CN104087644B - It is a kind of to improve the method for total triterpene and betulin content in white birch cell using hydrogen - Google Patents
It is a kind of to improve the method for total triterpene and betulin content in white birch cell using hydrogen Download PDFInfo
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- CN104087644B CN104087644B CN201410270442.8A CN201410270442A CN104087644B CN 104087644 B CN104087644 B CN 104087644B CN 201410270442 A CN201410270442 A CN 201410270442A CN 104087644 B CN104087644 B CN 104087644B
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Abstract
The invention belongs to bioengineering field, and in particular to a kind of to improve the method for total triterpene and betulin content in white birch cell using hydrogen.Using white birch suspension cell as material, it is inoculated in by 50g/L inoculum concentrations in 100mL B5 fluid nutrient mediums, adds 0.1~0.3mg/L6 BA and 0.5~4.0mg/L TDZ, sucrose 20g/L, acid hydrolyzed casein 1g/L, pH5.5~6.0.Being passed through within the 8th day hydrogen in cell suspension cultures makes its final concentration of 0.5 1.5mmol/L, is harvested after cultivating 12h, betulin content is up to 1.11mg/gDW, is control group 185.1%.Harvested after culture 24h, intracellular total triterpene contents reach 24.37mg/gDW, are the 181.32% of control group.Hydrogen has nontoxic advantage, facilitates the separation and purifying of metabolite.The invention provides a kind of new way to solve natural medicinal plants source in short supply and industrialization large-scale production white birch triterpene and betulin medicine.
Description
Technical field：
The present invention relates to the synthesis that hydrogen promotes secondary metabolites, it belongs to technical field of bioengineering.
Background technology：
Hydrogen is that nature is widely present and the simplest element of molecular structure, and 90% component is made of hydrogen in universe
's.Hydrogen is diatomic gas that is colourless, odorless, tasteless, having certain reproducibility.In submarine medicine field, hydrogen is extensive
During high deep hydrogen-oxygen mixing diving.The solubility of hydrogen cannot largely be absorbed, institute than relatively low by body
With people never have the effect for paying attention to hydrogen in higher organism body.The reproducibility of hydrogen is the most important chemical property of hydrogen,
We just learnt in middle school, and it is copper that hydrogen, which can reduce heated oxide copper,.But many people are not aware that, hydrogen also has only
Special biological property, past field of biology think that hydrogen belongs to physiological inert gas always, it is believed that hydrogen is not appoint
The gas of what biological effect.But from internationally famous magazine in 2007《Nature Medicine》Report, hydrogen have mystery
Selective antioxidation, patient only needs to breathe the hydrogen that 35min concentration is 2%, it is possible to effectively treats brain
Ischemical reperfusion injury.Breathing hydrogen can also treat newborn baby Following Cerebral Hypoxia-ischemia disease.Then, hydrogen is in botany, base
Become new research hotspot in terms of plinth medicine and clinical Study on Transformation.Think that the huge applications potentiality of hydrogen do not obtain really also
Understanding.
Up to the present, the research paper on hydrogen biomedicine already close to 500, just increases for average 2 days in the world
Add 1.Due to the field that hydrogen can not used for reference medically, as a kind of therapeutic gas, most effective way at present
It is by drinking hydrogen saturated water, clinical practice can be by breathing hydrogen and oxygen mixed gas and injection hydrogen saturated solution.
Experiment shows hydrogen water, H2In suction, and peritonaeum, the saturated brine of hydrogen is injected intravenously to disease caused by active oxygen
All there is protective effect.These research accumulated in evidence show, H2Various cells can be protected, tissue and organ are from oxygen
Change damage.The H in bacterium and algae2Metabolism many investigation have been carried out.The research of some early stages shows in some height etc.
H in plant2Develop and absorb hydrogenase existing for luminous leaf and hypothesis.However, few researchs are examined in higher plant
Look into the physiological action of hydrogen manufacturing and support the mechanism of this process.
In recent years, using Vitro Plant culture technique produce useful secondary metabolites research achieve it is very big into
Exhibition, such as paniculatum cell production alkannin, ginseng-cell culture production saponin, digitalis cell culture production alkaloid all reach
Industrialized production scale, with Catharanthus Roseus Cell culture production anti-cancer alkaloid had reached pilot scale it is horizontal (Liu Chunchao etc.,
1997；Hu Liyong, 2004).But the low yield phenomenon of secondary metabolite is to restrict the production of cell culture phytochemicals production technology
One of key problem of industryization application, it is to solve the problems, such as this base to understand and grasp plant cell Secondary Metabolic Regulation of Callus rule
Plinth.Although domestic and international researcher is directed to secondary metabolites low yield problem in plant cell culture and is studied, including high-quality thin
The selection and breeding of born of the same parents system, the optimization of condition of culture, culture technique is improved and active material synthesizes the (Guo Xiao such as the clone of key gene
It is red etc., 2005；Xu Maojun, 2009；Qian Dandan etc., 2011).But up to the present, in plant cell secondary metabolites low yield
Problem is not well solved yet.
Contain important secondary metabolite --- white birch in white birch (Betula platyphylla suk) leaf and bark
Triterpene (TBP), main Types include lupinane type, dammarane type, oleanane type triterpene material (Zhang, 2003), are pole
Have potentiality to be exploited anticancer, treatment angiocardiopathy, protect liver pioneer's medicine (Li Wei, 2000；Ye Yinying, 2000,2001).With
Nascent metabolism is compared, and Secondary Metabolism of Plant has very strong Modulatory character.For natural active matter in plant cell culture
Low yield problem, domestic and international researcher carried out substantial amounts of research and probe.In research and inquirement plant cell with secondary metabolite
Related mechanism is synthesized to not only help the regulation rule for grasping Secondary Metabolism of Plant and train to solving plant in production practices
Cell secondary metabolite low yield problem is supported to be of great significance.For the effect that white birch triterpene can be promoted to synthesize, it is white to study its regulation and control
The molecular mechanism that triterpene synthesizes in birch suspended culture cell, emphasis inquire into H2Influence to triterpene synthesis etc. is induced, establishes hydrogen
Regulate and control the technical system of white birch triterpene synthesis, can to solve the problems, such as in Production of Secondary Metabolites By Plant Cell Cultures that low yield provides
The technology leaned on and stablized.
Present disclosure：
To protect white birch natural resources, the resource production routine of Sustainable Development and Utilization is established, utilizes nontoxic hydrogen
Callus and suspended culture cell of the gas induction from white birch induction, carry out the synthesis and production of total triterpene and betulin,
At the same time quantitative detection is carried out using high performance liquid chromatography.Total triterpene and betulin class are mass produced to carry out industrialization method
Medicine provides new method.
Embodiment：
(1) culture of suspension cell
On superclean bench, axillary bud is cut, soaks 5min with 70% alcohol, then sterilized with 5% liquor natrii hypochloritis
10-20min, is then put in sterilized culture dish, is inoculated into the culture of the pre- various combinations for first passing through autoclave sterilization
(+3% sucrose+0.5~4.0mg/LBA, 0.2~4.0mg/L NAA of NT+1.0mg/L caseinhydrolysates) carries out callus group on base
The induction knitted.About 3-5 axillary bud of every bottle of inoculation.First optical culture is carried out after a week with light culture.After culture about 7 weeks, it is cured
Injured tissue.Callus is after squamous subculture, rapid propagation.
High cell growth speed is selected, loose and high total triterpene content of material callus, carries out subculture and suspend to train
Support.And be conducive to white birch suspension cell growth and secondary metabolite accumulation B5+0.1~0.3mg/L 6-BA+0.5~
Cultured cells system in 4.0mg/L TDZ suspension mediums.High yield white birch triterpene and betulin suspension cell line are obtained, is passed through
Filter, is dried, and is crushed, organic solvent (95% ethanol) extraction step, using liquid-phase chromatography method to betulin and betulin
Detection and quantitative analysis.
(2) extraction of betulin and measure
The method that the extraction of betulin and measure wait 2004 using model cassia twig etc. 2007, a pool.Extracts reagent：95% second
Alcohol；Extracting method：Ultrasonic extraction；Assay method：High performance liquid chromatography, Detection wavelength 210nm, mobile phase for acetonitrile and
Water (V:V=8:2), flow velocity 1.0mL/min.
This patent has the characteristics that：
1) hydrogen treat has nontoxic advantage, while production environment is from region, season, water quality, pest and disease damage, gas
The influence of the natural environments such as time.
2) with short production cycle, production process does not produce a large amount of slag and effluents, and safety non-pollution, has ecological benefits.
3) the white birch suspension cell of culture has the ability of the production many kinds of substance such as white birch triterpene and betulin, realizes
The utilization of white birch natural plant resource and its medicinal ingredient.
4) betulin being carried out using efficient liquid-phase chromatography method to be detected, method is simple, and Extraction solvent cleaning is nontoxic,
It is easy to operate.
5) it is in short supply to solve anti-AIDS and antitumor natural drug source, accelerate clinical large-scale application, there is provided a kind of
New way, has very high economic benefit and social benefit.
Embodiment
1) source of plant material
White birch is derived from the select tree in Northeast Forestry University's white birch reinforcing breeding garden.Axillary bud is selected, is inoculated into after disinfection in advance
(+3%+0.5~4.0mg/ of sucrose of NT+1.0mg/L caseinhydrolysates on culture medium by the various combinations of autoclave sterilization
LBA, 0.2~4.0mg/L NAA) carry out callus induction.Callus is after squamous subculture, rapid propagation.Select
The good callus of growth conditions will be transferred to fluid nutrient medium suspension culture after its multiple subculture.More than generation turn by 6
Connect, establish stable suspended culture cell system, the culture subculture cycle that suspends is 15d, and inoculum concentration is 5g cells in 100mL liquid
In culture medium.2) condition of culture
Using white birch suspension cell as experiment material, it is inoculated in 100mL B5 fluid nutrient mediums, cultivates by 50g/L inoculum concentrations
Condition is：120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%~50%, cultivation cycle 10d.Culture
Based component is B5 minimal mediums, adds 0.0.1-0.3mg/L 6-BA and 0.5-4mg/L TDZ, 20g/L sucrose, 1g/L sour waters
Solve casein, pH5.5~6.0,121 DEG C of autoclave sterilization 20min.
3) the preparation addition of hydrogen rich water
The hydrogen gas outlet insertion of SHC types hydrogen generator (being purchased from Shandong Sai Kesaisi hydrogen energy sources Co., Ltd) has been gone out
Half an hour in bacterium distilled water, distilled water are changed into 99.99% hydrogen rich water, add when white birch suspension cell culture was to the 8th day
Add in culture medium, final concentration is respectively 0.5-1.5mmol/L, 12 to 24h harvest cell after addition.
4) extraction of total triterpene and assay
Precision weighs 0.05g cell dry samples, adds 95% ethanol of 2ml 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 5% vanillic aldehydes of μ L-ice second
Acid and 800 μ L perchloric acid, 70 DEG C of water-bath 15min, on ice rapid cooling.Ethyl acetate measures it 551nm's after being settled to 5ml
Light absorption 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, its regression equation is y=45.036x+
0.0417, coefficient R 2=0.9983, oleanolic acid has good line in 0.005~0.025mg/mL content ranges
Sexual intercourse.
5) extraction of betulin and assay
The extraction of betulin waits 2004 method with reference to model cassia twig etc. 2007, a pool, and specific method is as follows：Precision claims
0.5g cell dry samples are taken, 25mL hydrochloric acid-ethanol solution (2: 8) is added, is heated to reflux 3h, lets cool, shake up and filter, precision measures
Subsequent filtrate 15mL, adds distilled water 15mL, is placed in 80 DEG C of water-baths and boils off ethanol, is then extracted 3 times, each 20mL with ether, closes
And ether extraction liquid and be evaporated in 40 DEG C of low temperature, add 1ml methanol dissolved residues, and it is filtered with 0.45 μm of organic filter membrane, this
For sample detection liquid, then it is detected using high performance liquid chromatography.
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.
Betulin linear relationship is investigated：Precision draw concentration be 0.5mg/mL betulin standard solution 0.2,
0.4th, 0.6,0.8,1mL, is respectively placed in 5mL volumetric flasks, adds 95% ethanol to be diluted to scale, shake up, precision draw 20 μ L into
Sample, measures its integrating peak areas value.Using concentration as abscissa, integrating peak areas value is ordinate, draws standard curve.White birch fat
Alcohol regression equation is：Y=3E+06x-128674.The result shows that betulin has well in the range of 0.2~1mg/ml
Linear relationship.
The effect of the present invention：The present invention promotes the ability of cell production triterpene, the white birch total triterpene of acquisition using hydrogen
Greatly improved with betulin content.By above-mentioned technical proposal, tested using white birch suspension cell as material, the result is that：
Harvested after hydrogen treat 12-24h, betulin content is up to 1.11mg/gDW, is non-hydrogenation control group 185.1%.Carefully
Intracellular total triterpene contents reach 24.37mg/gDW, are the 181.32% of non-hydrogenation control group.Therefore, this is that one kind has work very much
The white birch total triterpene of industry application prospect and the production method of betulin.
Attached drawing 1：White birch solid callus
Attached drawing 2：White birch suspension cell
Attached drawing 3：Betulin standard items go out peak figure
Attached drawing 4：H2Processing betulin goes out peak figure
Attached drawing 5：The standard curve of total triterpene contents measure (using oleanolic acid as standard items)
Attached drawing 6：The standard curve of betulin assay.
Claims (3)
1. it is a kind of using hydrogen improve white birch cell in the method for total triterpene and betulin content, it is characterised in that including with
Lower step：Step 1) is inoculated in 100mL B5 fluid nutrient mediums using white birch suspension cell as experiment material, by 50g/L inoculum concentrations
In, condition of culture is：120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%~50%, cultivation cycle is
10d, medium component are B5 minimal mediums, add 0.1-0.3mg/L 6-BA and 0.5~4.0mg/L TDZ, 20g/L sugarcane
Sugar, 1g/L acid hydrolyzed caseins, pH5.5~6.0,121 DEG C of autoclave sterilization 20min；Step 2) is in cell suspension cultures the 8th
It, which adds hydrogen rich water, makes its final concentration of 0.5-1.5mmol/L, and suspension cell is harvested after culture 12 to 24h.
2. the method for total triterpene and betulin content in white birch cell is improved using hydrogen according to claim 1, wherein
The white birch cell inoculation amount is 50g/L, shaking speed 120r/min.
3. the method for total triterpene and betulin content in white birch cell is improved using hydrogen according to claim 1, wherein
The hydrogen rich water is prepared by SHC type hydrogen generators, and suspending, culture adds on the 8th day, cultivation cycle 10
My god, and the final concentration of 0.5-1.5mmol/L of hydrogen rich water.
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| CN105255924A (en) * | 2015-09-14 | 2016-01-20 | 东北林业大学 | Betula platyphylla cycloartenol synthase gene BPX3 and application for regulation and control of betula platyphylla triterpene content |
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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) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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) |
Non-Patent Citations (1)
| Title |
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| 木本植物生长发育及繁殖过程中DNA甲基化模式重建;骆薇等;《中国农学通报》;20101231;第26卷(第24期);35-41 * |
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