CN107155887B - A method of imperialine is produced using Fritillaria unibracteata callus - Google Patents
A method of imperialine is produced using Fritillaria unibracteata callus Download PDFInfo
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- CN107155887B CN107155887B CN201710403578.5A CN201710403578A CN107155887B CN 107155887 B CN107155887 B CN 107155887B CN 201710403578 A CN201710403578 A CN 201710403578A CN 107155887 B CN107155887 B CN 107155887B
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- callus
- fritillaria
- alkaloid
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- imperialine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8966—Fritillaria, e.g. checker lily or mission bells
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Abstract
The present invention relates to natural resources of Chinese medicinal materials evaluation and exploration technology field, a kind of specifically method using Fritillaria unibracteata callus production Fritillaria unibracteata total alkaloid, comprising the following steps: (1) induction of Lax callus；(2) proliferation of callus and alkaloid accumulation culture；(3) callus extracts Fritillaria unibracteata alkaloid.The present invention is using Fritillaria unibracteata as the extraction raw material of fritillaria total alkaloids, its callus has the characteristics that breeding is fast, proliferation rate is high, it is accumulated by hormone regulating and controlling secondary metabolite, quick production Fritillaria unibracteata total alkaloid is achieved the purpose that, imperialine percentage composition is up to 0.32% in its callus after cultivating, and is medicinal material standard content specified in pharmacopeia more than 6 times.The method of the present invention is easy to operate, and fritillaria total alkaloids yield is high, is suitble to industrial production application.
Description
Technical field
The present invention relates to natural resources of Chinese medicinal materials evaluation and exploration technology fields, specifically a kind of to utilize Fritillaria unibracteata callus
The method for producing Fritillaria unibracteata total alkaloid.
Background technique
Fritillaria unibracteata (Fritillaria unibracteata Hsiao et K.C.Hsia) is Liliaceae perennial grass
This plant is one of main base plant of rare medicinal herbs bulbus fritillariae cirrhosae.Currently, using Fritillaria unibracteata dry bulb as main source
Bulbus fritillariae cirrhosae is the good merchantable brand in fritillaria, practises and claims " loose shellfish ", has effects that clearing heat and moistening lung, preventing phlegm from forming and stopping coughing, is the important for the treatment of cough
Good medicine.Fritillaria unibracteata slow growth in the natural environment, the period is long, sprouts from seed to medicinal material is grown up to, need 4-5 when
Between, far from the market demand is met, due to excavating for a long time, there is a serious shortage in the supply for wild resource, faces exhaustion.In order to solve Fritillaria unibracteata
Resource scarcity problem in terms of having numerous studies to concentrate on the culture of Fritillaria unibracteata tissue-culturing rapid propagation, is expected that by bulb regeneration to increase
Seedling resource realizes large-scale artificial cultivation, this shortens the production cycle to a certain extent, but faces many problems, such as group
It is not high to train seedling domestication success rate, and planting area is limited etc..With the continuous development that fritillaria alkaloid component is studied, directly from shellfish
It is more advantageous that alkaloid is extracted in female tissue culture bulb or tissue culture object, can be bred lot of materials in a short time and be come for extracting biology
Alkali, and do not limited by time and region.
A kind of suspend using cell mass is disclosed application No. is the Chinese invention patent of " 200910164379.9 " to cultivate
The method for producing bulbus fritillariae cirrhosae total alkaloid, this method are that culture production bulbus fritillariae cirrhosae is carried out after smashing callus, belong to fermentation
The problem of engineering scope, it is complicated that there is cultures, and process is not easily controlled.Also, Fritillaria unibracteata and bulbus fritillariae cirrhosae alkaloid component
It is had a certain difference in quality and quantity, tissue culture PRODUCTION TRAITS is carried out to Fritillaria unibracteata alkaloid and is of great significance, but at present
Have no the related method that fritillaria total alkaloids are produced using Fritillaria unibracteata.
Summary of the invention
The purpose of the present invention is existing in view of the above technology, a kind of utilization Fritillaria unibracteata callus production is provided
The method of imperialine, the method for the present invention prepare fritillaria total alkaloids using callus, with short production cycle, operation
Simple feature.
To achieve the above object, the technical solution adopted by the present invention is that: it is a kind of utilize Fritillaria unibracteata callus BULBUS FRITILLARIAE CIRRHOSAE
The method of alkali, step include:
(1) induction of callus: the bulb produced using laboratory by tissue cultures chooses tender squama as explant
Limb is cut into the fritter of 0.5cm, is inoculated in MS+NAA 1-2mg/L+6-BA 0.5-1mg/L+2,4-D1-1.5mg/L+ agar
On the culture medium of 5g/L+ sucrose 30g/L, control pH is 5.8, culture room temperature is 23 ± 1 DEG C, relative air humidity 50%-
60%, a large amount of callus are acquired within Fiber differentiation 30-35 days under the conditions of 12 hour/day of light application time, intensity of illumination 2000L；
(2) it is proliferated and is cultivated with alkaloid accumulation: the callus generated in step (1) being cut into 0.8 × 0.8cm, is inoculated with
Into the culture medium of MS+ZT1.5-3mg/L+NAA0-0.2mg/L+ agar 4.5g/L+ sucrose 30g/L, control cultivation temperature 23 ±
1 DEG C, relative air humidity 50%-60%, 12 hour/day of light application time, cultivate 30-35d under the conditions of intensity of illumination 2000L after obtain
Obtain the high culture of alkaloid；
(3) fritillaria alkaloid content determination: the callus after collection step (2) culture is placed on dry filter paper and inhales
Moisture is received, and is put into baking oven 40 DEG C drying to constant weight, is control with imperialine, extracts Fritillaria unibracteata imperialine, and examine
Survey its content.
In the present invention, the extracting method of Fritillaria unibracteata is extracted using the operating method of " Chinese Pharmacopoeia " (2005 editions).
The method of the present invention also provides a kind of Fritillaria unibracteata total alkaloid prepared using the above method.For art technology
For personnel, this hair is also belonged to using application of the Fritillaria unibracteata total alkaloid of the method for the present invention preparation in field of biological pharmacy
Bright protection scope.
The method have the benefit that: the present invention using Fritillaria unibracteata as the extraction raw material of fritillaria total alkaloids,
Its callus has the characteristics that breeding is fast, proliferation rate is high, is accumulated by hormone regulating and controlling secondary metabolite, has reached fast fast-growing
The purpose of Fritillaria unibracteata total alkaloid is produced, it is medicine that imperialine percentage composition, which is up to 0.32%, in the callus after culture
More than 6 times of medicinal material standard content specified in allusion quotation.The method of the present invention is easy to operate, and fritillaria total alkaloids yield is high, is suitble to industry
Production application.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
A method of Fritillaria unibracteata total alkaloid being prepared using Fritillaria unibracteata callus system, step includes:
(1) induction of callus: the bulb produced using laboratory by tissue cultures chooses tender squama as explant
Limb is cut into the fritter of 0.5cm, is inoculated in MS+NAA 1-2mg/L+6-BA 0.5-1mg/L+2,4-D1-1.5mg/L+ agar
On the culture medium of 5g/L+ sucrose 30g/L, control pH is 5.8, culture room temperature is 23 ± 1 DEG C, relative air humidity 50%-
60%, a large amount of callus are acquired within Fiber differentiation 30-35 days under the conditions of 12 hour/day of light application time, intensity of illumination 2000L；
(2) it is proliferated and is cultivated with alkaloid accumulation: the callus generated in step (1) being cut into 0.8 × 0.8cm, is inoculated with
Into the culture medium of MS+ZT1.5-3mg/L+NAA0-0.2mg/L+ agar 4.5g/L+ sucrose 30g/L, control cultivation temperature 23 ±
1 DEG C, relative air humidity 50%-60%, 12 hour/day of light application time, cultivate 30-35d under the conditions of intensity of illumination 2000L after obtain
Obtain the high culture of alkaloid；
(3) fritillaria alkaloid content determination: the callus after collection step (2) culture is placed on dry filter paper and inhales
Moisture is received, and is put into baking oven 40 DEG C drying to constant weight, No. 3 sieves of claying into power take three chloromethanes of 2g powder addition 40mL
Alkane-methanol (4:1) solution carries out the extraction of fritillaria alkaloid, and filter and remove residue is settled to 50ml.2-5ml solution is taken, is evaporated, is added
10ml chloroform soln dissolves to be measured.Using imperialine as reference substance (reference substance solution 0.2mg/ml), production standard is bent
Line measures test sample absorbance by ultraviolet specrophotometer in accordance with the law, and the content of sample is read from standard curve, and calculating is
It obtains (" Chinese Pharmacopoeia " 2,015).Imperialine percentage composition is up to 0.32% in callus after measuring step (2) culture,
It is medicinal material standard content specified in pharmacopeia more than 6 times.
The above embodiments are only used to illustrate and not limit the technical solutions of the present invention, although referring to above-described embodiment to this hair
It is bright to be described in detail, it will be apparent to an ordinarily skilled person in the art that: still the present invention can be modified or be waited
With replacement, without departing from the spirit or scope of the invention, or any substitutions, should all cover in power of the invention
In sharp claimed range.
Claims (1)
1. a kind of method using Fritillaria unibracteata callus production imperialine, which is characterized in that step includes:
(1) induction of callus: the bulb produced using laboratory by tissue cultures chooses tender cuiller piece as explant
It is cut into the fritter of 0.5cm, is inoculated in MS+NAA 1-2mg/L+6-BA0.5-1mg/L+2,4-D1-1.5mg/L+ agar 5g/L+ sugarcane
On the culture medium of sugared 30g/L, control pH is 5.8, culture room temperature is 23 ± 1 DEG C, relative air humidity 50%-60%, illumination
Time 12 hour/day, acquire a large amount of callus within Fiber differentiation 30-35 days under the conditions of intensity of illumination 2000L；
(2) it is proliferated and is cultivated with alkaloid accumulation: the callus generated in step (1) being cut into 0.8 × 0.8cm, is seeded to MS+
In the culture medium of ZT1.5-3mg/L+NAA0-0.2mg/L+ agar 4.5g/L+ sucrose 30g/L, 23 ± 1 DEG C of cultivation temperature of control,
It is given birth to after cultivating 30-35d under the conditions of relative air humidity 50%-60%, 12 hour/day of light application time, intensity of illumination 2000L
The high culture of alkaloids content；
(3) fritillaria alkaloid content determination: the callus after collection step (2) culture is placed on dry filter paper and absorbs water
Point, and be put into baking oven 40 DEG C drying to constant weight, it is control with imperialine, extracts Fritillaria unibracteata imperialine, and detect it
Content.
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| CN201710403578.5A CN107155887B (en) | 2017-06-01 | 2017-06-01 | A method of imperialine is produced using Fritillaria unibracteata callus |
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| CN107155887B true CN107155887B (en) | 2019-07-19 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108103176A (en) * | 2018-01-02 | 2018-06-01 | 中国药科大学 | Method based on metabolism group and transcription group association analysis screening fritillaria alkaloid synthesis key gene |
| CN116998407B (en) * | 2023-09-28 | 2023-12-05 | 西南林业大学 | Method for detoxification and rapid propagation of fritillaria fusiformis and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974478A (en) * | 2010-09-29 | 2011-02-16 | 成都大学 | Culture method for effectively improving total alkaloid content of Fritillaria cirrhosa D. Don |
| CN103283600A (en) * | 2013-05-31 | 2013-09-11 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly propagating dark purple fritillary by utilizing tissue culture technology |
-
2017
- 2017-06-01 CN CN201710403578.5A patent/CN107155887B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974478A (en) * | 2010-09-29 | 2011-02-16 | 成都大学 | Culture method for effectively improving total alkaloid content of Fritillaria cirrhosa D. Don |
| CN103283600A (en) * | 2013-05-31 | 2013-09-11 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly propagating dark purple fritillary by utilizing tissue culture technology |
Non-Patent Citations (4)
| Title |
|---|
| Morphogenesis in vitro of Fritillaria spp.;Marija Petric et al.;《Floriculture abd Ornamental Biotechnology》;20121231;第6卷(第1期);第78-89页 |
| 彭泽贝母愈伤组织的诱导及培养条件的优化研究;张寿文等;《全国第9届天然药物资源学术研讨会论文集》;20101231;第333页最后一段、第334页第1和3段 |
| 暗紫贝母组织培养基其生物碱积累研究;张波;《中国优秀硕士论文全文数据库农业科技辑》;20110915(第9期);第18-19页、第25页、第30页、第38页 |
| 暗紫贝母鳞茎器官培养生长特征和生物碱累积的研究;高山林等;《中国药科大学学报》;19921231;第23卷(第3期);第144-147页 |
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Effective date of registration: 20191025 Address after: No. 300, Bailong temple, Bailong District, Kunming City, Yunnan Province 650000 Co-patentee after: Yunnan Haochen Agricultural Technology Co., Ltd. Patentee after: Southwest Forestry University Address before: 650224 Southwest Forestry University, Bailong temple, Yunnan, Kunming Co-patentee before: YUNNAN CHECHUAN BIOTECHNOLOGY CO., LTD. Patentee before: Southwest Forestry University |
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Address after: 650000 No. 300, bailongsi, Bailong District, Kunming, Yunnan Patentee after: SOUTHWEST FORESTRY University Patentee after: Yunnan HaoChen Agriculture Co.,Ltd. Address before: 650000 No. 300, bailongsi, Bailong District, Kunming, Yunnan Patentee before: SOUTHWEST FORESTRY University Patentee before: Yunnan Haochen Agricultural Technology Co.,Ltd. |
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