage of producing, because callus produces ephedrine, the proportion of cell strengthens, and the cell free settling is in the bottom of liquid.Color is that the light yellow dark-brown that arrives does not wait.
(3) physiological and biochemical property
Can utilize multiple sugared source, only culture temperature is 25 ℃, is lower than 10 ℃ or be higher than 40 ℃ of cell poor growths even death.Only pH is 5.8, and cell all can be produced ephedrine and derivative in illumination and dark the cultivation, just the content difference of ephedrine and derivative.Peroxidase isozyme is an ephedra cell ZHJ-25 growth activity sign, and the ephedrine among phenylalanine transaminase and the ephedra cell ZHJ-25 and the metabolism productive rate of derivative are proportional.
The feature of ephedra cell is summarized as follows table:
The morphology and the growth conditions of table 1 ZHJ-25 Ephedra cell
| Ephedra cell ZHJ-25 | |
| Size | |
| 20—150μm | |
| Mobility | (-) |
| Compendency | A little less than |
| Aggregation | (+) |
| The adaptability of shear-stress | By force |
| The color of culture | Light yellow-dark-brown |
| Culture temperature | 22—26℃ |
The present invention has optimized the growth conditions of this cell, and can continuous or semi-continuous production ephedrine.The temperature of cultivating is 22-26 ℃.
The method steps of employing vegetable cell large scale culturing production ephedrine provided by the invention is as follows:
1. the acquisition of ephedra cell (Ephedra) ZHJ-25CGMCCNo.0359:
(ⅰ) the Chinese ephedra plant is cleaned with dish detergent, used distilled water flushing again three times.Put into common sterilizing agent as clorox, hydrogen peroxide in the aqueous solution of preparation such as mercuric chloride, was sterilized 10-30 minutes;
(ⅱ) stem of above-mentioned (ⅰ) being cleaned plant is cut into 0.5cm length, receives in the substratum that contains 1.0-10% (weight/volume) sugar, and was as shown in table 2,23-26 ℃ of heat insulating culture 5-30 days;
(ⅲ) gained callus after the growth of above-mentioned (ⅱ) is downcut from explant, transfer in the substratum of new preparation, carry out succeeding transfer culture.
2. preparation substratum, component is as shown in table 2
The substratum of table 2 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2.0—10.0 | Copper sulfate | 0.0001—0.001 |
| SODIUM PHOSPHATE, MONOBASIC | 0.3—5.0 | Zinc sulfate | 0.001—0.01 |
| Sal epsom | 0.5—3.5 | (EDTA)Fe | 0.1—2 |
| Sodium sulfate | 0.001—0.007 | Kinetin | 0.001—0.01 |
| Sulfate of ammoniac | 0.5—2.0 | Indolylacetic acid | 0.001—0.01 |
| Calcium chloride | 0.5—5.0 | Sucrose | 1.0—10.0(％w/v) |
In proportion all ingredients as shown in table 2 is put into a beaker, water is settled to 500 milliliters, with adjusting PH with base is 5.8, add 0.7% agar again and boil, divide the 50 milliliters of Erlenmeyer flasks of packing into, adorn 20 milliliters in every Erlenmeyer flask, sealing film seals, put it in the pressure sterilization pot, sterilized 15 minutes for 121 ℃, the cooling back is as solid medium.If do not add agar, transferred pH after packing get final product, sterilising conditions is same.
3. ephedrine production and succeeding transfer culture (three approach can be arranged)
(1) solid culture: (ⅰ) ephedra cell is inserted above-mentioned the sterilization and be equipped with in 50 milliliters of Erlenmeyer flasks of 20 milliliters of solid mediums.(ⅱ) cultivated 10-15 days, can change in the substratum of new preparation and carry out succeeding transfer culture at 25 ± 2 ℃.(ⅲ) cell cultures after 20-50 days, is taken out cultured cells, dry about 60 ℃, promptly get the raw material that contains ephedrine.Repeating step (ⅰ) and (ⅱ) can be with the ephedra cell cultured continuously.
(2) suspension culture: (ⅰ) cell of growing in the Erlenmeyer flask 10-15 days is changed over to the Erlenmeyer flask of 250 milliliters and 500 milliliters or 3 liters, the liquid nutrient medium of 100-1000 milliliters of tables 2 wherein is housed, the rotating speed of shaking table is 100-150rpm, cultivates 13-16 days at 25 ± 2 ℃.(ⅱ) with cell cultures under same condition, cultivate the raw material that can contain ephedrine in 20-25 days.Repeating step (ⅰ) can be with the ephedra cell cultured continuously.
(3) bioreactor culture: (ⅰ) utilize 3 liters to shake a ephedra cell that bottle carries out succeeding transfer culture and change in the one-level bio-reactor and cultivate, cultivated 10-15 days at 25 ± 2 ℃; (ⅱ) above-mentioned reactor cultured cells is changed over to, cultivated 20-50 days at 25 ± 2 ℃ than cultivating in the big bio-reactor of the former effective volume, can be with the ephedra cell cultured continuously.
Outside the substratum of the listed ephedra cell ZHJ-25 of table 2 provided by the invention is formed, also comprise used MS usually, N6, SH, B5, White, Heller, substratum such as E all can be used to cultivate ephedra cell.Ephedra cell of the present invention can utilize nitric nitrogen and ammonia-state nitrogen, so add saltpetre and sulfate of ammoniac in the substratum, the phosphoric acid of utilization and vitriol are the form addings with sulfate of ammoniac and SODIUM PHOSPHATE, MONOBASIC; The boron that utilizes, copper, zinc, trace elements such as iron are with copper sulfate, zinc sulfate, the form of Sodium Tetraborate and iron edetate etc. adds; The utilization of calcium ion is to add with the calcium chloride form, and the hormone of utilization is indolylacetic acid and kinetin etc.The carbon source of utilizing comprises monose, disaccharide, polysaccharide etc.In addition, add and contain fungus culture mediums 5-50% such as trace element, VITAMIN, polysaccharide and amino acid, but irritation cell secretion ephedrine, the productive rate of raising ephedrine.
The quantitative analysis of ephedrine
1. use the condition of the content of high pressure liquid chromatography quantitative analysis ephedrine of the present invention
Table 3 high pressure liquid chromatography quantitative analysis ephedrine
| Instrument | High pressure liquid chromatography (HPLC) |
| Post | TSKgel LS-410 (length 150.0mmol/L internal diameter 4.0mmol/L) |
| | 50℃ |
| Mobile phase | 0.5% sodium laurylsulfonate+0.1% phosphoric acid+35% acetonitrile solution |
| Flow velocity | 1ml/min |
| Volume injected | 20—400μg/ml |
| Detector | UV(210) |
2. the content of acid base titration quantitative analysis ephedra cell ZH-25 epheday intermedia alkali
Dried cell culture is ground, cross 100 purpose screen clothes, getting 1.0 gram samples addings is placed with in the tool plug Erlenmeyer flask of 100ml ether and 2ml 10% sodium hydroxide, airtight jolting 1 hour, after static about 2 hours, with the funnels filtration of having filter paper and 2 gram anhydrous sodium sulphate being housed, residue is again with a small amount of ether washing three times, merging filtrate.Filtrate is washed with saturated sodium-chloride, add the sulfuric acid standardized solution 20ml of 0.1M then, jolting 10 minutes, acid solution sodium hydroxide titration is an indicator with the methyl red.
The characteristic of ephedrine of the present invention
1. shape: ephedrine is colourless waxy solid; 2. fusing point: as shown in table 4;
3. molecular weight: 165; 4. molecular formula: C
10H
15NO; 5. molecular structural formula:
R1=H, R2=CH3 R1=R2=H R1=R2=CH36. specific optical rotation: as table 4
The fusing point of table 4 ephedrine and specific optical rotation value
| (-) ephedrine | (+) ephedrine | (-) N-methylephedrine | (-) norephedrine | (+) N methyl pseudoephedrine | (+) pseudonorephedrine | |
| Fusing point | 38.1 | 119 | 88 | 51 | 30 | 78 |
| Specific optical rotation [α] 20d | -6.3 | 51.2 | -29.2 | +48.1 | +38 |
7. stable: stable under acidic conditions, unstable under alkaline condition
8. solvability: (-) ephedrine is soluble in alcohol and water, dissolves in chloroform, ether, benzene, organic solvents such as toluene, can with mineral acid or alkali reaction; (+) pseudoephedrine then is insoluble in water, and alkalescence (-) ephedrine is slightly strong, is easy to combine salify with acid.
Advantage of the present invention
(1) stable content: natural ephedra is owing to the growth district difference, and the amount instability of contained ephedrine in its plant is subjected to the influence of disease and pest, bad climate condition etc., and cell cultures production ephedrine is not subjected to above-mentioned condition effect.(2) content height: the content of general ephedra epheday intermedia alkali is 0.8-1.75%, and the ephedrine in the ephedra cell culture is natural several times even tens times.(3) the protection environment is saved and is ploughed, but suitability for industrialized production: and the improper of natural plant excavated, and can cause the desertification in soil.Because about 2-3 years of the time that the artificial culture of Ephedra plant needs, plant can't be planted because of the soil poisons in the results back in addition, also makes idle land.And culture plant cell need not occupy cultivated land, and can carry out in factory building, can realize large-scale industrialization production.
Below in conjunction with drawings and Examples the present invention is further described in detail:
The adding of Fig. 1 phenylalanine is to the influence of ephedrine output
The adding of Fig. 2 fungal fermented filtrate is to the influence of ephedrine output
Embodiment 1 ephedra cell (Ephedra) ZHJ-25 CGMCC No.0359 cultivates and produces ephedrine
(1) in proportion all ingredients as shown in table 5 is put into a beaker, add sucrose, be settled to 500 milliliters, transferring pH with 1NKOH is 5.8, adds 0.7% agar again and boils, and is respectively charged into 50 milliliters of Erlenmeyer flasks, adorn 20 milliliters in every triangular flask, cover and seal film, with the 121 ℃ of sterilizations 15 minutes in Autoclave of the triangular flask that seals, cooling;
(2) ephedra cell (Ephedra) ZHJ-25 CGMCC No.0359 is inserted in the substratum of 50 liters of Erlenmeyer flasks in above-mentioned (ⅰ), cultivated 10-15 days at 25 ± 2 ℃;
(3) but a part of ephedra cell repeating step (ⅱ) reaches the purpose of succeeding transfer culture;
(4) above-mentioned part cell is continued cultivation after 20-50 days, take out about 60 ℃ dryings of cultured cells, must contain the raw material of ephedrine.The substratum of table 5 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/ |
| Saltpetre | |||
| 2 | Iron edetate | 0.1 | |
| SODIUM PHOSPHATE, MONOBASIC | 0.3 | Inositol | 0.005 |
| Sal epsom | 1 | Kinetin | 0.001 |
| Sodium sulfate | 0.001 | (IAA) indolylacetic acid | 0.0005 |
| Calcium chloride | 0.5 | Phenylalanine | 0.1 |
| Copper sulfate | 0.0001 | Sucrose | 3(％W/V) |
| Zinc sulfate | 0.01 | Agar | 0.7(％W/V) |
(ⅰ) in proportion various solution as shown in table 6 are put into a beaker, add sucrose, be settled to 500 milliliters, adding 1NNaOH accent pH is 5.8, adds 0.7% agar again and boils, be respectively charged into 100 milliliters of triangular flasks, adorn 30 milliliters in every triangular flask, cover and seal film, the triangular flask that seals was sterilized 15 minutes at 121 ℃, cooling (ⅱ) inserts ephedra cell in the triangular flask of (ⅰ), cultivates 10-15 days at 25 ± 2 ℃.(ⅲ) partly (ⅱ) but in the ephedra cell repeating step (ⅱ) cultivated, reach the purpose of succeeding transfer culture.(ⅳ) (ⅱ) part cell was continued cultivation after 20-50 days, it is dry to take out about 60 ℃ of the ephedra cells of cultivating, and promptly gets the raw material that contains ephedrine.The substratum of table 6 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 10.0 | Vitamins B | 0.5 |
| SODIUM PHOSPHATE, MONOBASIC | 3.0 | Iron edetate | 2 |
| Sal epsom | 3 | 2，4-D | 0.005 |
| Sodium sulfate | 0.007 | Kinetin | 0.01 |
| Sulfate of ammoniac | 3.0 | IAA | 0.005 |
| Calcium chloride | 3.0 | Sucrose | 1.75(％W/V) |
| Copper sulfate | 0.001 | Agar | 0.7(％W/V) |
| Zinc sulfate | 0.06 |
Embodiment 3 ephedra cells (Ephedra) ZHJ-25 CGMCC No.0359 cultivates and produces ephedrine
Press the formulated substratum of substratum shown in the table 7, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts containing 100 milliliters and newly joining in 250 milliliters of Erlenmeyer flasks of substratum of the bacterium of having gone out, be after cultivating 20-50 days on the shaking table of 100-120rpm at rotating speed, it is dry to take out about 60 ℃ of cultured cells, promptly gets the raw material that contains ephedrine.The substratum of table 7 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 5.0 | Iron edetate | 0.87 |
| SODIUM PHOSPHATE, MONOBASIC | 2.1 | Vitamins B | 0.045 |
| Sal epsom | 1.5 | IAA | 0.008 |
| Sodium sulfate | 0.003 | Kinetin | 0.0065 |
| Sulfate of ammoniac | 0.9 | Nicotinic acid | 0.078 |
| Calcium chloride | 1.75 | 2，4-D | 0.008 |
| Copper sulfate | 0.0006 | Sucrose | 3.0(％W/V) |
| Zinc sulfate | 0.05 | Agar | 0.7(％W.V) |
Embodiment 4 ephedra cells (Ephedra) ZHJ-25 CGMCC No.0359 cultivates and produces ephedrine
Press the formulated substratum of substratum shown in the table 8, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts in the 1L Erlenmeyer flask that contains 250 milliliters of new dispense liquid substratum of the bacterium of having gone out, be after cultivating 20-50 days on the shaking table of 100-120rpm at rotating speed, it is dry to take out about 60 ℃ of cultured cells, promptly gets the raw material that contains ephedrine.
The substratum of table 8 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 6.8 | Iron edetate | 0.75 |
| SODIUM PHOSPHATE, MONOBASIC | 1.75 | Inositol | 0.05 |
| Sal epsom | 2.5 | Kinetin | 0.0075 |
| Sodium sulfate | 0.0045 | Nicotinic acid | 0.05 |
| Calcium chloride | 0.75 | 2，4-D | 0.0078 |
| Copper sulfate | 0.0065 | Sucrose | 2.0(％W/V) |
| Zinc sulfate | 0.045 | Agar | 0.6(％W.V) |
The substratum of table 9 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2.5 | Iron edetate | 1.5 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Inositol | 0.056 |
| Sal epsom | 2.0 | Kinetin | 0.04 |
| Sodium Tetraborate | 0.005 | Nicotinic acid | 0.055 |
| Sulfate of ammoniac | 2.5 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Sucrose | 1.0(％W/V) |
| Zinc sulfate | 0.1 | Agar | 0.65(％W.V) |
Press the formulated substratum of substratum shown in the table 9, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts in the 3L Erlenmeyer flask that contains the new dispense liquid substratum of 1L of the bacterium of having gone out, be after cultivating 20-50 days on the shaking table of 100-120rpm at rotating speed, taking-up cultured cells drying promptly gets the raw material that contains ephedrine.
The adding of embodiment 6 phenylalanines is to the influence of ephedrine productive rate
The cell transfer that to grow 10-15 days is to the substratum shown in the table 10, till the content of ephedrine reaches maximum.Shown in Figure 1 is, adds the phenylalanines of different amounts in producing substratum, and the content of ephedrine increases to some extent.When the content of phenylpropyl alcohol peace acid had 0mg/L to increase to 20mg/L, the productive rate of ephedrine was increased to 860mg/L from 70mg/L.
The adding of embodiment 7 fungal fermented filtrates is to the influence of Content of Ephedrine With
The ephedra cell that to grow 10-15 days is transferred in the substratum shown in the table 11 and is cultivated, till the content of ephedrine reaches maximum.Fig. 2 is the variation of the ephedra cell epheday intermedia alkali content behind adding fungus culture medium and the mycelium.As can be seen from the figure, when fungal fermented filtrate and when 10% is increased to 50% (weight percent), the content of ephedrine is increased to 980mg/L from 50mg/L.
The substratum of table 10 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2.5 | Iron edetate | 1.5 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Inositol | 0.056 |
| Sal epsom | 2.0 | Kinetin | 0.04 |
| Sodium Tetraborate | 0.005 | | 0—20 |
| Sulfate of ammoniac | 2.5 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Sucrose | 2.5(％W/V) |
| Zinc sulfate | 0.1 | Agar | 0.65(％W/V) |
The substratum of table 11 ephedra cell ZHJ-25 is formed
Embodiment 8 ephedra cells (Ephedra) ZHJ-25 CGMCC No.0359 reactor is cultivated and is produced ephedrine
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 1.25 | Inositol | 0.056 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Kinetin | 0.04 |
| Sal epsom | 1.85 | Nicotinic acid | 0.055 |
| Sodium sulfate | 0.005 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Fungal fermented | 10—50(％W/V) |
| Zinc sulfate | 0.08 | Sucrose | 1.0(％W/V) |
| Iron edetate | 1.5 | Agar | 0.65(％W/V) |
Press the formulated substratum of substratum shown in the table 12, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts containing in the 3L Erlenmeyer flask that 1L newly joins table 12 liquid nutrient medium of the bacterium of having gone out, be after cultivating 10-15 days on the shaking table of 100-120rpm at rotating speed, changing cell over to effective volume is in 50 liters the bio-reactor (40 liters of nutrient solutions that the bacterium of having gone out is housed wherein are housed), cultivated 20-50 days, it is dry to take out about 60 ℃ of cultured cells, promptly gets the raw material that contains ephedrine.
Embodiment 9 ephedra cell reactors are cultivated and are produced ephedrine
(ⅰ) press the formulated substratum of substratum shown in the table 13, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts in the 3L Erlenmeyer flask that contains the new dispense liquid substratum of 1L of the bacterium of having gone out, is after cultivating 10-15 days on the shaking table of 100-120rpm at rotating speed; Be (40 liters of nutrient solutions wherein are housed) in 50 liters the aeration-agitation bio-reactor with (ⅰ) cell transfer to effective volume (ⅱ), cultivated 10-15 days; Be (240 liters of nutrient solutions wherein are housed) in 250 liters the aeration-agitation bio-reactor with (ⅱ) cell transfer to effective volume (ⅲ), cultivate and take out the cultured cells drying after 20-50 days, promptly get the raw material that contains ephedrine.
The substratum of table 12 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 1.25 | Inositol | 0.056 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Kinetin | 0.04 |
| Sal epsom | 1.85 | Nicotinic acid | 0.055 |
| Sodium sulfate | 0.005 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Fungal fermented | 10—50(％W/V) |
| Zinc sulfate | 0.08 | Sucrose | 1.0(％W/V) |
| Iron edetate | 1.5 | Agar | 0.65(％W/V) |
The substratum of table 13 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2.5 | Iron edetate | 1.5 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Inositol | 0.056 |
| Sal epsom | 2.0 | Kinetin | 0.04 |
| Sodium Tetraborate | 0.005 | | 0—20 |
| Sulfate of ammoniac | 2.5 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Sucrose | 2.5(％W/V) |
| Zinc sulfate | 0.1 | Agar | 0.65(％W/V) |
(ⅰ) press the formulated substratum of substratum shown in the table 14, the ZHJ-25 ephedra cell that to cultivate 10-15 days inserts in the 3L Erlenmeyer flask that contains the new dispense liquid substratum of 1L of the bacterium of having gone out, is after cultivating 10-15 days on the shaking table of 100-120rpm at rotating speed; Be in 200 liters the aeration-agitation bio-reactor, 180 liters of nutrient solutions to be housed wherein with (ⅰ) cell transfer to effective volume (ⅱ), cultivated 10-15 days; Be in 1 cubic metre the airlift bioreactor with (ⅱ) cell transfer (ⅲ), the nutrient solution continued growth 10-15 days of 980 liters of bacterium of having gone out wherein is housed to effective volume; (ⅳ) to be transferred to effective volume be to cultivate 20-50 days in 5 tons the airlift bioreactor to the ephedra cell that (ⅲ) cultivated, and takes out cultured cells, and drying promptly gets the raw material that contains ephedrine.The substratum of table 14 ephedra cell ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2.5 | Iron edetate | 1.5 |
| SODIUM PHOSPHATE, MONOBASIC | 2.5 | Inositol | 0.056 |
| Sal epsom | 2.0 | Kinetin | 0.04 |
| Sodium Tetraborate | 0.005 | Phenylalanine | 0—20 |
| Sulfate of ammoniac | 2.5 | 2，4-D | 0.003 |
| Calcium chloride | 2.5 | IAA | 0.003 |
| Copper sulfate | 0.001 | Sucrose | 2.5(％W/V) |
| Zinc sulfate | 0.1 | Agar | 0.65(％W/V) |
Claims (10)
1. method of producing ephedrine may further comprise the steps:
(1) ephedra cell (Ephedra) ZHJ-25CGMCCNo.0359 succeeding transfer culture, the preparation substratum, it is composed as follows:
Saltpetre 2.0-10.0MMOL/L copper sulfate 0.0001-0.001MMOL/L
SODIUM PHOSPHATE, MONOBASIC 0.3-5.0mmol/L zinc sulfate 0.001-0.01mmol/L
Sal epsom 0.5-3.5mmol/L iron edetate 0.1-2.0mmol/L
Sodium Tetraborate 0.001-0.007mmol/L kinetin 0.001-0.01mmol/L
Sulfate of ammoniac 0.5-2.0mmol/L indolylacetic acid 0.001-0.01mmol/L
Calcium chloride 0.5-5.0mmol/L sucrose 1.0-10.0%w/v puts into a beaker with above-mentioned all ingredients in proportion, water is settled to 500 milliliters, with adjusting PH with base is 5.8, add 0.7% agar again and boil, be respectively charged into 50 milliliters of Erlenmeyer flasks, adorn 20 milliliters in every Erlenmeyer flask, sealing film seals, put it in the pressure sterilization pot, sterilized 15 minutes for 121 ℃, the cooling back is as the substratum of succeeding transfer culture;
(2) succeeding transfer culture:
Adopt ephedra cell (Ephedra) ZHJ-25CGMCCNo.0359 to carry out ephedrine production and succeeding transfer culture.
2. by the described a kind of method of producing ephedrine of claim 1, it is characterized in that: also add the phenylalanine that concentration is 0.1-20mmol/L in the described substratum.
3. by the described a kind of method of producing ephedrine of claim 1, it is characterized in that: also add the fungal fermented filtrate of 10-50% weight percent in the described substratum, its concentration is 10-50w/v.
4. by the described a kind of method of producing ephedrine of any claim in the claim 1-3, it is characterized in that: the agar that also adds 0.6-0.7% weight percent in the described substratum.
5. by the method for alkali in described a kind of production of any claim in the claim 1-3, it is characterized in that: the vitamins B that also adds 0.4-0.5mmol/L in the described substratum.
6. by the described a kind of method of producing ephedrine of any claim in the claim 1-3, it is characterized in that: the nicotinic acid that also adds 0.078mmol/L in the described substratum.
7. by the described a kind of method of producing ephedrine of claim 1, it is characterized in that: described ephedrine production and succeeding transfer culture are: solid culture, suspension culture or bioreactor culture.
8. by the described a kind of method of producing ephedrine of claim 7, it is characterized in that: described solid culture is: (1) is inserted ephedra cell in sterilized 50 milliliters of triangular flasks that 20 milliliters of solid mediums are housed; (2) cultivated 10-15 days at 25 ± 2 ℃, change in the substratum of new preparation and carry out succeeding transfer culture; (3) ephedra cell was cultivated 20-50 days under same condition, taken out cultured cells, 60 ℃ of dryings obtain containing the raw material of ephedrine; Repeating step (1) and (2) obtain the ephedra cell of cultured continuously.
9. by the described a kind of method of producing ephedrine of claim 7, it is characterized in that: described suspension culture is: (1) changes the ephedra cell of growing in the triangular flask 10-15 days in the Erlenmeyer flask of sterilized 250 milliliters, 500 milliliters or 3000 milliliters, 100-1000 milliliters liquid nutrient medium wherein is housed, the rotating speed of shaking table is 100-150rpm, cultivates 13-16 days at 25 ± 2 ℃; (2) with ephedra cell under same condition, cultivated 20-50 days, obtain containing the raw material of ephedrine; Repeating step (1) obtains the ephedra cell of cultured continuously.
10. by the described a kind of method of producing ephedrine of claim 7, it is characterized in that: described bioreactor culture is: (1) utilizes 3 liters to shake a ephedra cell that bottle carries out succeeding transfer culture and change in the one-level bio-reactor and cultivate, and cultivates 10-15 days at 25 ± 2 ℃; (2) ephedra cell that above-mentioned reactor is cultivated changes over to than cultivating in the big bio-reactor of the former effective volume, cultivates 20-50 days at 25 ± 2 ℃, obtains ephedra cell.
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| CN98125186A CN1077600C (en) | 1998-12-04 | 1998-12-04 | Epherdrine producing method by using large-scale ephedra cell culture as material |
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|---|---|---|---|
| CN98125186A CN1077600C (en) | 1998-12-04 | 1998-12-04 | Epherdrine producing method by using large-scale ephedra cell culture as material |
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| CN1077600C true CN1077600C (en) | 2002-01-09 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087079A (en) * | 1992-07-01 | 1994-05-25 | 明治制果株式会社 | (-)-Ritodrine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087079A (en) * | 1992-07-01 | 1994-05-25 | 明治制果株式会社 | (-)-Ritodrine |
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