CN103952459A - New application of 5-azacytidine (5-AZAC) in promoting accumulation of secondary metabolites - Google Patents
New application of 5-azacytidine (5-AZAC) in promoting accumulation of secondary metabolites Download PDFInfo
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- CN103952459A CN103952459A CN201410211396.4A CN201410211396A CN103952459A CN 103952459 A CN103952459 A CN 103952459A CN 201410211396 A CN201410211396 A CN 201410211396A CN 103952459 A CN103952459 A CN 103952459A
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Abstract
The invention provides a new method for promoting accumulation of secondary metabolites and new application of 5-azacytidine, relating to the technical field of bioengineering. The invention is characterized in that a certain concentration of 5-azacytidine elicitor is added to improve accumulation of secondary metabolites total triterpene and oleanolic acid of white birches. The invention provides a new method for industrialized large-scale production of triterpene and oleanolic acid medicaments through a plant cell isolated culture technology.
Description
Technical field:
The present invention relates to 5-azacytidine (5-AZAC) and process the accumulation that promotes the total triterpene of secondary metabolite white birch and Oleanolic Acid, belong to technical field of bioengineering.
Background technology:
In the leaf of white birch and crust, contain important triterpene substance (Yin waits quietly, 2009).Large quantity research shows, the pharmacologically active of white birch triterpene is various, and demonstrated the mechanism of action (Sun etc. 2002 different from medicine in the past; Valentin etc. 2002), curative effect is high and toxicity is low, the effect (Soler etc. 1996 such as have antitumor, anti-AIDS and antibacterial, antiviral, lipopenicillinase, cholagogic and protect the liver; Zhukova etc. 2005; Kovalenko etc. 2007).At present existingly drop into clinical use taking Oleanolic Acid etc. as the medicine of main component, as potential new medicinal preparation, have broad application prospects.
In recent years, the research that utilizes Vitro Plant culture technique to produce useful secondary metabolites makes great progress, but the low yield phenomenon of secondary metabolite remains one of key problem of restriction Vitro Plant culture technique commercial application, therefore increasing investigator starts to pay close attention to the method that improves secondary metabolite accumulation volume.But up to the present, in vegetable cell, the low yield problem of secondary metabolite is well solved not yet.5-azacytidine is a kind of methylation inhibitor, is medically usually used in the treatment of the diseases such as tumour.The present invention utilizes 5-azacytidine processing, promotes the total triterpene of white birch secondary metabolite and Oleanolic Acid accumulation.
Content of the present invention:
The object of the present invention is to provide a kind of novel method of secondary metabolite accumulation and new purposes of 5-azacytidine of promoting.Its feature is to utilize interpolation finite concentration 5-azacytidine, the accumulation that improves the total triterpene of white birch secondary metabolite and Oleanolic Acid.The invention of present method, for utilizing vegetable cell Techniques of in Vitro Culture to carry out large-scale industrialization a kind of novel method of having produced triterpene and olea acids drug provision.
Embodiment:
One, experiment material and method
(1) white birch suspension cell culture condition
This research, taking white birch flower pesticide suspension cell as experiment material, is inoculated in 100mL B5 liquid nutrient medium by 50g/L inoculum size, and culture condition is: 120rpm, and 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%～50%, culture cycle is 10d.Medium component is B5 minimum medium, and additional hormone and concentration are 0.2-0.4mg/L6-BA and 0.6-0.8mg/L TDZ, sucrose 15-25g/L, acid hydrolyzed casein 1-2g/L, pH5.5～6.0,121 DEG C of autoclave sterilization 20min.
(2) interpolation of 5-azacytidine
A.5-AZAC promote white birch accumulation of triterpene
After white birch flower pesticide suspension cell growth is stable, add 0.1-0.3mmol/L5-azacytidine, after 96-120h, gather in the crops white birch triterpene content in working sample.
B.5-AZAC promote Oleanolic Acid accumulation
5-azacytidine taking final concentration as 0.10.1-0.3mmol/L is processed white birch flower pesticide suspension cell, respectively at 48-72h results after processing, the content of Oleanolic Acid in working sample.
(3) extraction of white birch triterpene and assay
Precision takes 0.05g cell dry sample, adds 2ml95% ethanol and soaks 24h.Ultrasonic 40min again after 70 DEG C of water-bath 1h, gets 100 μ L supernatant liquors in 10ml centrifuge tube and is placed in 70 DEG C of water bath methods.Add 200 μ L5% Vanillin-glacial acetic acids and 800 μ L perchloric acid, 70 DEG C of water-bath 15min, cooling rapidly on ice.Ethyl acetate is measured its light absorption value at 551nm (control group is 200 μ L5% Vanillin-glacial acetic acid+800 microlitre perchloric acid+4ml ethyl acetate) after being settled to 5ml.
The typical curve of total triterpene is drawn taking Oleanolic Acid as standard substance, and its regression equation is y=43.044x-0.6438, coefficient R
2=0.997, Oleanolic Acid has good linear relationship in 4～32mg/L content range.
(4) extraction of Oleanolic Acid and assay
The extracting method of Oleanolic Acid is as follows: precision takes 0.5g cell dry sample, add 25mL hydrochloric acid-ethanolic soln (2: 8), reflux 3h, let cool, shake up and filter, precision measures filtrate 15mL, adding distil water 15mL, is placed in 80 DEG C of water-baths and boils off ethanol, then uses extracted with diethyl ether 3 times, each 20mL, merge ether extraction liquid and in 40 DEG C of low temperature evaporates to dryness, add 1ml dissolve with methanol residue, and used the organic membrane filtration of 0.45 μ m, this is sample detection liquid, then utilizes high performance liquid chromatography to detect.
High performance liquid chromatography (HPLC) testing conditions: with the 600-717-2487 of Waters company chromatographic system, chromatographic column HiQ sil C18V4.6mm × 250mm; Moving phase is acetonitrile: water=9:1; 25 DEG C of column temperatures; Sensitivity 16AUFS; Flow velocity 1.0mL/min; Detect wavelength 210nm, sample introduction 20uL.
Oleanolic Acid linear relationship is investigated: accurate Oleanolic Acid standard solution that concentration is 0.5mg/mL 0.2,0.5,1,2,3,4, the 5mL of drawing, be placed in respectively 5mL volumetric flask, add 95% alcohol dilution to scale, shake up, the accurate 20 μ L sample introductions of drawing, measure its peak area integrated value.Taking concentration as X-coordinate, peak area integrated value is ordinate zou, drawing standard curve.Oleanolic Acid regression equation is: y=107x+33446, R
2=0.9992.Result shows, Oleanolic Acid has good linear relationship within the scope of 0.2-5.0 μ g.
Effect of the present invention:
Utilize above-mentioned embodiment, a kind of novel elicitor 5-azacytidine has promoted the accumulation of secondary metabolite in white birch flower pesticide suspension cell, and result is as follows:
(1) facilitation effect of secondary metabolite white birch accumulation of triterpene is shown, in the white birch flower pesticide suspension cell of 0.1-0.3mmol/L5-azacytidine processing, there is maximum value in white birch triterpene content, be 25.64747mg/gDW in the time of 96-120h, is 197% of control group content now.
(2). the facilitation effect to secondary metabolite Oleanolic Acid shows, in the white birch flower pesticide suspension cell of 0.1-0.3mmol/L5-azacytidine processing, content of oleanolic acid reaches maximum 1.0186mg/gDW at 48-72h, is 3.73 times of control group now.
Claims (6)
1. this patent proposition 5-azacytidine promotes the total triterpene of white birch and Oleanolic Acid to accumulate new purposes.Concrete operations technology is: taking white birch suspension cell as experiment material, be inoculated in 100mL B5 liquid nutrient medium by 50g/L inoculum size, culture condition is: 120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%～50%, culture cycle is 10d.Medium component is B5 minimum medium, and additional hormone and concentration are 0.2-0.4mg/L6-BA and 0.6-0.8mg/L TDZ, sucrose 15-25g/L, acid hydrolyzed casein 1-2g/L, pH5.5～6.0,121 DEG C of autoclave sterilization 20min.After white birch flower pesticide suspension cell growth is stable, add 5-azacytidine, interpolation final concentration is 0.10.1-0.3mmol/L.Content of oleanolic acid reaches maximum 1.0186mg/gDW at 48-72h, is 3.73 times of control group now.There is maximum value in white birch triterpene content, be 25.64747mg/gDW in the time of 96-120h, is 197% of control group content now.
2. according to the method described in claims 1, liquid nutrient medium is wherein B5 minimum medium, and additional hormone and concentration are 0.2-0.4mg/L6-BA and 0.6-0.8mg/L TDZ, sucrose 15-25g/L, acid hydrolyzed casein 1-2g/L.
3. according to the method described in claims 1, white birch anther cell inoculum size is wherein 5-10g fresh cell, is inoculated in the 250ml triangular flask containing 100ml nutrient solution shaking speed 120r/min.
4. according to the method described in claims 1, wherein 5-azacytidine solution, by 0.22 μ m non-velum filteration degerming, adds after suspension culture is stable, and culture cycle is 10 days, and 5-azacytidine final concentration is 0.1-0.3mmol/L.
5. according to the method described in claims 1, harvest time is according to the difference of product, and Oleanolic Acid is that after adding elicitor, 48-72h gathers in the crops, and white birch triterpene is that after adding elicitor, 96-120h gathers in the crops.
6. according to the method described in claims 1, wherein U-18496 process after content of oleanolic acid be up to 1.0186mg/gDW, be 3.73 times of control group now.White birch triterpene content is up to 25.64747mg/gDW, is 197% of control group content now.
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| CN201410211396.4A CN103952459B (en) | 2014-05-20 | 2014-05-20 | The method for promoting white birch total triterpene and oleanolic acid accumulation using 5 azacytidines (5 AZAC) |
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| CN201410211396.4A CN103952459B (en) | 2014-05-20 | 2014-05-20 | The method for promoting white birch total triterpene and oleanolic acid accumulation using 5 azacytidines (5 AZAC) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104472363A (en) * | 2014-12-05 | 2015-04-01 | 浙江理工大学 | Induction method for promoting accumulation of rosmarinic acid and danshinolic acid in salvia miltiorrhiza hairy roots |
Citations (3)
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|---|---|---|---|---|
| CN101824459A (en) * | 2009-12-23 | 2010-09-08 | 东北林业大学 | Method for promoting accumulation of triterpene in betula platyphylla suk. suspension cell by utilizing endophytic fungi elicitor |
| 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) |
| WO2013167751A1 (en) * | 2012-05-11 | 2013-11-14 | Vib Vzw | Triterpenoid sapogenin production in plant and microbial cultures |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824459A (en) * | 2009-12-23 | 2010-09-08 | 东北林业大学 | Method for promoting accumulation of triterpene in betula platyphylla suk. suspension cell by utilizing endophytic fungi elicitor |
| WO2013167751A1 (en) * | 2012-05-11 | 2013-11-14 | Vib Vzw | Triterpenoid sapogenin production in plant and microbial cultures |
| 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 (2)
| Title |
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| H.-A. ARFMANN ET AL.: "Effect of 5-Azacytidine on the Formation of Secondary Metabolites in Catharanthus roseus Cell Suspension Cultures", 《Z. NATURFORSCH》 * |
| 骆薇等: "木本植物生长发育及繁殖过程中DNA甲基化模式重建", 《中国农学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104472363A (en) * | 2014-12-05 | 2015-04-01 | 浙江理工大学 | Induction method for promoting accumulation of rosmarinic acid and danshinolic acid in salvia miltiorrhiza hairy roots |
| CN104472363B (en) * | 2014-12-05 | 2016-08-24 | 浙江理工大学 | Promote rosmarinic acid and the revulsion of salvianolic acid B accumulation in Hairy Root Cultures of Salvia miltiorrhiza |
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