CN107853174A - A kind of method for improving angelica keiskei koidz lateral bud survival rate - Google Patents
A kind of method for improving angelica keiskei koidz lateral bud survival rate Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses a kind of method for improving angelica keiskei koidz lateral bud survival rate, comprise the following steps：Lateral bud culture：Take the tender angelica keiskei koidz tender stem segmentses flowing water of children to rinse and remove stem section moisture after using aseptic process, the stem section after cutting is put into MS culture mediums and cultivated, in the range of the pH value for keeping culture medium is 5.5 6.5.Wherein, Multiplying culture：Stem section after the completion of lateral bud culture is put into culture medium, the concentration of culture medium mysoinositol is 86 110mg/L, the concentration of indolebutyric acid is 0.05 0.15mg/L, and incubation time is 5 7d.Using embodiment provided by the invention, improved by the method for planting after the culture to lateral bud culture medium and increment cultivation, improve lateral bud survival rate.
Description
Technical field
The present invention relates to angelica keiskei koidz lateral bud to cultivate field, more particularly to a kind of method for improving angelica keiskei koidz lateral bud survival rate.
Background technology
Angelica keiskei koidz alias lindenleaf torricellia bark or leaf, Ru Tongcai, big greenery, native pseudo-ginseng, scattered blood ginger, angelica keiskei koidz, chicken dish, big beef fat, fork flower
Sedum uizoon, Radix et Rhizoma Gynurae divaricatae, gynura divaricata, rich and honour dish, chrysanthemum pseudo-ginseng, in vain chicken dish, back of the body dish and Wen Youqing.Composite family Gynura Cass plant, with
All herbal medicine.Summer and autumn gather, and clean section, using fresh herb or dry.It is sweet, light, tremble with fear.Available for clearing heat and detoxicating, relieving rigidity of muscles and knitting bone, cool blood
Hemostasis.For Bronchopneumonia, infantile hyperpyrexia, pertussis, red eye, swell pain, rheumatic arthritis, uterine bleeding；Traumatic injury is controlled in external application,
Fracture, traumatism and bleeding, mastitis, sore malignant boil swell, burn and scald.
In the prior art, by carrying out medium culture to the lateral bud of angelica keiskei koidz, then carried out using bottle inoculation explant
Multiple Buds are formed and culture and then progress culture of rootage, then are transplanted, and in this process, lateral bud stem section is needed by multiple
Living environment transfer and also need to be cultivated using bottle, the damage of stem section can be caused, the lateral bud of angelica keiskei koidz in practical application
Survival rate only has 85%-92% or so, therefore the culture for being unfavorable for angelica keiskei koidz lateral bud during actual production uses, it is necessary to is survived
The raising of rate.
The content of the invention
The technical problems to be solved by the invention are to provide the method for improving angelica keiskei koidz lateral bud survival rate, by being trained to lateral bud
Support the method for planting after the culture and increment cultivation of base to improve, improve lateral bud survival rate.
To achieve the above object, the present invention provides following technical scheme, a kind of side for improving angelica keiskei koidz lateral bud survival rate
Method, methods described comprise the following steps：
Lateral bud culture：Take the tender angelica keiskei koidz tender stem segmentses flowing water of children to rinse and remove stem section moisture after using aseptic process, will
Each stem section with lateral bud is cut into 0.8-1.5cm length, and the stem section after cutting is put into MS culture mediums and cultivated, and keeps culture medium
PH value be 5.5-6.5 in the range of, the stem section after cutting first is subjected to the light culture of 1-3 days in the medium, during this period often
It adds agar, sucrose, vitamin C to culture medium, and keeps agar in culture medium, sucrose, ascorbic concentration difference
：Between 4.5-6.5g/L, 24-32g/L, 15-21mg/L, temperature is 23-26 DEG C, is being 2000lx-3000lx by intensity
Illumination condition under cultivate 22-30d；
Multiplying culture：Stem section after the completion of lateral bud culture is put into culture medium, the concentration of culture medium mysoinositol is 86-
110mg/L, the concentration of indolebutyric acid are 0.05-0.15mg/L, incubation time 5-7d；
Plantation：It is placed in air and takes exercise 2-3 days after cleaning stem section, transplant into greenhouse, is covered with film with moisture-heat preservation,
Holding humidity is between 70%-80%, temperature carries out sheltering from heat or light for 50%-60% between 23-26 degrees Celsius, is removed after 20d
Film, gathered in after 4-6 months.
Preferably, methods described also includes：
Extracting flavonoids：By angelica keiskei koidz complete stool natural air drying, powder is ground into, adds the petroleum ether that temperature is 42-70 DEG C, point
It is 1 not according to powder and petroleum ether mass ratio:2.5-4 and 1:5-7 is extracted, and the time extracted every time is 1.5-2H, is obtained
Extract powder；Extraction solution is added in powder is extracted, and flows back and filters using 60 DEG C -70 DEG C of water-bath, after being filtered
Powder；Powder after filtering is soaked using distilled water to extract, respectively with 0.3mol/L NaOH and 0.3mol/L
HCl handles 20-25 minutes, and between being cleaned to PH with distilled water and being 5.5-6.5；Eluted using graded ethanol, and do not received
Collection eluent merges concentration.
Preferably, the concentration of graded ethanol is respectively：35% ethanol, 55% ethanol, 75% ethanol, 95% ethanol and anhydrous
Ethanol, obtain respectively each concentration ethanol eluate merge concentration after, using 30% ethanol carry out constant volume.
Preferably, the extraction solution is respectively 60% ethanol, 60% methanol and distilled water.
Preferably, before using graded ethanol elution, add Silon and be stirred, and powder solution is added
In the core chromatographic column of Silon, then eluted using graded ethanol.
Preferably, the MS culture mediums just also include methyl α-naphthyl acetate that concentration is 0.02-0.03mg/L, dense outside minimal medium
Spend the benzyl aminoadenine for 0.02-0.03mg/L.
Preferably, air themperature is placed in be taken exercise 2-3 days in 20-22 DEG C of air after cleaning stem section, and keeps 90%
Air humidity.
Preferably, the eluent merged after concentrating is positioned in circulation reflux device together and carries out refluxing extraction, 60-70
Under the conditions of DEG C, refluxing extraction 3-4 times, each 30-40min.
The method provided by the invention for improving angelica keiskei koidz lateral bud survival rate, has the advantages that：
A kind of 1. application method for improving angelica keiskei koidz lateral bud survival rate provided by the invention, by being trained to the lateral bud of angelica keiskei koidz
Support and sucrose, agar and vitamin are added in base and is maintained at and must then carry out between concentration range and process is light culture
Increment culture and plantation, the survival rate of lateral bud can be improved by test of many times.
2. a kind of application method for improving angelica keiskei koidz lateral bud survival rate provided by the invention, is first extracted using petroleum ether
Powder is obtained, is then flowed back using water-bath and filters acquisition powder, respectively at the HCl with 0.3mol/L NaOH and 0.3mol/L
Manage 20-25 minutes, and between being cleaned to PH with distilled water and being 5.5-6.5；Eluted using graded ethanol, and Shou Ji not eluted
Liquid merges concentration, and the general flavone content of angelica keiskei koidz can be caused to improve 6%-10%.
3. a kind of application method for improving angelica keiskei koidz lateral bud survival rate provided by the invention, Air Temperature is placed in after cleaning stem section
Spend in the air for 20-22 DEG C and take exercise 2-3 days, and keep 90% air humidity, directly stem section is placed in air than ever
The existence of stem section can more be tempered.
Embodiment
To make the purpose, technical scheme and advantage of invention of greater clarity, below by embodiment, to the technology of the present invention
Scheme is further elaborated.However, it should be understood that specific embodiment described herein is only explaining skill of the present invention
Art scheme, it is not intended to limit the invention the scope of technical scheme.
Embodiment 1：
(1) culture medium is prepared：Prepare improvement MS inducing cultures：Add 1.5mg/L 6-BA, 0.1mg/L NAA, PH
It is worth for 5.5-6.5；
(2) lateral bud culture：Take the tender angelica keiskei koidz tender stem segmentses flowing water of children to rinse and remove stem section moisture after using aseptic process,
Each stem section with lateral bud is cut into 0.8cm length, the stem section after cutting is put into MS culture mediums and cultivated, keeps culture medium
In the range of pH value is 5.5-6.5, the stem section after cutting first is subjected to the light culture of 2 days in the medium, during this period daily to
Culture medium adds agar, sucrose, vitamin C, and keeps agar in culture medium, sucrose, ascorbic concentration to exist respectively：
Between 4.5g/L, 24g/L, 15mg/L, temperature is 23 DEG C, is cultivated under by intensity for 2100lx-2500lx illumination condition
22d。
Multiplying culture：Stem section after the completion of lateral bud culture is put into culture medium, the concentration of culture medium mysoinositol is 86mg/
L, the concentration of indolebutyric acid is 0.05mg/L, incubation time 5d；
Plantation：It is placed in air and takes exercise 2 days after cleaning stem section, transplant into greenhouse, is covered with moisture-heat preservation, protected with film
Hold humidity between 70%-80%, temperature carry out sheltering from heat or light for 50%-60% between 23 degrees Celsius, film is removed after 20d,
Lateral bud survival rate is gathered in after being 96%, 4-6 months.In addition, the air that air themperature is 20-22 DEG C is placed in after cleaning stem section
It is middle to take exercise 2-3 days, and keep 90% air humidity
Embodiment 2：
(1) culture medium is prepared：Prepare improvement MS inducing cultures：Add 1.5mg/L 6-BA, 0.1mg/L NAA, PH
It is worth for 6.0-6.5；
Lateral bud culture：Take the tender angelica keiskei koidz tender stem segmentses flowing water of children to rinse and remove stem section moisture after using aseptic process, will
Each stem section with lateral bud is cut into 1.2cm length, and the stem section after cutting is put into MS culture mediums and cultivated, keeps the PH of culture medium
It is worth in the range of 5.5-6.5, the stem section after cutting first to be carried out to the light culture of 3 days in the medium, during this period daily to training
Support base and add agar, sucrose, vitamin C, and keep agar in culture medium, sucrose, ascorbic concentration to exist respectively：6.5g/
L, between 32g/L, 21mg/L, temperature is 23-26 DEG C, and 30d is cultivated under by intensity for 3000lx illumination condition；
Multiplying culture：Stem section after the completion of lateral bud culture is put into culture medium, the concentration of culture medium mysoinositol is
110mg/L, the concentration of indolebutyric acid are 0.15mg/L, incubation time 7d；
Plantation：It is placed in air and takes exercise 3 days after cleaning stem section, transplant into greenhouse, is covered with moisture-heat preservation, protected with film
Hold humidity between 70%-80%, temperature carry out sheltering from heat or light for 50%-60% between 26 degrees Celsius, film is removed after 20d,
Lateral bud survival rate is gathered in after being 96%, 4-6 months.
(2) tissue cultures：It is placed in air and takes exercise 2-3 days after cleaning stem section, transplant into greenhouse, is covered with film to protect
Wet insulation, holding humidity is between 70%-80%, temperature carries out sheltering from heat or light for 50%-60% between 23-26 degrees Celsius,
Film is removed after 20d, is gathered in after 4-6 months.MS culture mediums are just 0.02- also including concentration outside minimal medium
0.03mg/L methyl α-naphthyl acetate, the benzyl aminoadenine that concentration is 0.02-0.03mg/L.
As shown in table 1, by increasing inositol, agar, sucrose, vitamin C and keep finite concentration, and increase inositol,
The H1-H6 of acquisition, lateral bud survival rate are respectively：94%th, 97%, 94,97%, 94,96%, do not have in common MS culture mediums
Inositol, agar, sucrose, it is ascorbic in the case of lateral bud survival rate between 85%-93%.
Table 1
Embodiment 3：
Extracting flavonoids：By angelica keiskei koidz complete stool natural air drying, powder is ground into, adds the petroleum ether that temperature is 42-70 DEG C, point
It is 1 not according to powder and petroleum ether mass ratio:2.5-4 and 1:5-7 is extracted, and the time extracted every time is 1.5-2H, is obtained
Extract powder；Extraction solution is added in powder is extracted, and flows back and filters using 60 DEG C -70 DEG C of water-bath, after being filtered
Powder；Powder after filtering is soaked using distilled water to extract, respectively with 0.3mol/L NaOH and 0.3mol/L
HCl handles 20-25 minutes, and between being cleaned to PH with distilled water and being 5.5-6.5；Eluted using graded ethanol, and do not received
Collection eluent merges concentration.
Petroleum ether is used to be extracted for solvent in apparatus,Soxhlet's, wherein, powder and petroleum ether mass ratio are 1:2.5-4 and
1:5-7, extraction solution are respectively 60% ethanol, 60% methanol and distilled water, to remove chlorophyll and dewax, degreasing, then certainly
Petroleum ether can be flung to, it is standby to obtain powder.Add extraction solution after, using water-bath flow back and be filtrated to get filter after powder, so
The removal of impurity is gone using distilled water immersion afterwards, and neutralisation treatment is carried out using NaOH and HCL, then carries out distilled water cleaning again.
Then eluted using graded ethanol, and not Shou Ji eluent merge concentration.The eluent after concentrating will be merged together
It is positioned in circulation reflux device and carries out refluxing extraction, under the conditions of 60-70 DEG C, refluxing extraction 3-4 times, each 30-40min.
Wherein, graded ethanol is respectively：35% ethanol, 55% ethanol, 75% ethanol, 95% ethanol and absolute ethyl alcohol, point
Do not obtain each concentration ethanol eluate merge concentration after, using 30% ethanol carry out constant volume.In addition, using gradient
Before ethanol elution, add Silon and be stirred, and powder solution is added in the core chromatographic column of Silon, so
Eluted afterwards using graded ethanol.
It is recognised that the illustrative embodiments that above-described embodiment uses only for explanation inventive principle, but this hair
Bright to be not limited only to this, those skilled in the art can make various improvement and change in the case where not departing from real situation of the present invention, this
A little improvement and change fall within protection scope of the present invention.
Claims (8)
- A kind of 1. method for improving angelica keiskei koidz lateral bud survival rate, it is characterised in that, methods described comprises the following steps：Lateral bud culture：Take the tender angelica keiskei koidz tender stem segmentses flowing water of children to rinse and remove stem section moisture after using aseptic process, will be each The individual stem section with lateral bud is cut into 0.8-1.5cm length, and the stem section after cutting is put into MS culture mediums and cultivated, keeps the PH of culture medium Be worth in the range of 5.5-6.5, the stem section after cutting first carried out to the light culture of 1-3 days in the medium, during this period daily to Culture medium adds agar, sucrose, vitamin C, and keeps agar in culture medium, sucrose, ascorbic concentration to exist respectively： Between 4.5-6.5g/L, 24-32g/L, 15-21mg/L, temperature is 23-26 DEG C, in the light by intensity for 2000lx-3000lx 22-30d is cultivated according under the conditions of；Multiplying culture：Stem section after the completion of lateral bud culture is put into culture medium, the concentration of culture medium mysoinositol is 86-110mg/ L, the concentration of indolebutyric acid is 0.05-0.15mg/L, incubation time 5-7d；Plantation：It is placed in air and takes exercise 2-3 days after cleaning stem section, transplant into greenhouse, is covered with moisture-heat preservation, kept with film Humidity is between 70%-80%, temperature carries out sheltering from heat or light for 50%-60% between 23-26 degrees Celsius, is removed after 20d thin Film, gathered in after 4-6 months.
- 2. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 1, it is characterised in that methods described is also Including：Extracting flavonoids：By angelica keiskei koidz complete stool natural air drying, powder is ground into, the petroleum ether that temperature is 42-70 DEG C is added, presses respectively It is 1 according to powder and petroleum ether mass ratio:2.5-4 and 1:5-7 is extracted, and the time extracted every time is 1.5-2H, is extracted Powder；Extraction solution is added in powder is extracted, and flows back and filters using 60 DEG C -70 DEG C of water-bath, powder after being filtered； Powder after filtering is soaked using distilled water to extract, respectively at the HCl with 0.3mol/L NaOH and 0.3mol/L Manage 20-25 minutes, and between being cleaned to PH with distilled water and being 5.5-6.5；Eluted using graded ethanol, and Shou Ji not eluted Liquid merges concentration.
- 3. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 2, it is characterised in that graded ethanol Concentration is respectively：35% ethanol, 55% ethanol, 75% ethanol, 95% ethanol and absolute ethyl alcohol, the second of each concentration is obtained respectively After alcohol eluen merges concentration, constant volume is carried out using 30% ethanol.
- 4. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 2, it is characterised in that the extraction is molten Liquid is respectively 60% ethanol, 60% methanol and distilled water.
- 5. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 1, it is characterised in that using gradient Before ethanol elution, add Silon and be stirred, and powder solution is added in the core chromatographic column of Silon, so Eluted afterwards using graded ethanol.
- A kind of 6. method for improving angelica keiskei koidz lateral bud survival rate according to claim 1, it is characterised in that the MS cultures Base just also includes the benzyl amino that the methyl α-naphthyl acetate, concentration that concentration is 0.02-0.03mg/L are 0.02-0.03mg/L outside minimal medium Adenine.
- 7. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 1, it is characterised in that after cleaning stem section It is placed in the air that air themperature is 20-22 DEG C and takes exercise 2-3 days, and keeps 90% air humidity.
- 8. a kind of method for improving angelica keiskei koidz lateral bud survival rate according to claim 2, it is characterised in that concentrated merging Eluent afterwards is positioned in circulation reflux device together carries out refluxing extraction, under the conditions of 60-70 DEG C, refluxing extraction 3-4 times, often Secondary 30-40min.
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