CN1101467C - Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method - Google Patents
Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method Download PDFInfo
- Publication number
- CN1101467C CN1101467C CN98125187A CN98125187A CN1101467C CN 1101467 C CN1101467 C CN 1101467C CN 98125187 A CN98125187 A CN 98125187A CN 98125187 A CN98125187 A CN 98125187A CN 1101467 C CN1101467 C CN 1101467C
- Authority
- CN
- China
- Prior art keywords
- ephedra
- cell
- zhj
- substratum
- preserving number
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to an ephedra cell induced from ephedra by means of a biologic technique and an induction method thereof. The induced ephedra cell ZHJ-25 of the present invention was preserved in the Chinese Microbe Preservation Center in the code of CCMCNO0359 on 23rd of september in 1998. In the induction method of the present invention, the ephedra is cleaned, sterilized and inoculated to a 1.0 to 10% weight / volume of sugar culture medium where heat preservation is carried out for 5 to 30 days at 23 to 26 DEG C, and an obtained ephedra callus is transferred to a culture medium for subculture. The proliferation quantity of the obtained ephedra cell of the present invention is 3 to 5 times that during inoculation, and the content of ephedrine is 0.1 to 20% of dry weight.
Description
The present invention relates to utilize biotechnology to induce ephedra cell system from ephedra.More specifically, the present invention relates to induce ephedra cell (Ephedra) ZHJ-25 by the Chinese ephedra plant, preserving number is the method for CGMCC No.0359 and the high yield clone that can produce ephedrine.
Mainly contain ephedrine in the ephedra, pseudoephedrine, L-N N-Methylephedrine, d-N methyl pseudoephedrine, L-demethyl ephedrine, d-demethyl pseudoephedrine etc.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 clinical specific treatment cough, asthma, spring fever, the Whooping cough etc. of being used for of elevation of blood pressure.In addition, it can lower the pain of cramp disease, also can make mydriatic.
At present, producing ephedrine both at home and abroad mostly is to utilize natural Chinese ephedra plant to extract, and utilizes this method as the Chifeng Pharmaceutics Factory of China Inner Mongol and more than ten tame ephedrine manufacturers.The production ephedrine is extracted in natural phant production 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 and ecotope has suffered destruction.Addressed Chinese ephedra plant community poorness in " discussion of Xinjiang Chinese ephedra plant land occupation condition " literary composition of Liu Guojun, at most only put down in writing 5-10 group on the ground at 10 * 5 square metres samples, what have has only 2-3 group.Report that in " artificial growth Herba Ephedrae alkaloid content rule " literary composition of Cheng Zhengming only eight factories that produce ephedrine have just been built up in Xinjiang, year consumption Chinese ephedra plant is nearly about 60,000 tons.The Chinese ephedra plant resources is about to exhausted in several years.It is reported that the Xinjiang Herba Ephedrae is through assay, the amount usable with industrial production value is only for accumulateing 1/3 of a surname's amount.Irrational excavating not only destroyed the grassland, and makes deserta also be subjected to serious harm.
The plant of plantation Ephedra generally needs the time in 3 years just can gather in the crops, and the soil of planting the Chinese ephedra plant has toxicity, need fall into disuse 1--2 and can plant other plant, need stand and uses a large amount of arable lands so plant Herba Ephedrae.
In view of above situation, be necessary the additive method of Development and Production ephedrine.
Learn that by literature search the method for utilizing the Chinese ephedra plant to induce ephedra cell to produce ephedrine in the present report both domestic and external has multiple, but the content of its ephedrine is general all about 0.17-0.6%.At Ramawal, K.G.etc.Effect of Amino Acid on Ephedrine Production in Ephedra geradianaCallous Culture Phylochemistry vol.18 (3); The content of report ephedrine is 0.5--0.6% in p484-485 one literary composition.
The objective of the invention is to overcome the defective of above-mentioned prior art, from the Chinese ephedra plant cell, separate and induce ephedra cell ZHJ-25, two of purpose is to provide the new way of a production ephedrine raw material, utilizes the method for biotechnology exploitation large-scale industrial production ephedrine.Promptly the method by tissue culture induces callus, can utilize this ZHJ-25 ephedra cell to carry out the suitability for industrialized production ephedrine.Three of purpose is to improve productive rate, inductive ephedra cell system of institute of the present invention, and the content of ephedrine is the 2.0--5.0% of dry weight, the proliferative amount of cell is 3--6 times.The object of the present invention is achieved like this: the title of this microorganism: ephedra cell (Ephedra) ZHJ-25
This ephedra cell (Ephedra) ZHJ-25 is deposited in Chinese microbial preservation center (it abbreviates CGMCC as) on September 23rd, 1998, and deposit number is CGMCC No.0359.
Ephedra cell (Ephedra) ZHJ-25, preserving number is the microbiological property of CGMCC No.0359: utilize the Chinese ephedra plant induction to produce the ephedra cell ZHJ-25 of raw ephedra alkali, this ephedra cell has following feature: the morphological specificity of (1) ephedra cell:
Containing 2, observe after 2 days in 23-26 ℃ of cultivation on the nutrient agar of 4-D and indolylacetic acid, can find out that cell is increasing, on original cell mass, breed and tiny granular cell, cultivate the cell mass that just can be observed the 0.5-5mm size after 3-5 days again. ephedra cell is spheroid and spheroid, and the diameter of cell is approximately about the 10-150 micron.(2) feature of Chinese ephedra callus
The cell of Chinese ephedra forms callus through propagation, the Chinese ephedra callus has presents aggregate, the state that presents dispersion shape that has. it is wavy that the callus edge presents the class particulate, little sticking. when suspension culture, present the tiny particulate state about 1mm, 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 invention provides and utilizing the ephedra cell of Chinese ephedra plant induction birth ephedrine produced thereby is the method for ZHJ-25, optimized the growth conditions of this clone, and through metabolic regulation, obtain the ephedra cell system of high yield, 3-6 when the proliferative amount of its cell is inoculation times, the content of ephedrine is the 0.1-20% of dry weight.
Used Chinese ephedra plant plant comprises ephedra sinica Ephdra sinica Stapf in the inventive method, ephedra equisetina Ephedra equisetinna Bunge, epheday intermedia Ephdra intermedia Schrenk, Ephedra przewalskii Ephdra przewalskii Stapf; Also comprise Herba Ephedrae monospermae, blue branch Chinese ephedra, sea grape, thin sub-Chinese ephedra etc.
The present invention utilizes Chinese ephedra plant induction ephedra cell (Ephedra) ZHJ-25, and preserving number is that the step of CGMCC No.0359 is as follows: (i) the Ephedra plant is cleaned with dish detergent, is 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 minute; The stem of (ii) above-mentioned (i) being cleaned plant is cut into 0.5cm length, receives to contain 1.0-10% (weight/volume) sugar
Substratum (as shown in table 1) in, at 23-26 ℃ of heat insulating culture 5-30 days; (iii) gained Chinese ephedra callus after the growth is (ii) downcut from explant, transfer to the training of new preparation table 1
Support in the base at 23-26 ℃ of heat insulating culture 12-15 days.
The substratum of table 1 ephedra cell (Ephedra) ZHJ-25 is formed
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 2-10.0 | Copper sulfate | 0.0001-0.001 |
| SODIUM PHOSPHATE, MONOBASIC | 0.3-3.0 | Zinc sulfate | 0.01-0.1 |
| Sal epsom | 1-3 | Iron edetate | 0.1-2 |
| Sodium Tetraborate | 0.001-0.007 | Kinetin | 0.001-0.01 |
| Sulfate of ammoniac | 0.5-3.0 | IAA | 0.0005-0.005 |
| Calcium chloride | 0.5-3.0 | Sucrose | 1.0-10(％W/V) |
In the described substratum compositing formula, comprise that also adding concentration is the phenylalanine of 0.1-20mmol/L.
In the described substratum compositing formula, also comprise the fungal fermented filtrate that adds the 10%-50% weight percent, the concentration of its fungal fermented filtrate is 10-50w/v.
In the described substratum compositing formula, also comprise the agar that adds the 0.6-0.7% weight percent.
Advantage of the present invention:
The invention provides a kind of new ephedra cell (Ephedra) ZHJ-25, preserving number is CGMCC No.0359, and the induction method of this ephedra cell, this method can obtain the ephedra cell system of high yield, 3-5 when the proliferative amount of its cell is inoculation doubly, utilize high pressure lipuid chromatography (HPLC) and nineteen ninety version the Pharmacopoeia of the People's Republic of China in method such as acid base titration ephedra cell of the present invention is measured, the content of gained ephedrine is 0.1-20%.This method can realize industrialization, and the ephedrine produced thereby of making a living provides a new raw material supply approach.
Below in conjunction with drawings and Examples the present invention is further detailed:
Fig. 1 is the growth curve of ephedra cell
The Chinese ephedra plant is cleaned with dish detergent, used distilled water flushing again three times.Put into the ethanol aqueous solution 15 minutes of 70% (V/V), aqueous sodium hypochlorite solution 10-30 minute of putting into 30% (V/V) again.It is long that plant after the sterilization is cut into about 0.5cm, receives in the substratum (table 2 prescription) that contains 1% (weight/volume) sugar, at 23-26 ℃ of heat insulating culture 5-30 days, as seen has tiny callus to grow from explant.Downcut callus, transfer in the new preparation substratum (composition is as shown in table 2), under same culture condition, cultivated 12--15 days, can obtain good Chinese ephedra callus.
The substratum of table 2 ephedra cell ZHJ-25 is formed
Embodiment 2,3,4,5,6 ephedra cell (Ephedra) ZHJ-25, and preserving number is inducing of CGMCC No.0359
| Reagent name | Concentration mmol/L | Reagent name | Concentration mmol/L |
| Saltpetre | 5.0 | Copper sulfate | 0.0001 |
| SODIUM PHOSPHATE, MONOBASIC | 3.0 | Zinc sulfate | 0.06 |
| Sal epsom | 1.5 | Iron edetate | 0.1 |
| Sodium Tetraborate | 0.005 | Kinetin | 0.001 |
| Sulfate of ammoniac | 2.0 | IAA (indolylacetic acid) | 0.0005 |
| Calcium chloride | 2.0 | Sucrose | 1.0(％W/V) |
Ephedra cell induces process and inductive condition as described in the embodiment 1 by the Chinese ephedra plant, the substratum difference when just ephedra cell is induced, existing illustrate respectively as follows:
The substratum of table 3 ephedra cell ZHJ-25 is formed
Embodiment 7 ephedra cells (Ephedra) ZHJ-25, preserving number are the variation of CGMCC No.0359 proliferation rate with the process of cultivation
Ephedra cell is cultivated in solid growth culture media, be about to ephedra cell in every 8-15 days and transfer to and carry out succeeding transfer culture in the growth medium of new preparation, guarantee the cells physiological activity.Fig. 1 is in the ephedra cell process of growth, sampling in per three days, claim the fresh weight amount of cell, the variation of weight when calculating cell with respect to inoculation.As can be seen from Figure 1, at 8-15 days, it is maximum that cell proliferation rate reaches, and prolongs vegetative period, and cell activity reduces, and proliferation rate reduces.Its proliferation rate calculates by following formula:
Embodiment 8 ephedra cells (Ephedra) ZHJ-25, preserving number contains 2 for CGMCC No.0359 cultivation production ephedrine (i) ephedra cell is seeded in, in the plant cell culture medium of 4-D and indolylacetic acid, 23-26 ℃ of training
Supported the cultivation that promptly gets ephedra cell 10-30 days; After (ii) passing through 10-30 days cultivation, the color of ephedra cell becomes dark-brown, takes out ephedra cell at low temperatures
Drying is ground into fine powder then, crosses 100 purpose stainless steel meshs, as sample.
Claims (5)
1. an ephedra cell (Ephedra) ZHJ-25, preserving number is CGMCC No.0359.
2. induce the described ephedra cell of claim 1 (Ephedra) ZHJ-25 for one kind, preserving number is the method for CGMCCNo.0359, comprises the following steps to carry out in proper order:
(1) the 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, sterilized 10-30 minute;
(2) stem with above-mentioned (1) clean plant is cut into 0.5cm length, receives in the substratum that contains 1.0-10% (weight/volume) sugar, at 23-26 ℃ of heat insulating culture 5-30 days;
(3) gained Ephedra callus after the growth of (2) is downcut from explant, transfer in the substratum of new preparation and carry out succeeding transfer culture, under 23-26 ℃ of temperature, cultivated 12-15 days;
Described step, (2) and, the culture medium compositing formula of the ephedra cell (3): reagent name concentration mmol/L reagent name concentration mmol/L potassium nitrate 2-10.0 copper sulphate 0.0001-0.001 sodium dihydrogen phosphate 0.3-3.0 zinc sulfate 0.01-0.1 magnesium sulfate 1-3 iron edetate 0.1-2 Boratex 0.001-0.007 kinetin 0.001-0.01 sulfate of ammoniac 0.5-3.0 IAA 0.005-0.005 calcium chloride 0.5-3.0 sucrose 1.0-10, (%w/v).
3. by described ephedra cell (Ephedra) ZHJ-25 that induces of claim 2, preserving number is that the method for CGMCCNo.0359 is characterized in that: in the described substratum compositing formula, comprise that also adding concentration is the phenylalanine of 0.1-20mmol/L.
4. by described ephedra cell (Ephedra) ZHJ-25 that induces of claim 2, preserving number is that the method for CGMCCNo.0359 is characterized in that: in the described substratum compositing formula, also comprise the fungal fermented filtrate that adds the 10%-50% weight percent, the concentration of its fungal fermented filtrate is 10-50w/v.
5. be that the method for CGMCCNo.0359 is characterized in that by described ephedra cell (Ephedra) the ZHJ-25 preserving number of inducing of claim 2: in the described substratum compositing formula, also comprise the agar that adds the 0.6-0.7% weight percent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98125187A CN1101467C (en) | 1998-12-04 | 1998-12-04 | Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98125187A CN1101467C (en) | 1998-12-04 | 1998-12-04 | Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1256309A CN1256309A (en) | 2000-06-14 |
| CN1101467C true CN1101467C (en) | 2003-02-12 |
Family
ID=5229072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98125187A Expired - Fee Related CN1101467C (en) | 1998-12-04 | 1998-12-04 | Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1101467C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100562577C (en) * | 2006-03-01 | 2009-11-25 | 新疆大学 | Conversion and the acquisition transgenic yeast engineering bacteria of the total DNA of ion-beam mediated Chinese ephedra in yeast |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1134930A (en) * | 1996-02-21 | 1996-11-06 | 新疆医学院 | Salicylic dextrogyric pseudoephedrine and synthesis method thereof |
-
1998
- 1998-12-04 CN CN98125187A patent/CN1101467C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1134930A (en) * | 1996-02-21 | 1996-11-06 | 新疆医学院 | Salicylic dextrogyric pseudoephedrine and synthesis method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1256309A (en) | 2000-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20090068715A1 (en) | Method of producing bio-ethanol | |
| CN108913625B (en) | Salt-tolerant streptomycete, microbial inoculum thereof and application of microbial inoculum thereof in promoting plant growth | |
| CN112725186B (en) | Breeding method of high-temperature-resistant chlorella for aquaculture | |
| CN107467075B (en) | Application of bacillus pumilus as rice growth promoter | |
| CN115725419B (en) | Phosphorus-dissolving blueberry endophytic trichoderma and application thereof | |
| CN107549018A (en) | Chinese mugwort tissue culture method | |
| CN109402009B (en) | Method for screening nitrogen-fixing blue algae antagonistic to rhizoctonia solani and application thereof | |
| CN1600082A (en) | Tissue culture technique for peony | |
| CN1101467C (en) | Ephedra cell ZHJ-25 CGMCC No.0359 and its induction method | |
| CN103451116A (en) | Pseudomonas fluorescens Y13, and preparation method and applications thereof | |
| CN110257277A (en) | A kind of composite bacteria agent ZLM-11 and its application | |
| CN1058290C (en) | Arnebia euchroma (Royle) Johnst. cell cultivation and prodn. process by solid two step method | |
| CN110373334B (en) | Trichoderma asperellum HN082102 and application thereof | |
| CN112831449A (en) | Seed expansion method for carrying out Nostoc sphaeroides cultivation section generation by utilizing temperature rise | |
| CN116355808B (en) | Bacillus aerophilus and application thereof | |
| CN110183260A (en) | A kind of plant nutrition liquid and its application using fermented liquid for biologically producing hydrogen preparation | |
| CN117025398B (en) | Protozoan flagellate NJAU-W1 for promoting tomato growth and preventing and controlling bacterial wilt and application thereof | |
| CN114774311B (en) | Flavobacterium and application thereof in promoting crop growth and improving crop cold resistance | |
| CN108531428A (en) | A kind of preparation method of fast-growing frankia bacterium powder | |
| CN108707560A (en) | A kind of microbial liquid bacterial manure and its preparation method and application | |
| CN116144531B (en) | Phosphate-dissolving bacteria for promoting crop growth and application thereof | |
| CN112369424B (en) | Seed coating agent and coating method for reducing black spot of rhizoma atractylodis | |
| CN116514602B (en) | Liquid microbial fertilizer and application method thereof | |
| CN117802012B (en) | Ammonia philic bacteria, bacterial agent and application thereof | |
| CN107629970A (en) | A kind of microorganism and application for being used to prevent and treat ginseng crythrodermia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |