CN101974478B - Culture method for effectively improving total alkaloid content of Fritillaria cirrhosa D. Don - Google Patents
Culture method for effectively improving total alkaloid content of Fritillaria cirrhosa D. Don Download PDFInfo
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- CN101974478B CN101974478B CN2010102964569A CN201010296456A CN101974478B CN 101974478 B CN101974478 B CN 101974478B CN 2010102964569 A CN2010102964569 A CN 2010102964569A CN 201010296456 A CN201010296456 A CN 201010296456A CN 101974478 B CN101974478 B CN 101974478B
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 37
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 35
- 241001547127 Fritillaria cirrhosa Species 0.000 title abstract description 8
- 238000012136 culture method Methods 0.000 title abstract 2
- 241000935235 Fritillaria meleagris Species 0.000 claims abstract description 54
- 239000002243 precursor Substances 0.000 claims abstract description 25
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 238000009630 liquid culture Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000008929 regeneration Effects 0.000 claims description 16
- 238000011069 regeneration method Methods 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 10
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 10
- 239000006035 Tryptophane Substances 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 229960004799 tryptophan Drugs 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 6
- 230000003203 everyday effect Effects 0.000 claims description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 230000004069 differentiation Effects 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000004615 ingredient Substances 0.000 abstract description 3
- 239000002028 Biomass Substances 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 230000001850 reproductive effect Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 description 14
- 239000000126 substance Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 229930000044 secondary metabolite Natural products 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 238000004114 suspension culture Methods 0.000 description 3
- 206010011224 Cough Diseases 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 230000000144 pharmacologic effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 230000000954 anitussive effect Effects 0.000 description 1
- 229940124584 antitussives Drugs 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
- 208000013116 chronic cough Diseases 0.000 description 1
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 230000003419 expectorant effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
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- AHLPHDHHMVZTML-BYPYZUCNSA-N ornithyl group Chemical class N[C@@H](CCCN)C(=O)O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a culture method for effectively improving the total alkaloid content of Fritillaria cirrhosa D. Don, which is characterized in that: in the method, precursor-feeding technology is adopted, and in the liquid culture process of young regenerated bulbs of Fritillaria cirrhosa D. Don, the total alkaloid content of Fritillaria cirrhosa D. Don cultures is improved by adding amino acid precursors. The experimental results show that the total alkaloid content of final obtained Fritillaria cirrhosa D. Don cultures can reach 0.212 percent after 25-day culturing, which is 52.5 percent higher than that of the control group without adding amino acid precursors. The invention not only effectively improves the total alkaloid content of Fritillaria cirrhosa D. Don in liquid culture, but also significantly increases the reproductive rate of biomass of Fritillaria cirrhosa D. Don, thereby achieving the purpose of rapidly producing the effective ingredients of unibract fritillary bulbs better.
Description
Technical field
The present invention relates to a kind of cultural method that improves leaf roll bulb of fritillary total alkaloid content, particularly relate to a kind of through adding the cultural method that the specific amino acids precursor effectively improves leaf roll bulb of fritillary total alkaloid content in the liquid medium within.
Background technology
The leaf roll bulb of fritillary (Fritillaria cirrhosa D.Don) is the Liliaceae per nnial herb, and its bulb is that the medicinal material Unibract Fritillary Bulb is produced in famous and precious river.The Chinese medicinal materials Unibract Fritillary Bulb is used to treat lung-heat type cough, and deficiency of Yin labor is coughed, and chronic cough of deficiency lung does not more wait disease, and effect is remarkable, and annual consumption is all increasing year by year, is that main ingredient or the Chinese patent medicine that comprises Unibract Fritillary Bulb have kind more than 130 with the Unibract Fritillary Bulb at present.For a long time; The Unibract Fritillary Bulb medicinal material mainly relies on and excavates natural crude drugs is main; Factor such as destroyed because of excessive collection and habitat, its standing stock are sharply descended, people further increase the demand of Unibract Fritillary Bulb medicinal material in addition; Thereby finally causing its price constantly soaring, the breach in medicinal material market is increasing.
In order to obtain the Unibract Fritillary Bulb medicinal material fast, to satisfy the heavy demand in Unibract Fritillary Bulb medicinal material market, people begin to adopt biotechnological means to realize the quick production of Unibract Fritillary Bulb medicinal material.Current employing tissue culture technique carries out the quick breeding of the leaf roll bulb of fritillary succeeds, has accomplished leaf roll bulb of fritillary callus induction and researchs such as fast breeding, bulb regeneration, cell mass suspension culture and the quick cultivation of leaf roll bulb of fritillary tissue cultured seedling like people such as Wang Yuehua.Through leaf roll bulb of fritillary Determination of Total Alkaloid result is shown, the total alkaloid content of the leaf roll bulb of fritillary total alkaloid content that tissue culture is produced in the wild leaf roll bulb of fritillary bulb.People such as alpine forest study the chemical ingredients and the pharmacological action of Unibract Fritillary Bulb group training thing; Detected result shows; The bulb that obtains through tissue culture has consistent chemical ingredients with the commodity bulb, all has 13 kinds of chemical substances such as vegeto-alkali, organic acid, saponin amino acid; Show that through The pharmacological results group training tendril-leaved fritillary bulb and wild tendril-leaved fritillary bulb have Antitussive and Expectorant Effect equally, and drug toxicity is all lower, relatively safety.
In biotechnology research, also do not see and adopt the precursor technology of feeding to significantly improve the report of the total alkaloid content of the leaf roll bulb of fritillary in the liquid culture at present the leaf roll bulb of fritillary.It is in the medicinal plant tissue culture that precursor is fed, and improves the content of secondary metabolite in the cell through adding secondary metabolite synthetic precursor substance.This is because the secondary metabolite in the vegetable cell produces through a series of metabolic processes in the plant materials, in tissue culture procedures, suitable metabolic intermediate is added in the substratum, can promote a large amount of generations of secondary metabolite effectively.Vegeto-alkali is produced through a series of biochemical reactions by a-amino acids such as phenylalanine(Phe), tyrosine, tryptophane, Methionin, ornithines in the intravital synthetic major part of plant.Therefore the main active principle of the leaf roll bulb of fritillary is an alkaloids substance, is feasible in theory through adding the bulb bearing again total alkaloid content of the leaf roll bulb of fritillary that amino acid precursor improves liquid culture in the liquid medium within.
Summary of the invention
The object of the present invention is to provide a kind of cultural method of effective raising leaf roll bulb of fritillary total alkaloid content, this method is effectively to improve the total alkaloid content in the leaf roll bulb of fritillary culture through in the bulb bearing again liquid culture process of the leaf roll bulb of fritillary, adding the amino acid precursor thing.
For achieving the above object, the solution that the present invention adopts comprises the following steps:
(1) the bulb bearing again acquisition of the leaf roll bulb of fritillary: will induce the tight callus that begins preliminary differentiation of generation to be cut into the fritter of 1.5～2.5cm, and be transferred to the bulb bearing again substratum MS+6-BA of generation 1～2.0mgL
-1+ NAA0.1～0.3mgL
-1+ sucrose 20～60gL
-1+ agar 5.5～7.0gL
-1In cultivate, the pH value that is adopted is 5.5～6.5,15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx; After cultivating about 40d, can obtain the good regeneration bulb of raised growth;
(2) the bulb bearing again liquid culture of the leaf roll bulb of fritillary: the leaf roll bulb of fritillary regeneration bulb that to cut the diameter that (1) step obtains be 3～6mm, do not sprout is transferred to the liquid nutrient medium MS+6-BA2.0mgL that is added with the amino acid precursor thing
-1+ NAA0.2～1.0mgL
-1+ sucrose 20～60gL
-1In cultivate, pH 5.5～6.5, inoculum size is 30～50gL
-1, 15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx, shaking speed 110～130rmin
-1, behind cultivation 24～28d, promptly obtaining the high regeneration bulb of total alkaloid content, above-mentioned amino acid precursor thing is any one in phenylalanine(Phe), tryptophane, the tyrosine, add-on is 0.25～1.00mmolL
-1
The regeneration bulb of above-mentioned cultivation is leached from liquid nutrient medium, wipe away dried surface-moisture with dry filter paper after, take by weighing weight, be fresh weight; Its living weight propagation multiple=(Yield fresh weight-inoculum size fresh weight)/inoculum size fresh weight.
The regeneration bulb of above-mentioned cultivation is leached from liquid nutrient medium, wipe away dried surface-moisture with dry filter paper after, place 60 ℃ baking oven to dry to constant weight, measure absorbancy with spectrophotometry at the 412nm place, can calculate total alkaloid content in the culture.
The present invention adopts the precursor technology of feeding; In the bulb bearing again liquid culture of the leaf roll bulb of fritillary, come effectively to improve the total alkaloid content in the leaf roll bulb of fritillary culture through the metabolic intermediate that in nutrient solution, adds alkaloids substances such as phenylalanine(Phe), tyrosine or tryptophane; The amino acids material that is added in addition can also be participated in intracellular protein synthesis and served as carbon source and nitrogenous source; Particularly tryptophane can be transformed into indolylacetic acid (claiming plant hormone again) under the effect of aminotransferase and decarboxylase, thereby the growth of vegetable cell is produced promoter action.The growth cycle of leaf roll bulb of fritillary regeneration bulb liquid culture is 24～28 days among the present invention; Cultivate that bulb bearing again total alkaloid content is the highest after 25 days can be 0.212%, improved 52.5% than the total alkaloid content that does not add the amino acid precursor control group 0.139%.
The present invention adopts the regeneration bulb to carry out suspension culture as outer plant materials, can overcome to adopt callus repeatedly being prone to cause the problem that callus makes a variation and regenerative power is lost behind the succeeding transfer culture as what outer plant materials was cultivated existence in the conventional suspension culture; And adopt the regeneration bulb to keep the integrity of leaf roll bulb of fritillary organ and the production system of secondary metabolite effectively, thereby help under the prerequisite that improves the culture living weight, guaranteeing active constituent content as outer plant materials.Therefore can improve leaf roll bulb of fritillary regeneration bulb liquid culture total alkaloid content fast with the inventive method; It is long to have solved the growth cycle that the rare traditional Chinese medicine Unibract Fritillary Bulb exists aborning preferably; Problems such as medicinal material output and active constituent content are low, thus can realize utilizing the purpose of fermentation engineering scale operation rare traditional Chinese medicine Unibract Fritillary Bulb.
Embodiment
Embodiment 1
(1) the bulb bearing again acquisition of the leaf roll bulb of fritillary: will induce the tight callus that begins preliminary differentiation of generation to be cut into the fritter of 1.5cm, and be transferred to the substratum MS+6-BA1mgL that produces bulb
-1+ NAA0.2mgL
-1+ sucrose 40gL
-1+ agar 6.5gL
-1In cultivate, the pH value that is adopted is 6.0,15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx; After cultivating about 40d, can obtain the good regeneration bulb of raised growth;
(2) the bulb bearing again liquid culture of the leaf roll bulb of fritillary: the tender regeneration bulb of leaf roll bulb of fritillary children that to cut the diameter that (1) step obtains be 3～6mm, do not sprout is transferred to the liquid nutrient medium MS+6-BA 2.0mgL that is added with tryptophane
-1+ NAA 0.4mgL
-1+ sucrose 30gL
-1In cultivate, pH value 6.5, inoculum size are 50gL
-1, 15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx, shaking speed 110～130rmin
-1, said tryptophane is pressed 0.50mmolL
-1Add, promptly add the 0.50mmol tryptophane in every liter of substratum;
(3) mensuration of culture total alkaloid content: the leaf roll bulb of fritillary regeneration bulb that will cultivate after 25 days leaches from liquid nutrient medium; After wiping away dried surface-moisture with dry filter paper; Place 60 ℃ baking oven to dry to constant weight; Measure absorbancy at the 412nm place with spectrophotometry, and the total alkaloid content that calculates in the culture is 0.212%, exceeded 52.5% than the control group total alkaloid content that does not add any amino acid precursor 0.139%.Its living weight propagation multiple is 2.45, has exceeded 8.4% than the control group living weight propagation multiple 2.26 that does not add any amino acid precursor.Suitable amino acid precursor is added in explanation in leaf roll bulb of fritillary liquid nutrient medium not only can significantly improve the bulb bearing again total alkaloid content of the leaf roll bulb of fritillary, and can also produce promoter action to a certain degree to its living weight propagation multiple.
Embodiment 2
The amino acid precursor thing that is added in this embodiment (2) step is a tryptophane, and add-on is 1.00mmolL
-1Other step is with embodiment 1; Cultivating the total alkaloid content that records after 25 days in the culture is 0.140%; Be starkly lower than the bulb bearing again total alkaloid content 0.212% of the leaf roll bulb of fritillary among the embodiment 1, explain that there is the best concentration of adding in the adding of amino acid precursor thing in leaf roll bulb of fritillary liquid nutrient medium, adding different precursor concentration has tangible influence to the bulb bearing again total alkaloid content of the leaf roll bulb of fritillary.
Embodiment 3
The amino acid precursor thing that is added in this embodiment (2) step is a phenylalanine(Phe), and add-on is 0.25mmolL
-1, other step is with embodiment 1, and the total alkaloid content that after cultivating 25 days, records in the culture is 0.173%, has exceeded 22.4% than the control group total alkaloid content that does not add any amino acid precursor 0.139%.
Embodiment 4
The substratum that produces bulb in this embodiment (1) step is MS+6-BA 1.5mgL
-1+ NAA0.3mgL
-1+ sucrose 40gL
-1+ agar 6.5gL
-1, the amino acid precursor thing that is added in (2) step is a tyrosine, add-on is 0.75mmolL
-1, liquid nutrient medium is MS+6-BA2.0mgL
-1+ NAA 0.7mgL
-1+ sucrose 30gL
-1, other is with embodiment 1, and the total alkaloid content that after cultivating 25 days, records in the culture is 0.170%, has exceeded 22.3% than the control group total alkaloid content that does not add any amino acid precursor 0.139%.
In the three seed amino acid precursors, tryptophane helps to improve the total alkaloid content of the leaf roll bulb of fritillary most.
Claims (1)
1. a cultural method that effectively improves leaf roll bulb of fritillary total alkaloid content is characterized in that comprising the following steps:
(1) the bulb bearing again acquisition of the leaf roll bulb of fritillary: will induce the tight callus that begins preliminary differentiation of generation to be cut into the fritter of 1.5～2.5cm, and be transferred to the bulb bearing again substratum MS+6-BA of generation 1～2.0mgL
-1+ NAA0.1～0.3mgL
-1+ sucrose 20～60gL
-1+ agar 5.5～7.0gL
-1In cultivate, the pH value that is adopted is 5.5～6.5,15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx; After 38～42d cultivates, can obtain the good regeneration bulb of raised growth;
(2) the bulb bearing again liquid culture of the leaf roll bulb of fritillary: the leaf roll bulb of fritillary regeneration bulb that to cut the diameter that (1) step obtains be 3～6mm, do not sprout is transferred to the liquid nutrient medium MS+6-BA2.0mgL that is added with the amino acid precursor thing
-1+ NAA 0.2～1.0mgL
-1+ sucrose 20～60gL
-1In cultivate, pH 5.5～6.5, inoculum size is 30～50gL
-1, 15～23 ℃ of culture temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx, shaking speed 110～130rmin
-1, behind cultivation 24～28d, promptly obtaining the high regeneration bulb of total alkaloid content, above-mentioned amino acid precursor thing is any one in phenylalanine(Phe), tryptophane, the tyrosine, add-on is 0.25～1.00mmolL
-1
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| CN103468633B (en) * | 2013-09-24 | 2015-02-25 | 薛刚 | Inducing method for improving cell synchronization of tendril-leaved fritillary bulbs |
| CN103477991A (en) * | 2013-10-17 | 2014-01-01 | 黄振忠 | Primary culture medium specially used for tissue culture seedling culture of radix sophorae subprostratae |
| CN103621406B (en) * | 2013-11-27 | 2015-12-09 | 成都大学 | A kind of with the cultural method of the Bulbus Fritillariae Cirrhosae root Bulbus Fritillariae Cirrhosae callus that is explant |
| CN107155887B (en) * | 2017-06-01 | 2019-07-19 | 西南林业大学 | A method of imperialine is produced using Fritillaria unibracteata callus |
| 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 |
| CN109757377A (en) * | 2019-03-01 | 2019-05-17 | 南京工业大学大丰海洋产业研究院 | A kind of cultural method for accelerating fritillaria thunbergii reproduction speed |
| CN109757378B (en) * | 2019-03-13 | 2022-05-31 | 南京工业大学大丰海洋产业研究院 | Method for rapidly propagating fritillaria thunbergii seedlings by using intermittent immersion system |
| CN111492977B (en) * | 2020-05-18 | 2022-09-13 | 成都大学 | Induced culture method of bulbus fritillariae cirrhosae round bulbs |
| CN112273227A (en) * | 2020-10-15 | 2021-01-29 | 永州职业技术学院 | Culture medium for improving survival rate of explants and preparation method and application thereof |
| CN112931210B (en) * | 2021-03-18 | 2022-06-21 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
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| CN101638669A (en) * | 2009-09-09 | 2010-02-03 | 成都大学 | Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture |
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| CN101638669A (en) * | 2009-09-09 | 2010-02-03 | 成都大学 | Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture |
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| Title |
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| 王跃华等.离体培养条件对卷叶贝母鳞茎再生的影响.《中药材》.2010,第33卷(第6期),854－856. * |
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