CN106134976A - Mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene is utilized to improve the method for the content of vallesiachotamine in Herba Catharanthi Rosei suspension cell - Google Patents
Mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene is utilized to improve the method for the content of vallesiachotamine in Herba Catharanthi Rosei suspension cell Download PDFInfo
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Abstract
The present invention relates to (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene as mixing elicitor, collaborative promotion Herba Catharanthi Rosei suspension cell synthesis vallesiachotamine.Catharanthus Roseus Cell is at B5+IAA0.5+6-BA1.0In culture medium, under the conditions of 25 DEG C, illumination cultivation is after 12 days, add mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, gather after continuing to cultivate 1-7 days under same condition of culture, from culture, extract alkaloid, utilize the changes of contents of HPLC method detection vallesiachotamine.Result shows, after adding mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, it is possible to the secondary metabolite being obviously promoted in Herba Catharanthi Rosei suspension cell generates vallesiachotamine, after the 5th day, the content of vallesiachotamine reaches maximum, for 75.2 times of matched group.The inducing action of mixing elicitor is better than and is used alone elicitor, illustrates that (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, as mixing elicitor, have the effect coordinating to promote the secondary metabolite synthesis vallesiachotamine in Catharanthus Roseus Cell.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene of adding in Herba Catharanthi Rosei suspension cell culture liquid to the method improving vallesiachotamine yield.
Background technology
Herba Catharanthi Rosei (Catharanthus roseus) it is a kind of important medicinal plants of Apocynaceae, originate in Mediterranean, India, America, the torrid zone.The history of Chinese cultivated Herba Catharanthi Rosei is the longest, mainly cultivates in the Changjiang river areas to the south, and it is the most universal that Guangdong, Guangxi, Yunnan etc. save (autonomous region) cultivation.Containing more than 130 kind of alkaloid in Herba Catharanthi Rosei plant, these alkaloids have pharmacologically active, vinblastine (vinblastine) mostly
It is one of current most widely used natural plants antitumor drug with vincristine (vincristine)；Reserpine (serpentine) can be used for treating the disease such as hypertension, arrhythmia；Ajmalicine (ajmalicine) has sedation；Catharanthine (catharanthine) has the effects such as antibacterial anti hemorrhagic, diuresis, blood sugar lowering；Wherein vallesiachotamine (vallesiachotamine) has the effects such as antitumor, antiinflammatory, analgesia, and additionally it also has the activity of suppression monoamine oxidase A.
At present, most of vinca alkaloidses are mainly obtained by extraction from Herba Catharanthi Rosei plant, but content that these alkaloids are in Herba Catharanthi Rosei body is extremely low and chemical constitution structure is complicated, and the content that vallesiachotamine is in Herba Catharanthi Rosei plant is lower, can not meet far away the needs in market.At present, vallesiachotamine is mainly with crossing chemical synthesis acquisition, but cost is high, environmental pollution is big.Utilize the secondary metabolite of Herba Catharanthi Rosei suspension cell cultivated to synthesize vallesiachotamine, there is low cost, the advantage such as pollution-free, meet country about building the requirement of " resource-conserving, environmentally friendly " society, be a kind of method having very much application prospect.Utilize plant cell mainly to use two-step method, i.e. cell first to cultivate a period of time on growth medium to produce secondary metabolite, to accumulate Biomass, then be transferred in production medium promote that cell synthesizes secondary metabolite in a large number.But, two-step method applies inconvenience in the industrial production, changes during culture medium the most time-consuming, easily causes again cell contamination.But, at the late log phase of Herba Catharanthi Rosei suspension cell growth, by the method adding elicitor in culture medium, promote the one-step method of the synthesis of secondary metabolite vallesiachotamine, there is easy and simple to handle, the advantage of low cost.
Summary of the invention
It is an object of the invention to provide and a kind of utilize Herba Catharanthi Rosei suspension cell to the method producing vallesiachotamine, join in Herba Catharanthi Rosei suspension cell culture liquid as mixing elicitor with (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, make the content of vallesiachotamine be significantly improved.
The method of the present invention is the 12nd day (late log phase of cell growth) at Catharanthus Roseus Cell conventional culture methods, then in culture fluid, add mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, wherein, the amount respectively 25-100 mmol/L(optimal dose adding Argylene and (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate is 50 mmol/L) and 45-180 μm ol/L(optimal dose be 90 μm ol/L), continuing to cultivate 1-7 days under the conditions of 25 DEG C, the culture medium used is B5+IAA0.5+6-BA1.0Culture medium.Finally gather Catharanthus Roseus Cell, extract vinca alkaloids, utilize HPLC method to detect vallesiachotamine changes of contents.
The present invention, by selecting the (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate of debita spissitudo and Argylene as mixing elicitor, stimulates Herba Catharanthi Rosei suspension cell, makes the content of vallesiachotamine in Catharanthus Roseus Cell culture be significantly improved.In the experimental group adding mixing elicitor, the content of vallesiachotamine reaches 0.367 mg/g, is 72.5 times of blank group respectively, is individually added into 31.7 times of Argylene experimental group, is individually added into 3.9 times of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate experimental group.The present invention is easy and simple to handle, and the content of vallesiachotamine is the highest, it is adaptable to industrialized production.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates that culture extracts.
Fig. 2 is 50 mM Argylenes and 90 μMs of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate individually and when being used in mixed way, on the impact of vallesiachotamine content in Herba Catharanthi Rosei suspension cell.
Detailed description of the invention
Experiment material
Herba Catharanthi Rosei plant cell suspension cultures.
The preparation of culture medium
Conventionally preparation obtains B5+IAA0.5+6-BA1.0Culture medium.
The preparation of elicitor
0.924 g Na2S2O3·5H2O is dissolved in 50 ml deionized waters, then 0.16 g AgNO3It is dissolved in 50 ml deionized waters.Then AgNO3Solution is poured in hypo solution lentamente, topples over, and stirring, obtains the silver thiosulfate solution that concentration is 9.2 μm ol/L the most rapidly.Weigh 39.2 mg (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate, be dissolved in 10 ml dehydrated alcohol, obtain the (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate solution that concentration is 0.018 μm ol/L.
Cultural method
Each enforcement is at B5+IAA respectively0.5+6-BA1.0In culture medium, cultivate 12 days under illumination condition, according to variable concentrations requirement, add the elicitor of different content.Under the conditions of 25 DEG C, 110 r/min, continue under illumination condition to cultivate 1-7 days.Vinca alkaloids is extracted, by the change of HPLC method detection alkaloid from Herba Catharanthi Rosei culture.Result is the most as shown in Figure 1 and Figure 2.
From figure 1 it appears that promote the increase of vallesiachotamine content after adding elicitor.As can be seen from Figure 2, compared with matched group, the addition of Argylene can promote the synthesis of vallesiachotamine, its facilitation adds 4 times relative to matched group, and at first day of induction, facilitation effect reaches maximum, and afterwards along with the prolongation of induction time, its facilitation effect gradually weakens.It addition, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate is after induction Herba Catharanthi Rosei suspension cell 5 days, the content of vallesiachotamine reaches maximum, and for 19.2 times of matched group, its facilitation effect has declined afterwards.But, using (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene as mixing elicitor use time, the content ratio of vallesiachotamine is used alone (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate group more significantly raising, from the 1st day to the 5th day, along with the prolongation of induction time, the content of vallesiachotamine increased sharply, after induction 5 days, the content of vallesiachotamine reaches maximum, and for 75.2 times of matched group, the content of vallesiachotamine decreases afterwards.The present invention using (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene as mixing elicitor use time, ratio is used alone Argylene or (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate has more significantly inducing effect, illustrates that (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene have the collaborative effect stimulating vallesiachotamine synthesis.
Table 1 Herba Catharanthi Rosei suspension cell culture based formulas
Table 2 HPLC chromatogram condition
Claims (5)
1. one kind utilizes mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene to improve the method for vallesiachotamine content in Herba Catharanthi Rosei suspended cell culture, it is characterized in that Herba Catharanthi Rosei suspension cell is cultivated 10-15 days according to a conventional method, then in culture fluid, add (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene, the amount adding (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate is 45-180 μm ol/L, and the amount of Argylene is 25-100
Mmol/L, continues to cultivate 1-7 days.
The most in accordance with the method for claim 1, it is characterized in that the addition of (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate is 90 μm ol/L, the addition of Argylene is 50 mmol/L.
3., according to the method described in claim 1 or 2, it is characterized in that continuing to cultivate 5 days after adding elicitor.
4., according to the method described in claim 1 or 2, it is characterized in that used medium is B5+IAA0.5+6-BA1.0Culture medium.
5. according to the method described in claim 1 or 3, it is characterized in that condition of culture be 25 DEG C, illumination, 110 r/min.
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| CN201510158054.5A CN106134976A (en) | 2015-04-07 | 2015-04-07 | Mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene is utilized to improve the method for the content of vallesiachotamine in Herba Catharanthi Rosei suspension cell |
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| CN201510158054.5A CN106134976A (en) | 2015-04-07 | 2015-04-07 | Mixing elicitor (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and Argylene is utilized to improve the method for the content of vallesiachotamine in Herba Catharanthi Rosei suspension cell |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277197A (en) * | 2017-01-24 | 2018-07-13 | 桂林莱茵生物科技股份有限公司 | A method of improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell |
| CN109122323A (en) * | 2018-10-22 | 2019-01-04 | 覃家日 | The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum |
| CN109526738A (en) * | 2018-12-13 | 2019-03-29 | 湖南美可达生物资源股份有限公司 | Elicitor combines the method that induction promotes macleaya cordata P6H gene and DBOX gene expression with mechanical damage |
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| CN1799335A (en) * | 2005-12-19 | 2006-07-12 | 东北林业大学 | Method for improving vinblastine content in madagascar periwinkle |
| CN1800189A (en) * | 2005-12-19 | 2006-07-12 | 东北林业大学 | Method for separating vindoline , catharanthine, vinblastine and vincristine from Vinca rosea |
| CN103081736A (en) * | 2013-01-10 | 2013-05-08 | 宁波大学 | Harvest pretreatment method of fast improving output of catharanthus roseus alkaloid |
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2015
- 2015-04-07 CN CN201510158054.5A patent/CN106134976A/en active Pending
Patent Citations (3)
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| CN1799335A (en) * | 2005-12-19 | 2006-07-12 | 东北林业大学 | Method for improving vinblastine content in madagascar periwinkle |
| CN1800189A (en) * | 2005-12-19 | 2006-07-12 | 东北林业大学 | Method for separating vindoline , catharanthine, vinblastine and vincristine from Vinca rosea |
| CN103081736A (en) * | 2013-01-10 | 2013-05-08 | 宁波大学 | Harvest pretreatment method of fast improving output of catharanthus roseus alkaloid |
Non-Patent Citations (1)
| Title |
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| AMES P.KUTNEY等: "ALKALOID PRODUCTION IN CATHARANTHUS ROSEUS CELL CULTURES: ISOLATION AND CHARACTERIZATION OF ALKALOIDS FROM ONE CELL LINE", 《PHYROCHEMISTRY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277197A (en) * | 2017-01-24 | 2018-07-13 | 桂林莱茵生物科技股份有限公司 | A method of improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell |
| CN108277197B (en) * | 2017-01-24 | 2020-03-24 | 桂林莱茵生物科技股份有限公司 | Method for increasing mogroside V content in suspension cells of momordica grosvenori |
| CN109122323A (en) * | 2018-10-22 | 2019-01-04 | 覃家日 | The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum |
| CN109526738A (en) * | 2018-12-13 | 2019-03-29 | 湖南美可达生物资源股份有限公司 | Elicitor combines the method that induction promotes macleaya cordata P6H gene and DBOX gene expression with mechanical damage |
| CN109526738B (en) * | 2018-12-13 | 2021-10-01 | 湖南美可达生物资源股份有限公司 | Method for inducing and promoting expression of macleaya cordata P6H gene and DBOX gene by combination of elicitor and mechanical injury |
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Application publication date: 20161123 |