CN106479954A - A kind of culture medium for Radix Glycyrrhizae isolated cells - Google Patents

A kind of culture medium for Radix Glycyrrhizae isolated cells Download PDF

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CN106479954A
CN106479954A CN201611014877.1A CN201611014877A CN106479954A CN 106479954 A CN106479954 A CN 106479954A CN 201611014877 A CN201611014877 A CN 201611014877A CN 106479954 A CN106479954 A CN 106479954A
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culture medium
radix glycyrrhizae
isolated cells
phytohormone
culture
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夏雪城
裘先临
马丹
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TIANJIN BOAI BIO-PHARMACEUTICAL Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
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    • C12N2500/00—Specific components of cell culture medium
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/76—Undefined extracts from plants
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides

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Abstract

The present invention has extensively screened the nutrient substance as Glycyrrhiza cell growth by laboratory facilities, determines inventive formulation according to Glycyrrhiza cell own metabolism characteristic and practical effect.This formula component based on MS culture medium, and with the addition of caseinhydrolysate, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and the Phenylalanine of higher dosage, it is found surprisingly that on this basis and contribute to lifting cell culture density by adding the Chinese crude drug of particular types and composition.Based on discovery beneficial above, the present invention passes through the Chinese medicine composition that laboratory facilities preferred Herba lygodii, pocket grass etc. are originally used for human disease treatment, gained culture medium surprisingly obtains prominent culture effect so that Glycyrrhiza cell culture density is obviously improved, and its speed of growth is faster simultaneously.The present invention obtains superior technique effect with the technological means of novelty, and cost is relatively low simultaneously, be easily achieved, and therefore has a good application prospect.

Description

A kind of culture medium for Radix Glycyrrhizae isolated cells
Technical field
The present invention relates to plant cell culture technology, the nutritional condition further to plant cell design and in particular to A kind of culture medium for Radix Glycyrrhizae isolated cells.
Background technology
Plant cell culture technology is with vitro plant cell or protoplast as object, with cell concentration amplification and target generation Thank to the culture process for the purpose of product accumulation.For the cell culture for the purpose of obtaining specific metabolite, it is main Process procedure is not only in that the amplification of cell concentration, and meanwhile, the induction synthesis of target metabolic product is even more important to culture benefit.? In the incubation of plant cell, condition of culture and nutritional condition are the most important factor affecting yield, and wherein condition of culture is Refer to the control of the environmental conditions such as temperature, dissolved oxygen, illumination, pH, and nutritional condition refers mainly to the culture medium of plant cell.Due in envelope Close the sole nutrition source that culture medium in culture environment is plant cell, therefore the growth water to plant cell for the composition of culture medium Flat, metabolite classification has extremely important impact, particularly with the cell culture for the purpose of obtaining specific metabolite For, culture medium is often the key factor affecting culture efficiency.
Radix Glycyrrhizae is the important traditional medicine of China, has saying of " ten side nine grass ".One of its main component licoflavone, There are multiple biological activities, in addition to the effect such as antiinflammatory, antibacterial, resistance state, also have at aspects such as antioxidation, anticancer, anti-cancers bright Aobvious biological activity, can prevent and treat the diseases such as viral hepatitis, hyperlipidemia, cancer and acquired immune deficiency syndrome (AIDS), become Radix Glycyrrhizae research at present Focus.In recent years, the Radix Glycyrrhizae market demand sharply increases, and Licorice there is a serious shortage in the supply, therefore adopt cell large-scale culture come The effective ingredient producing Radix Glycyrrhizae is extremely urgent.At present, although existing many researcheres launch around Glycyrrhiza cell suspension medium Research, but still not ideal enough especially in terms of its nutrition supply system construction in terms of culture effect.
In the research of Glycyrrhiza cell culture, how to improve in culture mainly effectively the content of medicinal ingredient flavone and Quality, amplification culture scale, finally realize industrialized production, be the research emphasis in this field.Domestic and international researcher is in Radix Glycyrrhizae The aspects such as callus culture, cell suspension cultures, fermentation culture are done a lot of work, but the culture effect of current Glycyrrhiza cell The yield of fruit and total flavones still has to be hoisted.
Content of the invention
It is contemplated that for the technological deficiency of prior art, providing a kind of culture medium for Radix Glycyrrhizae isolated cells, with Solve the not good technical problem of Glycyrrhiza cell culture effect in prior art.
The invention solves the problems that another technical problem be prior art Glycyrrhiza cell incubation in licoflavone product Amount is relatively low.
For realizing above technical purpose, the present invention employs the following technical solutions：
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate 80～150 μ Mol/L, salicylic acid 1～10mg/L, Phenylalanine 20～50mg/L, tyrosinase 15～10mg/L, cinnamic acid 5～10mg/L, acetic acid Sodium 5～10mg/L, L-Cysteine 0.5～2mg/L, caseinhydrolysate 500～800mg/L, leucine 0.1～5mg/L.
Preferably, the content of wherein (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate is 200 μm of ol/L.
Preferably, wherein salicylic content is 10mg/L.
Preferably, the also fungus polysaccharide containing 30mg/L of this culture medium.
Preferably, the Herba lygodii squeezing juice also containing 1g/L for this culture medium.
Preferably, the pocket grass squeezing juice also containing 1g/L for this culture medium.
Preferably, this culture medium contains the composition of MS culture medium, also contain phytohormone 2.4-D0.5mg/L simultaneously, plant Thing hormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 200 μm of ol/L of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate, salicylic acid 10mg/L, phenylpropyl alcohol Propylhomoserin 35mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.5mg/L, fungus polysaccharide 30mg/L, Herba lygodii squeezing juice 1g/L, pocket grass squeezing juice 1g/L.
In above technical scheme, described MS culture medium can be prepared according to the general technology general knowledge of this area, when So can also be using the MS culture medium of formula in detail below：NH4NO31650mg/L, KNO31900mg/L, CaCl2·2H2O 440mg/L, MgSO4·7H2O 370mg/L, KH2PO4170mg/L, KI 0.83mg/L, H3BO36.2mg/L, MnSO4·4H2O 22.3mg/L, ZnSO4·7H2O 8.6mg/L, Na2MoO4·2H2O 0.25mg/L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, FeSO4·7H2O 27.8mg/L, Na2-EDTA·2H2O 37.3mg/L, inositol 100mg/L, Nicotinic acid 0.5mg/L, vitamin B6 0.5mg/L, vitamin B1 0.1mg/L, glycine 2mg/L, water surplus.
In above technical scheme, Herba lygodii, pocket grass refer to its fresh herb respectively.Heretofore described squeezing Juice, refers to the Fresh sap of each raw material using physical method acquisition, including but not limited to the routine side such as squeezing, pulverizing centrifugation Method.
The present invention has extensively screened the nutrient substance as Glycyrrhiza cell growth by laboratory facilities, according to Glycyrrhiza cell certainly Body metabolic characteristic and practical effect determine inventive formulation.This formula component based on MS culture medium, and with the addition of The caseinhydrolysate of higher dosage, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Phenylalanine, are found surprisingly that on this basis by adding specific kind The Chinese crude drug of class and composition contributes to lifting cell culture density.Based on discovery beneficial above, the present invention passes through laboratory facilities Preferably Herba lygodii, pocket grass etc. are originally used for the Chinese medicine composition of human disease treatment, and gained culture medium surprisingly obtains , so that Glycyrrhiza cell culture density is obviously improved, its speed of growth is faster simultaneously for prominent culture effect.The present invention is with novelty Technological means obtain superior technique effect, cost is relatively low simultaneously, be easily achieved, and therefore has a good application prospect.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessively unnecessary details, Will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 80 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 1mg/L, Phenylalanine 20mg/L, tyrosinase 15 mg/L, cinnamic acid 5mg/L, sodium acetate 5mg/L, L-Cysteine 0.5mg/L, caseinhydrolysate 500mg/L, leucine 0.1mg/L.
Embodiment 2
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 150 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 10mg/L, Phenylalanine 50mg/L, tyrosine 10mg/L, cinnamic acid 10mg/L, sodium acetate 10mg/L, L- half Guang ammonia Sour 2mg/L, caseinhydrolysate 800mg/L, leucine 5mg/L.
Embodiment 3
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 200 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 5mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.6mg/L.
Embodiment 4
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 115 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 10mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.6mg/L.
Embodiment 5
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 115 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 5mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.6mg/L, fungus polysaccharide 30mg/L.
Embodiment 6
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 115 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 5mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.6mg/L, Herba lygodii squeezing juice 1g/L.
Embodiment 7
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 115 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 5mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.6mg/L, pocket grass squeezing juice 1g/L.
Embodiment 8
A kind of culture medium for Radix Glycyrrhizae isolated cells, this culture medium has the composition of MS culture medium, also contains plant simultaneously Hormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, 200 μm of ol/ of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate L, salicylic acid 10mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, cinnamic acid 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.5mg/L, fungus polysaccharide 30mg/L, Herba lygodii squeezing juice 1g/L, lotus The careless squeezing juice 1g/L of bag.
Embodiment 9
The present embodiment is for evaluating the culture medium that above example 1～8 is provided as thin to Radix Glycyrrhizae during synthetic medium The impact of born of the same parents' total flavonoid yield, experimental result is as shown in table 1.
The impact to Glycyrrhiza cell total flavonoid yield for the culture medium prepared by table 1 various embodiments above
When as can be seen here, by the use of culture medium provided by the present invention as synthetic medium culture Glycyrrhiza cell, total to it Yield of flavone has definite facilitation.
Above embodiments of the invention are described in detail, but described content have been only presently preferred embodiments of the present invention, Not in order to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should It is included within protection scope of the present invention.

Claims (7)

1. a kind of culture medium for Radix Glycyrrhizae isolated cells, it is characterised in that this culture medium has the composition of MS culture medium, is gone back simultaneously Containing phytohormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone NAA 0.5mg/L, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate 80～150 μm of ol/L, salicylic acid 1～10mg/L, Phenylalanine 20～50mg/L, tyrosinase 15～10mg/L, cinnamic acid 5～ 10mg/L, sodium acetate 5～10mg/L, L-Cysteine 0.5～2mg/L, caseinhydrolysate 500～800mg/L, leucine 0.1 ～5mg/L.
2. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that wherein jasmonic first The content of ester is 200 μm of ol/L.
3. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that wherein salicylic Content is 10mg/L.
4. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that this culture medium also contains There is the fungus polysaccharide of 30mg/L.
5. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that this culture medium also contains There is the Herba lygodii squeezing juice of 1g/L.
6. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that this culture medium also contains There is the pocket grass squeezing juice of 1g/L.
7. a kind of culture medium for Radix Glycyrrhizae isolated cells according to claim 1 is it is characterised in that this culture medium contains The composition of MS culture medium, also contains phytohormone 2.4-D 0.5mg/L, phytohormone 6-BA 0.5mg/L, phytohormone simultaneously NAA 0.5mg/L, 200 μm of ol/L of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate, salicylic acid 10mg/L, phenylalanine-3,4-quinone 5mg/L, tyrosine 7mg/L, Cortex Cinnamomi Sour 8mg/L, sodium acetate 8mg/L, L-Cysteine 1.2mg/L, caseinhydrolysate 650mg/L, leucine 2.5mg/L, funguses are many Sugared 30mg/L, Herba lygodii squeezing juice 1g/L, pocket grass squeezing juice 1g/L.
CN201611014877.1A 2016-11-15 2016-11-15 A kind of culture medium for Radix Glycyrrhizae isolated cells Pending CN106479954A (en)

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CN113403252A (en) * 2021-06-07 2021-09-17 湖南省中医药研究院 Eucommia ulmoides suspension cell culture method for improving content of effective components

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Application publication date: 20170308