CN105838749A - Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone - Google Patents
Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone Download PDFInfo
- Publication number
- CN105838749A CN105838749A CN201610353013.6A CN201610353013A CN105838749A CN 105838749 A CN105838749 A CN 105838749A CN 201610353013 A CN201610353013 A CN 201610353013A CN 105838749 A CN105838749 A CN 105838749A
- Authority
- CN
- China
- Prior art keywords
- flavone
- cell
- herba dendrobii
- culture
- unicellular
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone. The method is the one for producing Dendrobium huoshanense flavone by utilizing a cell cultivation reactor for suspension cultivation of Dendrobium huoshanense cells, and comprises the following steps: (1) single cell preparation, (2) single cell system cultivation, (3) cell mass scale-up cultivation, and (4) extraction of Dendrobium huoshanense flavone: when the cell density realizes 60,000-80,000 cells per ml, the cultivation temperature is lowered to 7-10 DEG C, cultivation is continued for 5-7 days. Dendrobium huoshanense cells are collected and Dendrobium huoshanense flavone is extracted. The method can greatly shorten the cell cultivation cycle and improve the yield of Dendrobium huoshanense flavone products, and is simple and stable in technology, so as to be suitable for industrial production.
Description
Technical field
The present invention relates to Herba Dendrobii technical field, particularly relate to a kind of suspended culture cell and produce Herba Dendrobii Huang
The method of ketone.
Background technology
Herba Dendrobii flavonoid is one of main component of Herba Dendrobii, and flavone compound refers to two to be had
The phenyl ring (A-Yu B-ring) of phenolic hydroxyl group passes through a series of compounds that central authorities' thricarbon atom is interconnected,
Its basic parent nucleus is 2-phenyl chromone.Flavone compound structure often connects have phenolic hydroxyl group, methoxyl group,
The functional group such as methyl, isopentene group.Additionally, it is the most normal and sugar is combined into glycosides.Herba Dendrobii flavone has the heart
Vascular system is active, antibacterial and antiviral activity, anti-tumor activity, resisting oxidation free radical activity, antiinflammatory,
Analgesic activities, liver-protecting activity, have blood pressure lowering, blood fat reducing, defying age, raising immunity of organisms spasmolytic and resistance
The pharmacological actions such as state.Along with social development, people are more and more higher to healthy concern, and its demand also can be got over
Come the biggest, but the extracting method of tradition Herba Dendrobii flavone is with the root of Herba Dendrobii platymiscium, stem, leaf etc.
Plant extracts gained, and it is profound to Herba Dendrobii that the product of this technique gained can not meet future market
The demand of processing.
Along with the development of plant cell engineering technology, carry out artificial culture amplification with cell and extract the metabolism of plant
Product, can not be suitable to industrialization by limitations affect such as land area, climatic environment, region, pest and disease damages
Produce.Utilize cell engineering to produce the technology of Herba Dendrobii flavone, at home and abroad early have research, also take
Obtained many achievements, but there is a lot of difficult problem problem in process of production, such as complex process, Biomass speedup
Low, target product yield is low, production cycle length etc..About utilizing airlift agitation tank suspension culture Herba Dendrobii
Cell produce Herba Dendrobii flavone research it is not yet reported that, therefore a kind of efficiently to produce Herba Dendrobii yellow in exploitation
The method of ketone natural product is the most significant.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of technique is simple, stable, is suitable for industrialized production
Suspended culture cell produces the method for Herba Dendrobii flavone.
The present invention goes to solve above-mentioned technical problem by techniques below means: a kind of suspended culture cell produces
The method of Herba Dendrobii flavone, comprises the following steps:
(1) unicellular preparation: carry out aseptic seeding with solid medium after taking the sterilization of Herba Dendrobii capsule,
Take out after sprouting into protocorm, use enzymatic isolation method separation to prepare unicellular, and centrifugal filtration remove impurity, collect
Unicellular；
(2) monoclonal is cultivated: unicellular be inoculated in a MS fluid medium training by collect
Support；
(3) cell mass amplification culture: cultured monoclonal is inoculated in the 2nd MS fluid medium
Middle cultivation；In described 2nd MS fluid medium 2-3 times that P element content is MS fluid medium,
Described 2nd MS fluid medium is also added with the Ge element of 1-2mg/L, 0.02-0.1mg/L selenium element and
The acid hydrolyzed casein of 0.1-0.2g/L, pH value is 5.8 ± 0.4.
Use the 2nd MS fluid medium of the present invention, can extend relative to MS fluid medium and train in batches
Support cell growth time more than 2 times, improve cell density more than 2 times.Ge element, selenium element, acid hydrolysis
Casein can dramatically increase the fresh weight of Herba Dendrobii suspension cell, improve protein and chlorophyllous content, makes
Cellular anti-oxidant activity is significantly raised.
(4) Herba Dendrobii flavone is extracted: when cell density reaches 60000-80000/ml, reduce and cultivate
Temperature, to 7-10 DEG C, continues to cultivate to 5-7 days, uses centrifugal collection Herba Dendrobii cell；Extract Huoshan stone again
Dry measure used in former times flavone.
Owing to flavone is secondary metabolite, grow not coupling with cell, reduce cultivation temperature to this temperature model
In enclosing, make cell not increase, the generation of flavone product can be promoted.
Preferably, the culture medium that described step (1) sowing uses is the solid culture being suitable for orchid growth
Base.
Preferably, described solid medium is 1/2MS culture medium.In 1/2MS culture medium, a great number of elements halves,
It is suitable for germination of arethusa seeds.
Preferably, in described step (2), unicellular inoculum concentration is 25 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, and concussion is cultivated, temperature 22 ± 2 DEG C, when cell density reaches 10000-20000
/ mL, gets final product amplification culture.This range of light intensities contributes to the morphogenesis of cell and vigor promotes, too high then
Waste, too low, do not reach effect.This colour temperature is warm light, and royal purple light more equalizes, beneficially metabolite
Synthesis.This temperature range is the temperature that Herba Dendrobii growth equalizes the most with metabolism.Under this density, cell
Population growth be in finger logarithm period, the period that i.e. vigor is the most vigorous.
Preferably, in described step (3), monoclonal inoculum concentration is 30 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, airlift agitation suspension culture, and regulation Ventilation Rate and stir speed (S.S.) maintain dissolved oxygen to exist
30%.
Preferably, described 2nd MS fluid medium 0.1-2mg/L NAA, 0.1-2mg/L have been also added with
6-BA, 50-100mg/L pectase, 10-50mg/L cellulase, pH value is 5.8 ± 0.4.Add plant
Growth regulator and biological enzyme formulation in culture medium, can active cell merisis, and prevent cell aggregation
Caking, it is ensured that nutrition and the supply of molten oxygen and carbon dioxide are uniformly, fully.
Preferably, described Ge element derives from GeO2Or organic germanium.
Preferably, described selenium element derives from Na2SeO3Or organic selenium.
It is an advantage of the current invention that: first Herba Dendrobii aseptically sowing seeds is sprouted the protocorm formed by the present invention
Stem, utilize that enzymatic isolation method isolates in high vigor protocorm is unicellular, is trained monoclonal further,
After carry out cell mass amplification culture, in amplification culture, add plant growth regulator and biological enzyme formulation and arrive
In culture medium, energy active cell merisis, and prevent cell aggregation from luming, it is ensured that nutrition and dissolved oxygen and two
Carbonoxide supply is uniformly, fully.The present invention can be greatly shortened cell culture period, improves product yield,
And technique is simple, stable, it is suitable for industrialized production.
Detailed description of the invention
The detection method of flavones content of the present invention
1, determination of total flavonoids, the general flavone content extracting embodiment is analyzed, specific analytical method
As follows.The preparation of reference substance solution: take control substance of Rutin 20mg, accurately weighed, put in 50ml measuring bottle,
Add 60% appropriate amount of ethanol, put in 80 DEG C of water-baths and heat, make dissolving, let cool, with 60% ethanol dilution to scale,
Shake up.Precision measures 25ml, puts in 50ml measuring bottle, is diluted with water to scale, shakes up, and obtains (every 1ml
In the 0.2mg Han rutin).The drafting of standard curve: accurately draw 0.2mg/ml rutin solution 0.4,0.8,
1.2,1.6,2.0,2.4ml, be respectively placed in 10mL test tube, replace rutin solution to be zero with 30% ethanol
Pipe, respectively adds 30% ethanol to 2.4ml, adds people 5%NaNO respectively2Solution 0.4ml, shakes up, and stands 6min；
Add 10%Al (NO3)3Solution 0.4ml, shakes up, and stands 6min；Add 4%NaOH solution 4ml,
Shake up, stand 15min, add to scale with 30% ethanol；Absorbance is measured, with absorbance at 510nm
It is that abscissa draws standard curve for vertical coordinate and rutin content.By absorbance X to concentration Y (mg/ml)
Carry out regression Calculation, obtain standard curve equation: Y=0.9778X+0.001R2=0.9991.
The mensuration of sample: weigh the Herba Dendrobii flavone sample 50mg that this experiment prepares, be diluted to solution extinction
Value after (10-100 times) dilution, is measured in standard curve range as stated above.Each sample is surveyed
Trying 10 times, the value in the range of line taking is averaged.
Embodiment 1
The present embodiment discloses a kind of method that suspended culture cell produces Herba Dendrobii flavone, comprises the following steps:
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 23g/L, and light intensity is 2000lux, and colour temperature is 4000K, and concussion is cultivated, and adjusting pH value is 5.4,
Temperature 20 DEG C, when cell density reaches 10000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in that containing the 2nd MS, (P element in this MS culture medium is normal
2 times of rule MS fluid medium)+0.1mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+0.1mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+50mg/L neutrality pectase
The Ge element of+10mg/L neutral cellulase+1mg/L (derives from GeO2)+0.02mg/L selenium element (source
In Na2SeO3)+0.1g/L fluid medium 30L gas-lifting type stirred fermentor in, fresh cell inoculum concentration
For 28g/L, light intensity is 2000lux, and colour temperature is 4000K, airlift agitation suspension culture, regulation ventilation speed
Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar content
Keep pH 5.4 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS liquid training of 2 times of concentration
Support base；
(4) reach 60000/ml when cell density, after measured cell fresh weight 0.48g/mL in every milliliter of culture fluid,
Protein content 11mg/g, every gram of fresh cell Determination of Chlorophyll content 0.21mg/g in every gram of fresh cell, every gram fresh carefully
SOD activity 297U/g in born of the same parents, reduction cultivation temperature to 7 DEG C, continues to cultivate 5 days, uses 1000rpm low
Speed is centrifugal, 10min, collects Herba Dendrobii cell, is completed by the Herba Dendrobii cell collected latter 70 DEG C and is dried
To constant weight, and claying into power, room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, be placed in continuous circumfluence extraction device, added
80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h, profit
Remove chlorophyll with 45 micrometer Millipore membrane filtrations, further supernatant is carried out rotary evaporation concentration, then do
Dry to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.The Huoshan obtained according to the assay method of total flavones
In Herba Dendrobii flavone powder, the content of total flavones is 0.114g, and yield is 0.23%.
The present invention concrete mode used of sterilizing is 75% alcohol disinfecting 1min, 0.1%HgCl2Sterilization 15min,
Sterilized water washs 3 times.Following example all use the method to carry out disinfection.
The enzymatic isolation method of the present invention separates and uses prior art, specifically at 20uM sucrose, and 10uM MgCl2,
In 20uM pH7.8Tris HCl buffer, use pectase, cellulase to cell mass room temperature treatment 4-8h.
And centrifugal under the conditions of 500rpm, 10min, clean after collect unicellular.Following example all use the party
Method is entered to separate.
Embodiment 2
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 25g/L, and light intensity is 3000lux, and colour temperature is 5000K, and concussion is cultivated, and adjusting pH value is 5.8,
Temperature 22 DEG C, when cell density reaches 15000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
3 times of conventional MS fluid medium)+1mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+1mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase
The Ge element of+30mg/L neutral cellulase+1.5mg/L (derives from GeO2)+0.05mg/L selenium element (
Come from Na2SeO3) the 30L gas-lifting type stirred fermentor of fluid medium of acid hydrolyzed casein of+0.13g/L
In, fresh cell inoculum concentration is 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, and airlift agitation suspends
Cultivating, regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulate feed supplement liquid
Stream dosage keeps sugar content to keep pH 5.8 at 3% and NaOH solution stream dosage；Feed supplement liquid is 2 times of concentration
The 2nd MS fluid medium；
(4) reach 70000/ml when cell density, after measured cell fresh weight 0.56g/mL in every milliliter of culture fluid,
Protein content 12.4mg/g, every gram of fresh cell Determination of Chlorophyll content 0.21mg/g in every gram of fresh cell, every gram fresh
SOD activity 337U/g in cell, reduction cultivation temperature, to 9 DEG C, continues to cultivate 6 days, uses 1000rpm
Low-speed centrifugal, 10min, collects Herba Dendrobii cell, is completed by the Herba Dendrobii cell collected latter 70 DEG C and do
Dry to constant weight, and clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, be placed in continuous circumfluence extraction device, added
80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h, profit
Remove chlorophyll with 45 micrometer Millipore membrane filtrations, further supernatant is carried out rotary evaporation concentration, then do
Dry to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.The Huoshan obtained according to the assay method of total flavones
In Herba Dendrobii flavone powder, the content of total flavones is 0.185g, and yield is 0.37%.
Embodiment 3
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 27g/L, and light intensity is 4000lux, and colour temperature is 6000K, and concussion is cultivated, and adjusting pH value is 6.2,
Temperature 24 DEG C, when cell density reaches 20000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
2 times of conventional MS fluid medium)+2mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+2mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+100mg/L neutrality pectase
Ge element (deriving from the organic germanium)+0.1mg/L selenium element (source of+50mg/L neutral cellulase+2mg/L
In Na2SeO3)+0.2g/L acid hydrolyzed casein fluid medium 30L gas-lifting type stirred fermentor in,
Fresh cell inoculum concentration is 32g/L, and light intensity is 4000lux, and colour temperature is 6000K, airlift agitation suspension culture,
Regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulate feed supplement liquid stream dosage
Sugar content is kept to keep pH 6.2 at 3% and NaOH solution stream dosage；Feed supplement liquid is the second of 2 times of concentration
MS fluid medium；
(4) reach 80000/ml when cell density, after measured cell fresh weight 0.58g/mL in every milliliter of culture fluid,
Protein content 12.3mg/g, every gram of fresh cell Determination of Chlorophyll content 0.19mg/g in every gram of fresh cell, every gram fresh
SOD activity 313U/g in cell.Reduction cultivation temperature, to 10 DEG C, continues to cultivate 7 days, uses 1000rpm
Low-speed centrifugal, 10min, collects Herba Dendrobii cell, is completed by the Herba Dendrobii cell collected latter 70 DEG C and do
Dry to constant weight, and clay into power, room temperature Seal and preservation is standby.After cell reaches this density, cannot continue
Add density, reach the agitation limit of 30L airlift fermentor.
2, extract the stage
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, and be placed in continuous circumfluence extraction device, add
Enter 80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h,
Utilize 45 micrometer Millipore membrane filtrations to remove chlorophyll, further supernatant is carried out rotary evaporation concentration, then
It is dried to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.Obtain suddenly according to the assay method of total flavones
In the Herba Dendrobii flavone powder of mountain, the content of total flavones is 0.193g, and yield is 0.39%.
Embodiment 4
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 24g/L, and light intensity is 2500lux, and colour temperature is 4500K, and concussion is cultivated, and adjusting pH value is 5.6,
Temperature 21 DEG C, when cell density reaches 12000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
3 times of MS fluid medium)+0.5mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+0.7mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase+20mg/L is neutral
Ge element (deriving from organic germanium)+0.06mg/L selenium element (deriving from organic selenium) of cellulase+1.8mg/L
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.16g/L, fresh cell connects
Amount of planting is for 29g/L, and light intensity is 2500lux, and colour temperature is 4500K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.9 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) reach 60000/ml when cell density, after measured cell fresh weight 0.52g/mL in every milliliter of culture fluid,
Protein content 11.7mg/g, every gram of fresh cell Determination of Chlorophyll content 0.21mg/g in every gram of fresh cell, every gram
SOD activity 302U/g in fresh cell, reduction cultivation temperature, to 8 DEG C, continues to cultivate 6 days, uses 1000rpm
Low-speed centrifugal, 10min, collects Herba Dendrobii cell, is completed by the Herba Dendrobii cell collected latter 70 DEG C and do
Dry to constant weight, and clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, and be placed in continuous circumfluence extraction device, add
Enter 80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h,
Utilize 45 micrometer Millipore membrane filtrations to remove chlorophyll, further supernatant is carried out rotary evaporation concentration, then
It is dried to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.Obtain suddenly according to the assay method of total flavones
In the Herba Dendrobii flavone powder of mountain, the content of total flavones is 0.189g, and yield is 0.38%.
Embodiment 5
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 25g/L, and light intensity is 3000lux, and colour temperature is 4400K, and concussion is cultivated, and adjusting pH value is 5.7,
Temperature 22 DEG C, when cell density reaches 18000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
2 times of conventional MS fluid medium)+1mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+1.2mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+60mg/L neutrality pectase
The Ge element of+25mg/L neutral cellulase+25mg/L neutral cellulase+1.8mg/L (derives from
GeO2) liquid culture of acid hydrolyzed casein of+0.08mg/L selenium element (deriving from organic selenium)+0.17g/L
In the 30L gas-lifting type stirred fermentor of base, fresh cell inoculum concentration is 30g/L, and light intensity is 2000lux, color
Temperature is 5200K, airlift agitation suspension culture, and regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%,
Molten CO23%, regulation feed supplement liquid stream dosage keeps sugar content to keep pH at 3% and NaOH solution stream dosage
5.8；Feed supplement liquid is the 2nd MS fluid medium of 2 times of concentration；
(4) 60000/ml, after measured cell fresh weight in every milliliter of culture fluid are reached when cell density
Protein content 11.4mg/g, every gram of fresh cell Determination of Chlorophyll content in 0.55g/mL, every gram of fresh cell
0.17mg/g, SOD activity 311U/g in every gram of fresh cell, reduction cultivation temperature, to 7 DEG C, continues cultivation 6
My god, use 1000rpm low-speed centrifugal, 10min, collect Herba Dendrobii cell, the Herba Dendrobii that will collect
Cell completes latter 70 DEG C and is dried to constant weight, and clays into power, and room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, and be placed in continuous circumfluence extraction device, add
Enter 80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h,
Utilize 45 micrometer Millipore membrane filtrations to remove chlorophyll, further supernatant is carried out rotary evaporation concentration, then
It is dried to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.Obtain suddenly according to the assay method of total flavones
In the Herba Dendrobii flavone powder of mountain, the content of total flavones is 0.201g, and yield is 0.40%.
Embodiment 6
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 26g/L, and light intensity is 3000lux, and colour temperature is 5000K, and concussion is cultivated, and adjusting pH value is 6.1,
Temperature 20 DEG C, when cell density reaches 12000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
3 times of MS fluid medium)+0.3mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+1.4mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+60mg/L neutrality pectase+30mg/L is neutral
The Ge element of cellulase+30mg/L neutral cellulase+2mg/L (derives from GeO2)+0.05mg/L selenium unit
The 30L gas-lifting type stirring of the fluid medium of the acid hydrolyzed casein of element (deriving from organic selenium)+0.18g/L
In fermentation tank, fresh cell inoculum concentration is 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, and gas lift stirs
Mix suspension culture, regulation Ventilation Rate and stir speed (S.S.) and maintain molten O230%, molten CO23%, regulation
Feed supplement liquid stream dosage keeps sugar content to keep pH 5.8 at 3% and NaOH solution stream dosage；Feed supplement liquid is 2
2nd MS fluid medium of times concentration；
(4) 60000/ml, after measured cell fresh weight in every milliliter of culture fluid are reached when cell density
Protein content 12.2mg/g, every gram of fresh cell Determination of Chlorophyll content in 0.57g/mL, every gram of fresh cell
0.21mg/g, SOD activity 301U/g in every gram of fresh cell, reduction cultivation temperature, to 8 DEG C, continues to cultivate 6 days,
Use 1000rpm low-speed centrifugal, 10min, collect Herba Dendrobii cell, the Herba Dendrobii cell that will collect
Completing latter 70 DEG C and be dried to constant weight, and clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, and be placed in continuous circumfluence extraction device, add
Enter 80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h,
Utilize 45 micrometer Millipore membrane filtrations to remove chlorophyll, further supernatant is carried out rotary evaporation concentration, then
It is dried to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.Obtain suddenly according to the assay method of total flavones
In the Herba Dendrobii flavone powder of mountain, the content of total flavones is 0.196g, and yield is 0.39%.
Embodiment 7
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 26g/L, and light intensity is 4000lux, and colour temperature is 6000K, and concussion is cultivated, and adjusting pH value is 6.2,
Temperature 23 DEG C, when cell density reaches 13000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
2 times of MS fluid medium)+1.3mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+0.8mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase+35mg/L is neutral
The Ge element of cellulase+1.6mg/L (derives from GeO2)+0.1mg/L selenium element (deriving from organic selenium)
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.17g/L, fresh cell connects
Amount of planting is for 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.6 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) 70000/ml, after measured cell fresh weight in every milliliter of culture fluid are reached when cell density
Protein content 12.2mg/g, every gram of fresh cell Determination of Chlorophyll content in 0.54g/mL, every gram of fresh cell
0.18mg/g, SOD activity 322U/g in every gram of fresh cell, reduction cultivation temperature, to 8 DEG C, continues to cultivate 7 days,
Use 1000rpm low-speed centrifugal, 10min, collect Herba Dendrobii cell, the Herba Dendrobii cell that will collect
Completing latter 70 DEG C and be dried to constant weight, and clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, be placed in continuous circumfluence extraction device, added
80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h, profit
Remove chlorophyll with 45 micrometer Millipore membrane filtrations, further supernatant is carried out rotary evaporation concentration, then do
Dry to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.The Huoshan obtained according to the assay method of total flavones
In Herba Dendrobii flavone powder, the content of total flavones is 0.145g, and yield is 0.29%.
Comparative example 1
Choose the Seeded growth Herba Dendrobii tissue cultured seedling of 8 months, tissue cultured seedling is cleaned and completes after draining, then 70 DEG C
Being dried to constant weight, and clay into power, room temperature Seal and preservation is standby.
Weighed the Herba Dendrobii powder 50.0g being dried of 20 mesh sieves, be placed in continuous circumfluence extraction device, added
80% ethanol 3L reflux, extract, 1h.Take the extraction solution in flask, add appropriate decolorizing with activated carbon 1h, profit
Remove chlorophyll with 45 micrometer Millipore membrane filtrations, further supernatant is carried out rotary evaporation concentration, then do
Dry to constant weight, it is thus achieved that solubility Herba Dendrobii flavone coarse powder.The Huoshan obtained according to the assay method of total flavones
In Herba Dendrobii flavone powder, the content of total flavones is 0.156g, and yield is 0.31%.
By above-mentioned cultivation and extraction, the judgement cell culture method of the present invention that can will be apparent from is carefully
Born of the same parents cultivate the Herba Dendrobii flavone of production can be close to the even more than Seeded growth same period 8 months on yield
The Herba Dendrobii flavone that tissue cultured seedling produces.
The method of the present invention is not intended to the production of Herba Dendrobii flavone, applies also for other edible or medicinal dendrobiums
The production of platymiscium flavone.The oneth MS fluid medium of the present invention is conventional MS fluid medium or P
Element is the culture medium of conventional MS fluid medium P element 2-3 times.
The foregoing is only the preferred embodiment of the invention, not in order to limit the invention,
Any amendment, equivalent and the improvement etc. made within all spirit in the invention and principle, all should
Within being included in the protection domain of the invention.
Claims (8)
1. the method that a suspended culture cell produces Herba Dendrobii flavone, it is characterised in that include following step
Rapid:
(1) unicellular preparation: carry out aseptic seeding with solid medium after taking the sterilization of Herba Dendrobii capsule,
Take out after sprouting into protocorm, use enzymatic isolation method separation to prepare unicellular, and centrifugal filtration remove impurity, collect
Unicellular；
(2) monoclonal is cultivated: unicellular be inoculated in a MS fluid medium training by collect
Support；
(3) cell mass amplification culture: cultured monoclonal is inoculated in the 2nd MS fluid medium
Middle cultivation；In described 2nd MS fluid medium 2-3 times that phosphorus element content is MS fluid medium,
Described 2nd MS fluid medium is also added with the Ge element of 1-2mg/L, 0.02-0.1mg/L selenium element and
The acid hydrolyzed casein of 0.1-0.2g/L, pH value is 5.8 ± 0.4；
(4) Herba Dendrobii flavone is extracted: when cell density reaches 60000-80000/ml, reduce and cultivate
Temperature, to 7-10 DEG C, continues to cultivate 5-7 days, uses centrifugal collection Herba Dendrobii cell；Extract Herba Dendrobii again
Flavone.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, its
Being characterised by, the culture medium that described step (1) sowing uses is to be suitable for the solid medium of orchid growth.
A kind of suspended culture cell the most according to claim 2 produces the method for Herba Dendrobii flavone, its
Being characterised by, described solid medium is 1/2MS solid medium.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, its
Being characterised by, in described step (2), unicellular inoculum concentration is 25 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, and concussion is cultivated, temperature 22 ± 2 DEG C, when cell density reaches 10000-20000
/ mL, gets final product amplification culture.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, and it is special
Levying and be, in described step (3), monoclonal inoculum concentration is 30 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, temperature 22 ± 2 DEG C, airlift agitation suspension culture, regulation Ventilation Rate and stirring speed
Rate maintains dissolved oxygen 30%.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, its
Be characterised by, described second liquid MS culture medium has been also added with 0.1-2mg/L NAA, 0.1-2mg/L 6-BA,
50-100mg/L pectase, 10-50mg/L cellulase, pH value is 5.8 ± 0.4.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, its
Being characterised by, described Ge element derives from GeO2Or organic germanium.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii flavone, and it is special
Levying and be, described selenium element derives from Na2SeO3Or organic selenium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610353013.6A CN105838749A (en) | 2016-05-20 | 2016-05-20 | Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610353013.6A CN105838749A (en) | 2016-05-20 | 2016-05-20 | Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105838749A true CN105838749A (en) | 2016-08-10 |
Family
ID=56594424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610353013.6A Pending CN105838749A (en) | 2016-05-20 | 2016-05-20 | Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105838749A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109928946A (en) * | 2019-04-02 | 2019-06-25 | 贵州师范学院 | A method of ventilation suspended culture cell produces product medicinal herbs flavones |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250827A (en) * | 2010-10-13 | 2011-11-23 | 黎云祥 | E.wushanense suspended cell culture system, and establishing method and application thereof |
| CN103468632A (en) * | 2013-09-17 | 2013-12-25 | 南京通泽农业科技有限公司 | Method for producing flavone from hovenia acerba suspension cells |
| CN104450817A (en) * | 2014-10-13 | 2015-03-25 | 皖西学院 | Method for producing flavone of anoectochilus roxburghii by suspension cultivation of cells |
-
2016
- 2016-05-20 CN CN201610353013.6A patent/CN105838749A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250827A (en) * | 2010-10-13 | 2011-11-23 | 黎云祥 | E.wushanense suspended cell culture system, and establishing method and application thereof |
| CN103468632A (en) * | 2013-09-17 | 2013-12-25 | 南京通泽农业科技有限公司 | Method for producing flavone from hovenia acerba suspension cells |
| CN104450817A (en) * | 2014-10-13 | 2015-03-25 | 皖西学院 | Method for producing flavone of anoectochilus roxburghii by suspension cultivation of cells |
Non-Patent Citations (3)
| Title |
|---|
| 吴佳雯等: "《乐清市铁皮石斛总黄酮含量的测定》", 《安徽农业科学》 * |
| 徐国华等: "《硒对铁皮石斛拟原球茎生长及抗氧化系统的影响》", 《南京师大学报（自然科学版）》 * |
| 魏明等: "《锗对霍山石斛类原球茎悬浮培养细胞生长和多糖合成及氧化还原态的影响》", 《生物工程学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109928946A (en) * | 2019-04-02 | 2019-06-25 | 贵州师范学院 | A method of ventilation suspended culture cell produces product medicinal herbs flavones |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102552335A (en) | Traditional Chinese medicine health care product, its preparation method and its application | |
| CN108165497A (en) | A kind of Monascus Strains breeding method of high yield Mo Nakelin K | |
| CN105906641B (en) | A kind of method of suspended culture cell production Dendrobidium huoshanness alkaloid | |
| CN102191214B (en) | Preparation method for resveratrol-producing cell and special-purpose suspension medium thereof | |
| CN101715733A (en) | Tissue culturing method of protocorms of dendrobium candidum | |
| CN104876945B (en) | A kind of alkaloid dimer and preparation method thereof and the application as antivirotic | |
| CN105838749A (en) | Method for utilizing suspension cultivation cells to produce Dendrobium huoshanense flavone | |
| CN102960246A (en) | Tissue culturing method for effectively improving general flavone content of tartary buckwheat | |
| CN105557312A (en) | Method for cultivating Cordyceps products by taking Tenebrio molitor (L.) larvae as carriers | |
| CN101245334A (en) | Technique for suspension cultivation of algam dendrobium nobile embryoid of medicinal effective composition of native plant strain | |
| CN102293122A (en) | Cultivating method for cordyceps militaris by using manyprickle acathopanax root | |
| CN105861589A (en) | Method for producing dendrobium huoshanense polysaccharide through suspension culture cells | |
| CN105613285B (en) | A kind of method of rosmarinic acid contents in quick raising Radix Salviae Miltiorrhizae | |
| CN105493899A (en) | Method for cultivating cordyceps sinensis products with mealworm pupae as carriers | |
| CN104396758A (en) | Dendrobium officinale embryoid culture process | |
| CN106478399B (en) | Derivative in hydroxy anthraquinones category and its application | |
| CN106282032B (en) | Penicillium citrinum LB and the application in phillygenol is prepared in bioconversion forsythin | |
| CN104450817A (en) | Method for producing flavone of anoectochilus roxburghii by suspension cultivation of cells | |
| CN104082134A (en) | Dendrobium embryoid suspension culture process | |
| CN109400444A (en) | Inhibit the sesquiterpenoids and preparation method thereof of plant pathogenic fungi | |
| CN107043795B (en) | Method for producing camptothecin and 10-hydroxycamptothecin by using camptotheca acuminata suspension cells | |
| CN104404100A (en) | Method for producing anoectochilus roxburghii polysaccharose through suspension cultivation of cell | |
| CN104450787A (en) | Method for producing alkaloid of anoectochilus roxburghii by suspension cultivation of cells | |
| CN105838625A (en) | Ophiocordyceps sinensis bacterial strain and preparation method of ophiocordyceps sinensis mycelium powder | |
| CN106754620B (en) | A method of promote polysaccharide effective constituents in the lobate layer bacterium of currant to accumulate using plant source cigarette water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160810 |
|
| RJ01 | Rejection of invention patent application after publication |