CN1417343A - Plant callus particls suspending culture to produce rhodiola glycoside - Google Patents
Plant callus particls suspending culture to produce rhodiola glycoside Download PDFInfo
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- CN1417343A CN1417343A CN 01133396 CN01133396A CN1417343A CN 1417343 A CN1417343 A CN 1417343A CN 01133396 CN01133396 CN 01133396 CN 01133396 A CN01133396 A CN 01133396A CN 1417343 A CN1417343 A CN 1417343A
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Abstract
Alpine rhodiola callus particle in 15% is first inoculated to liquid MS culture liquid with 6-benzyl aminopurine and indolebutyric acid, and cultured under conditions of regulated cane sugar content, regulated pH value and stirring speed for 15-20 days. Then, it is transferred to MS culture liquid with added 2,4-D and cultured under conditions of regulated cane sugar content, regulated pH value and stirring speed for 6-9 days. After culture, alpine rhoidola callus particle is collected and extracted to obtain salidroside.
Description
The present invention relates to utilize vegetable cell or callus particle suspension culture technique to produce the secondary metabolite field, relate in particular to the method for the Radix Rhodiolae callus particulate suspension culture system production rhodioloside that utilizes a kind of structure consolidation.
Rhodioloside (salidroside) is a kind of Rhodida plant, and its underground part contains glycoside compound-rhodioloside, and molecular structural formula is as follows.Molecular structural formula has antifatigue, anti-hypoxia, resisting microwave radiation and the anti-ageing function of waiting for a long time, and can strengthen the ability that human body is resisted extraneous pessimal stimulation, is a kind of environmental adaptation unit medicine that DEVELOPMENT PROSPECT is arranged very much.If the drug main that utilizes this compound to develop at present relies on from the root of natural Rhodida plant and rhizome and extracts (average content is 0.5%), need to consume a large amount of natural plant resources, cause the natural resources of this rare species reducing (bright Haiquan etc., 1988 significantly; Ding Shuli etc., 1992; Jiang Minglan etc., 1994,1995).In order to protect this rare species, attempting in recent years Rhodida plant is carried out artificial culture and tissue culture.But, the salidroside content under the artificial cultivation condition low (being lower than 0.2%), the seedling root rot is serious, and need take big area arable land, popularization (Jiang Xiqiang etc., 1994 of limiting this technology; Qin Jiamei etc., 1994; Xu Jianfeng etc., 1996).Xu etc. (1997,1998) once induced and had set up a kind of callus particle suspension culture system of Radix Rhodiolae, and the rhodioloside output that obtains is 70-105mg/L.
The object of the present invention is to provide the higher callus particle suspension culture system of a kind of rhodioloside output.Simultaneously, adopt this method neither to consume the natural resources of rhodiola plant, do not occupy cultivated land again, reduced destruction, realized Sustainable utilization of resources ecotope.
In order to achieve the above object, the technical solution used in the present invention is: the Radix Rhodiolae callus particle that with inoculum size is 15% (v/v) is in the additional 5mg/L 6-benzyl aminopurine (6-Benzylaminopurine of liquid Murashige-Skoog (MS) minimum medium, BA) and 2.5mg/L Yin tremble butyric acid (Indole-3-butyric acid, IBA), 4.5% sucrose, pH is 5.7, intensity of illumination 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are to cultivate 15-20 days under the grown cultures condition of 100rpm/min, forward total nitrogen content again to and be basic MS culture medium half the additional 4mg/L of liquid MS medium 2,4-D, ((2,4-Dichlorophenoxy) acetic acid), 1.5% sucrose, pH are 4.2, intensity of illumination 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are that the production culture condition of 150rpm/min was cultivated 6-9 days down, collect the callus particle, extract rhodioloside.
Advantage of the present invention is:
1, the particulate state callus with the structure consolidation of Radix Rhodiolae is the raw material production rhodioloside.Raw material relies on plant tissue culture technique to obtain, and the Radix Rhodiolae natural resources is not had destruction, and saves and plough, and has realized the protection and the sustainable use of natural resources.
2, the callus particle with this kind Radix Rhodiolae is that material carries out suspension culture, callus grain pattern consolidation, and big or small homogeneous, not fragmentary during suspension culture, do not produce foam, viscosity is little, reduces to cultivate difficulty.
3, growth rhythm and the rhodioloside accumulation characteristic of cultivating at the callus particle suspension of this Radix Rhodiolae, this cultural method effectively raises rhodioloside output.
4, adopt the measure of adding Jujubogenin tyrosol to improve rhodioloside output significantly.
5, the present invention is applicable to scale operation.
Below embodiments of the invention are described in further detail: the Radix Rhodiolae callus particle that with inoculum size is 15% (v/v) is in the additional 5mg/L 6-benzyl aminopurine (6-Benzylaminopurine of liquid Murashige-Skoog (MS) minimum medium, BA) and 2.5mg/L Yin tremble butyric acid (Indole-3-butyric acid, IBA), 4.5% sucrose, pH is 5.7, intensity of illumination 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are to cultivate 15-20 days under the grown cultures condition of 100rpm/min, forward total nitrogen content again to and be basic MS culture medium half the additional 4mg/L of liquid MS medium 2,4-D, ((2,4-Dichlorophenoxy) acetic acid), 1.5% sucrose, pH are 4.2, intensity of illumination 150 μ E/m
2/ S, light application time 16 hours/day and stirring velocity are that the production culture condition of 150rpm/min was cultivated 6-9 days down, collect the callus particle, extract rhodioloside.
Described can also be that the Radix Rhodiolae callus particle of 15% (v/v) is in the additional 5mg/L 6-benzyl aminopurine (6-Benzylaminopurine of liquid Murashi ge-Skoog (MS) minimum medium with inoculum size, BA) and the 2.5mg/L Yin butyric acid (Indole-3-butyricacid that trembles, IBA), add the Jujubogenin tyrosol (tyrosol) of 4mM in 4.5% sucrose, medium pH is 5.7, intensity of illumination is 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are under the culture condition of 100rpm/min, cultivate 15-20 days, collect the callus particle, extract rhodioloside.
Described Radix Rhodiolae callus particle is to be to induce a kind of green that obtains under the condition of 1/3-1/2 or punctation, the diameter callus particle at the 2-5 millimeter is arranged at the additional 3-6mg/L BA of solid MS minimum medium and 1-3mg/L IBA and BA/IBA ratio by the cotyledon of Radix Rhodiolae.The growth cycle that Radix Rhodiolae callus particle suspension is cultivated is 20-25 days, and its biomass accumulation is asynchronous with the rhodioloside accumulation, and maximum biomass is accumulated in 15-20 days behind the subculture, the 3-5 of the highest accumulation volume behind subculture of rhodioloside days.
Described Radix Rhodiolae callus particulate inoculum size is 15%, places earlier under the grown cultures condition to cultivate 15-20 days, changes to immediately to produce under the culture condition to cultivate 6-9 days again, collects the callus particle, extracts rhodioloside.
It is raw material that the present invention induces the callus particle of the structure consolidation of generation with a kind of cotyledon by Radix Rhodiolae, and it is carried out fluid suspension culture respectively under grown cultures condition and production culture condition.Can also utilize the measure of adding Jujubogenin tyrosol to improve rhodioloside output.
Described a kind of cotyledon by Radix Rhodiolae is induced the callus particle of the structure consolidation of generation to be green or punctation is arranged, the structure consolidation, and diameter is at the particle of 2-5 millimeter.The growth cycle of its suspension culture is 20-25 days, and its biomass accumulation is asynchronous with the rhodioloside accumulation: maximum biomass is accumulated in 15-20 days behind the subculture, the 3-5 of the highest accumulation volume behind subculture of rhodioloside days.
Described grown cultures condition, refer to the additional 5mg/L BA of liquid MS minimum medium and, 2.5mg/LIBA, 4.5% sucrose; PH is 5.7; Intensity of illumination is 150 μ E/m
2/ s, light application time 16 hours/day; Agitation speed is 100rpm/min.
Described production culture condition refers to 2 of the additional 4mg/L of liquid MS minimum medium, 4-D, 1.5% sucrose; PH is 4.2; Intensity of illumination is 150 μ E/m
2/ s, light application time 16 hours/day; Agitation speed is 150rpm/min.
Described training mode refers to that inoculum size is that the callus particle of 15% (v/v) places earlier under the grown cultures condition and cultivated 15-20 days, topple over and fall liquid nutrient medium, change to immediately to produce to cultivate under the bar and cultivated 6-9 days again, results collection callus particle extracts rhodioloside.
The measure that Jujubogenin tyrosol (tyrosol) is added in described utilization improves rhodioloside output, method is to add the Jujubogenin tyrosol (tyrosol) of 4mM in the liquid MS medium under the grown cultures condition, cultivated 15-20 days, collect the callus particle, extract rhodioloside, can make output improve 29 times.
Claims (4)
1, a kind of plant callus particls suspending culture to produce rhodiola glycoside, it is characterized in that: the Radix Rhodiolae callus particle that with inoculum size is 15% (v/v) is in the additional 5mg/L 6-benzyl aminopurine (6-Benzylaminopurine of liquid Murashige-Skoog (MS) minimum medium, BA) and 2.5mg/L Yin tremble butyric acid (lndole-3-butyric acid, IBA), 4.5% sucrose, pH is 5.7, intensity of illumination 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are to cultivate 15-20 days under the grown cultures condition of 100rpm/min, forward total nitrogen content again to and be basic MS culture medium half the additional 4mg/L of liquid MS medium 2,4-D, ((2,4-Dichlorophenoxy) aceticacid), 1.5% sucrose, pH are 4.2, intensity of illumination 150 μ E/m
2/ S, light application time 16 hours/day and stirring velocity are that the production culture condition of 150rpm/min was cultivated 6-9 days down, collect the callus particle, extract rhodioloside.
2, according to the described plant callus particls suspending culture to produce rhodiola glycoside of claim 1, it is characterized in that: the Radix Rhodiolae callus particle that can also be 15% (v/v) with inoculum size is in the additional 5mg/L 6-benzyl aminopurine (6-Benzylaminopurine of liquid Murashige-Skoog (MS) minimum medium, BA) and the 2.5mg/L Yin butyric acid (Indole-3-butyricacid that trembles, IBA), add the Jujubogenin tyrosol (tyrosol) of 4mM in 4.5% sucrose, medium pH is 5.7, intensity of illumination is 150 μ E/m
2/ s, light application time 16 hours/day and stirring velocity are under the culture condition of 100rpm/min, cultivate 15-20 days, collect the callus particle, extract rhodioloside.
3, according to claim 1 or 2 described plant callus particls suspending culture to produce rhodiola glycoside, it is characterized in that: Radix Rhodiolae callus particle is to be to induce a kind of green that obtains under the condition of 1/3-1/2 or punctation, the diameter callus particle at the 2-5 millimeter is arranged at the additional 3-6mg/L BA of solid MS minimum medium and 1-3mg/L IBA and BA/IBA ratio by the cotyledon of Radix Rhodiolae.The growth cycle that Radix Rhodiolae callus particle suspension is cultivated is 20-25 days, and its biomass accumulation is asynchronous with the rhodioloside accumulation, and maximum biomass is accumulated in 15-20 days behind the subculture, the 3-5 of the highest accumulation volume behind subculture of rhodioloside days.
4, according to the described plant callus particls suspending culture to produce rhodiola glycoside of claim 1, it is characterized in that: Radix Rhodiolae callus particulate inoculum size is 15%, place earlier under the grown cultures condition and cultivated 15-20 days, change to immediately to produce under the culture condition and cultivated again 6-9 days, collect the callus particle, extract rhodioloside.
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| CN 01133396 CN1417343A (en) | 2001-10-31 | 2001-10-31 | Plant callus particls suspending culture to produce rhodiola glycoside |
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| CN 01133396 CN1417343A (en) | 2001-10-31 | 2001-10-31 | Plant callus particls suspending culture to produce rhodiola glycoside |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914122A (en) * | 2010-08-24 | 2010-12-15 | 北京农学院 | Method for preparing salidroside by utilizing UGT72B14 |
| CN101121941B (en) * | 2007-03-26 | 2011-09-14 | 吉林师范大学 | Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system |
| CN106479955A (en) * | 2016-12-21 | 2017-03-08 | 江西宜信堂医疗科技有限公司 | A kind of rhodiola root cell culture medium |
| CN106520665A (en) * | 2016-11-15 | 2017-03-22 | 天津市博爱生物药业有限公司 | Culture medium for rhodiola rosea cell |
-
2001
- 2001-10-31 CN CN 01133396 patent/CN1417343A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101121941B (en) * | 2007-03-26 | 2011-09-14 | 吉林师范大学 | Method for producing salidroside by using agrobacterium rhizogenes to inherit and transfer rhodiola sachdlinensis and constructing capillaceous root cultural system |
| CN101914122A (en) * | 2010-08-24 | 2010-12-15 | 北京农学院 | Method for preparing salidroside by utilizing UGT72B14 |
| CN106520665A (en) * | 2016-11-15 | 2017-03-22 | 天津市博爱生物药业有限公司 | Culture medium for rhodiola rosea cell |
| CN106479955A (en) * | 2016-12-21 | 2017-03-08 | 江西宜信堂医疗科技有限公司 | A kind of rhodiola root cell culture medium |
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