CN103952459B - The method for promoting white birch total triterpene and oleanolic acid accumulation using 5 azacytidines (5 AZAC) - Google Patents
The method for promoting white birch total triterpene and oleanolic acid accumulation using 5 azacytidines (5 AZAC) Download PDFInfo
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- CN103952459B CN103952459B CN201410211396.4A CN201410211396A CN103952459B CN 103952459 B CN103952459 B CN 103952459B CN 201410211396 A CN201410211396 A CN 201410211396A CN 103952459 B CN103952459 B CN 103952459B
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- white birch
- oleanolic acid
- total triterpene
- triterpene
- azacitidine
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Abstract
The invention provides a kind of new method for promoting secondary metabolite to accumulate and the new application of 5 azacytidines, it is related to technical field of bioengineering.Its feature is, using the azacytidine elicitor of finite concentration 5 is added, to improve the accumulation of white birch secondary metabolite total triterpene and oleanolic acid.The invention of this method, to be carried out using plant cell Techniques of in Vitro Culture, industrialization mass produces triterpene and olive acids medicine provides a kind of new method.
Description
Technical field：
Promote secondary metabolite white birch total triterpene and oleanolic acid the present invention relates to 5-azacitidine (5-AZAC) processing
Accumulation method, belong to technical field of bioengineering.
Background technology：
Contain important triterpene substance in the leaf and crust of silver birch (Yin waits quietly, 2009).Numerous studies show, white birch
The pharmacological activity of triterpene is various, and shows mechanism of action (Suns etc. 2002 different from conventional medicine；Valentin etc.
2002), curative effect is high and toxicity is low, with the effect such as antitumor, anti-AIDS and antibacterial, antiviral, lipid-loweringing, cholagogic and liver protection
(Soler etc. 1996；Zhukova etc. 2005；Kovalenko etc. is 2007).Have at present with oleanolic acid etc. as main component
Medicine puts into Clinical practice, as potential new medicinal preparation, has broad application prospects.
In recent years, the research for producing useful secondary metabolites using Vitro Plant culture technique, which is achieved, very big enters
Exhibition, however the low yield phenomenon of secondary metabolite be still restrict Vitro Plant culture technique commercial application key problem it
One, therefore increasing researcher begins to focus on the method for improving secondary metabolite accumulation.But up to the present, plant
The low yield problem of secondary metabolites is not well solved yet in cell.5-azacitidine is a kind of methylation inhibitor, doctor
It is usually used in the treatment of the diseases such as tumour on.The present invention promotes white birch secondary metabolite total three using 5-azacitidine processing
Terpene and oleanolic acid accumulation.
Present disclosure：
It is an object of the invention to provide a kind of method that utilization 5-azacitidine promotes secondary metabolite accumulation.It is special
Point is, using finite concentration 5-azacitidine is added, to improve the accumulation of white birch secondary metabolite total triterpene and oleanolic acid.
The invention of this method, to carry out industrialization large-scale production triterpene and olive acids medicine using plant cell Techniques of in Vitro Culture
Thing is provided a method that.
Embodiment：
First, experiment material and method
(1) white birch suspension cell culture condition
This research is inoculated in the training of 100mL B5 liquid using white birch flower pesticide suspension cell as experiment material by 50g/L inoculum concentrations
Support in base, condition of culture is：120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%~50%, culture week
Phase is 10d.Medium component is B5 minimal mediums, addition 0.2-0.4mg/L6-BA and 0.6-0.8mg/L TDZ, 15-25g/
L sucrose, 1-2g/L acid hydrolyzed caseins, pH5.5~6.0,121 DEG C of autoclave sterilization 20min.
(2) addition of 5-azacitidine
A.5-AZAC white birch accumulation of triterpene is promoted
After white birch flower pesticide suspension cell growth is stable, 0.1-0.3mmol/L 5-azacitidines are added, in 96-120h
After harvest, white birch triterpene content in determination sample.
B.5-AZAC oleanolic acid accumulation is promoted
White birch flower pesticide suspension cell is handled with final concentration of 0.1-0.3mmol/L 5-azacitidine, after treatment 48-
72h is harvested, the content of oleanolic acid in determination sample.
(3) extraction of white birch triterpene and assay
Precision weighs 0.05g cell dry samples, adds the ethanol of 2ml 95% and soaks 24h.It is ultrasonic again after 70 DEG C of water-bath 1h
40min, takes 100 μ L of supernatant liquid to be placed in 70 DEG C of water bath methods in 10ml centrifuge tubes.Add 200 μ L5% vanillic aldehydes-glacial acetic acid
With 800 μ L perchloric acid, 70 DEG C of water-bath 15min, on ice rapid cooling.Ethyl acetate is settled to the suction that it is determined after 5ml in 551nm
Light value (control group is 200 μ L5% vanillic aldehydes -+800 microlitres of glacial acetic acid perchloric acid+4ml ethyl acetate).
The standard curve of total triterpene is drawn by standard items of oleanolic acid, and its regression equation is y=43.044x-
0.6438, coefficient R2=0.997, oleanolic acid has good linear relationship in 4~32mg/L content ranges.
(4) extraction of oleanolic acid and assay
The extracting method of oleanolic acid is as follows：Precision weighs 0.5g cell dry samples, add 25mL hydrochloric acid-ethanol solution (2:
8) 3h, is heated to reflux, is let cool, shakes up and filters, precision measures filtrate 15mL, plus distilled water 15mL, is placed in 80 DEG C of water-baths and steams
Ethanol is removed, is then extracted 3 times with ether, each 20mL, merges ether extraction liquid and is evaporated in 40 DEG C of low temperature, plus 1ml methanol is molten
Solve residue, and by its use 0.45 μm of organic membrane filtration, this be sample detection liquid, then examined using high performance liquid chromatography
Survey.
High performance liquid chromatography (HPLC) testing conditions：With Waters companies 600-717-2487 chromatographic systems, chromatographic column HiQ
sil C18V 4.6mm×250mm；Mobile phase is acetonitrile：Water=9：1；25 DEG C of column temperature；Sensitivity 16AUFS；Flow velocity 1.0mL/
min；Detection wavelength 210nm, sample introduction 20uL.
Oleanolic acid linear relationship is investigated：Precision draw concentration for 0.5mg/mL oleanolic acid standard solution 0.2,
0.5th, 1,2,3,4,5mL, is respectively placed in 5mL volumetric flasks, plus 95% ethanol is diluted to scale, shakes up, and precision is drawn 20 μ L and entered
Sample, determines its integrating peak areas value.Using concentration as abscissa, integrating peak areas value is ordinate, draws standard curve.Olive
Sour regression equation is：Y=107x+33446, R2=0.9992.As a result show, oleanolic acid has in 0.2-5.0 μ g ranges
Good linear relationship.
The effect of the present invention：
Using above-mentioned embodiment, a kind of elicitor 5-azacitidine promotes secondary generation in white birch flower pesticide suspension cell
Thank to the accumulation of product, it is as a result as follows：
(1) facilitation effect to secondary metabolite white birch accumulation of triterpene shows, 0.1-0.3mmol/L 5-azacitidines
In the white birch flower pesticide suspension cell of processing, white birch triterpene content occurs maximum 25.64747mg/gDW in 96-120h, is not
The 197% of the control group content handled through 5-azacitidine.
(2) facilitation effect to secondary metabolite oleanolic acid shows, the processing of 0.1-0.3mmol/L 5-azacitidines
White birch flower pesticide suspension cell in, content of oleanolic acid reaches peak 1.0186mg/gDW in 48-72h, is to be not added with 5- nitrogen
3.73 times of miscellaneous cytidine control group.
Claims (1)
1. a kind of method that utilization 5-azacitidine promotes secondary metabolite white birch total triterpene and oleanolic acid accumulation, its feature
It is to comprise the following steps：Step 1) using white birch flower pesticide suspension cell as experiment material, it is inoculated in 100mL by 50g/L inoculum concentrations
In B5 fluid nutrient mediums, condition of culture is：120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%~
50%, cultivation cycle is 10d, and medium component is B5 minimal mediums, addition 0.2-0.4mg/L 6-BA and 0.6-0.8mg/L
TDZ, 15-25g/L sucrose, 1-2g/L acid hydrolyzed caseins, 5.5~6.0,121 DEG C of autoclave sterilization 20min of pH；Step 2)
After white birch flower pesticide suspension cell growth is stable, addition 5-azacitidine makes its final concentration of 0.1-0.3mmol/L, oleanolic acid
Harvested after processing 48-72h, white birch total triterpene is harvested after processing 96-120h；Described white birch anther cell inoculum concentration is 5-
10g fresh cells, are inoculated in the 250ml triangular flasks of the nutrient solution containing 100ml, shaking speed 120r/min；Described 5- azepine born of the same parents
Glycosides solution is degerming by 0.22 μm of non-velum filteration, is added after the culture that suspends is stable, cultivation cycle is 10 days, and 5- azepine born of the same parents
The final concentration of 0.1-0.3mmol/L of glycosides.
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| CN102943104A (en) * | 2012-11-16 | 2013-02-27 | 东北林业大学 | Method for improving content of betulin and oleanolic acid in birch cell by utilizing MeJA (methyl-jasmonate) and SA (salicyl acid) |
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| CN101824459B (en) * | 2009-12-23 | 2015-05-20 | 东北林业大学 | Method for promoting accumulation of triterpene in betula platyphylla suk. suspension cell by utilizing endophytic fungi elicitor |
| US9994883B2 (en) * | 2012-05-11 | 2018-06-12 | Vib Vzw | Triterpenoid sapogenin production in plant and microbial cultures |
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| CN102943104A (en) * | 2012-11-16 | 2013-02-27 | 东北林业大学 | Method for improving content of betulin and oleanolic acid in birch cell by utilizing MeJA (methyl-jasmonate) and SA (salicyl acid) |
Non-Patent Citations (1)
| Title |
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| Effect of 5-Azacytidine on the Formation of Secondary Metabolites in Catharanthus roseus Cell Suspension Cultures;H.-A. Arfmann et al.;《Z. Naturforsch》;19851231;第40卷;第21-25页 * |
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Granted publication date: 20171024 Termination date: 20180520 |