CN104031970B - Method for increasing content of total triterpenes and oleanolic acid in white birch cells by use of SNP (sodium nitroprusside) - Google Patents
Method for increasing content of total triterpenes and oleanolic acid in white birch cells by use of SNP (sodium nitroprusside) Download PDFInfo
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- CN104031970B CN104031970B CN201410260611.XA CN201410260611A CN104031970B CN 104031970 B CN104031970 B CN 104031970B CN 201410260611 A CN201410260611 A CN 201410260611A CN 104031970 B CN104031970 B CN 104031970B
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Abstract
The invention belongs to the field of bioengineering, and particularly relates to a method for increasing the content of total triterpenes and oleanolic acid in white birch cells by use of SNP (sodium nitroprusside). With white birch anther suspension cells as experimental materials, the method comprises the following steps: inoculating the experimental materials to 100mL of B5 liquid medium according to an inoculation amount of 50g/L, wherein culture conditions are as follows: 120rpm, 25 DEG C, illumination intensity of 2,000lx, photoperiod of 16h, humidity of 40-50% and culture period of 10d, and the medium component is a B5 minimal medium added with hormone and 0.2mg/L6-BA and 0.6mg/L TDZ, 20g/L sucrose and 1g/L acid hydrolyzed casein; performing high-temperature high-pressure sterilization for 20min at 121 DEG C and pH value of 5.5-6.0; adding SNP in the 8th day of cell suspension culture so that the final concentration is 1mmol/L; culturing for 12h and harvesting, wherein the maximum content of oleanolic acid is 1.3089mg/g DW which is 569% of that of the control group; culturing for 48h and harvesting, wherein the content of total triterpenes in the cells reaches 23.86mg/g DW which is 143.20% of that of the control group. The invention provides a new means against the shortage of natural plant drug sources and for industrial large-scale production of white birch triterpenes and oleanolic acid drugs.
Description
Art
The invention belongs to bioengineering field is and in particular to a kind of improve total triterpene and Qi Dun in Betula platyphylla Suk. cell using snp
The method of tartaric acid content.
Background technology
There is in Japanese birch bark important triterpene substance, triterpene substance can be by suppressing cell proliferation, inducing cell
The mode such as apoptosis or suppression angiogenesis plays antitumor action, and triterpene substance also has the effect of antiinflammatory, antiviral in addition.
Its value in terms of medical and health of discovery with triterpene compound microbic activity progressively manifests, and has with oleanolic acid at present
Put into Clinical practice Deng the medicine for main component.However, the arboreal growth cycle is long, if obtain Betula platyphylla Suk. triterpene trees need to be carried out
Felling or strip off the skin, destroy resource, its complex structure in addition, manual method combined coefficient is low, the factor such as cost height and become restriction
It can not carry out the main cause of clinical large-scale application.The culture plant cell skill set up based on cellular omnipotency
Art produces for plant amedica and provides a new way.Have due to producing natural plant active substance using culture plant cell method
The not impact of the natural conditions such as climate, pest and disease damage, and it is not take up soil, not consumption of natural resource, with short production cycle and people
For controlled the advantages of, thus be considered as solve plant amedica produce and natural plant resource sustainable development between contradiction one kind
New bio technology.Numerous studies show that no is the key molecule that Secondary metabolites composite signal is transduceed in network, tool
Play an important role.But have not yet to see and promote the calluss of Betula platyphylla Suk. flower pesticide and suspension culture thin using donor substance snp of no
Born of the same parents carry out synthesizing the report of Betula platyphylla Suk. triterpene and oleanolic acid.
Content of the invention:
For protecting Betula platyphylla Suk. natural resourcess, set up the resource production routine of Sustainable Development and Utilization, using from Betula platyphylla Suk. flower pesticide
The calluss of induction and suspended culture cell, carry out synthesis and the production of total triterpene and oleanolic acid, utilize efficient liquid simultaneously
Phase chromatography carries out detection by quantitative.New for carrying out providing of industrialization method large-scale production total triterpene and olive acids medicine
Method.
Technical scheme is: on superclean bench, cut tassel, with 70% alcohol-pickled 5min, then with 5% hypochlorous acid
Sodium solution sterilization 20min, is then put in sterilized culture dish, selects the tassel of suitable size, with dissecting under anatomical lens
Pin strip off tassel, takes out flower pesticide, is inoculated into (nt+1.0mg/l in the pre- culture medium of various combinations first passing through autoclave sterilization
Caseinhydrolysate+3% sucrose+0.5～4.0mg/l2,4-d, 0.2～4.0mg/l kt) carry out the induction of calluss.Every bottle
About 30-35 flower pesticide of inoculation.First carry out optical culture with light culture after one week.After culture about 7 weeks, obtain Anther Culture wound healing group
Knit.Calluss, after successive transfer culture, are bred rapidly.
Select high cell growth speed, the loose and high calluss of total triterpene content of material, carry out subculture and the training that suspends
Support.And be conducive to white birch suspension cell growth and secondary metabolite accumulation b5+0.1～0.3mg/l6-ba+0.5～
Cultured cells system in 1.0mg/l tdz suspension medium.Obtain high yield Betula platyphylla Suk. triterpene and oleanolic acid suspension cell line, pass through
Filter, dries, and pulverizes, organic solvent (95% ethanol) extraction step, using liquid-phase chromatography method to betulin and oleanolic acid
Detection and quantitative analyses.
This patent has the following characteristics that
1) production environment is not affected by natural environment such as region, season, water quality, pest and disease damage, weathers.
2) with short production cycle, production process does not produce a large amount of slag and effluents, safety non-pollution, has ecological benefits.
3) the white birch suspension cell cultivated have produce the many kinds of substance such as Betula platyphylla Suk. triterpene and oleanolic acid ability it is achieved that
Betula platyphylla Suk. natural plant resource and its exploitation of medicinal ingredient.
4) carry out oleanolic acid using efficient liquid-phase chromatography method to be detected, method is simple, Extraction solvent cleaning is nontoxic,
Easy to operate.
5) be solution anti-AIDS and antitumor natural drug source is in short supply, accelerates clinical large-scale application, provides one kind
New way, has very high economic benefit and social benefit.
Specific embodiment
1) source of plant material
Betula platyphylla Suk. is taken from Northeast Forestry University Betula platyphylla Suk. and strengthens the select tree that blooms in breeding garden.Select tassel, take out flower pesticide, sterilization
It is inoculated into (nt+1.0mg/l caseinhydrolysate+3% sucrose in the pre- culture medium of various combinations first passing through autoclave sterilization afterwards
+ 0.5～4.0mg/l2,4-d, 0.2～4.0mg/l kt) carry out the induction of calluss.Calluss after successive transfer culture,
Rapid propagation.Select the good calluss of growth conditions and be transferred to fluid medium suspension culture by after its multiple subculture.Warp
After 6 switchings more than generation, set up stable suspended culture cell system, suspension culture subculture cycle is 15d, inoculum concentration is that 5g is thin
Born of the same parents are in 100ml fluid medium.
2) condition of culture
With Betula platyphylla Suk. flower pesticide suspension cell as experiment material, it is inoculated in 100ml b5 fluid medium by 50g/l inoculum concentration,
Condition of culture is: 120rpm, 25 DEG C, intensity of illumination 2000lx, photoperiod 16h, humidity 40%～50%, and cultivation cycle is 10d.
Medium component is b5 minimal medium, and additional hormone and concentration are 0.2mg/l6-ba and 0.6mg/l tdz, sucrose 20g/l,
Acid hydrolyzed casein 1g/l, ph5.5～6.0,121 DEG C of autoclave sterilization 20min.
3) elicitor adds
Snp (sodium nitroprusside) is purchased from sigma company of the U.S., through 0.22 μm after being dissolved in sterile purified water
Filtering with microporous membrane, is added in culture medium when Betula platyphylla Suk. flower pesticide suspension cell culture was to the 8th day, final concentration is respectively 1mmol/
L, 12h after interpolation, 48h harvesting.
4) extraction of total triterpene and assay
Precision weighs 0.05g cell dry sample, adds 2ml95% ethanol and soaks 24h.Ultrasonic again after 70 DEG C of water-bath 1h
40min, takes 100 μ l supernatant to be placed in 70 DEG C of water bath methods in 10ml centrifuge tube.Add 200 μ l5% vanillin-glacial acetic acid
With 800 μ l perchloric acid, 70 DEG C of water-bath 15min, cool down rapidly on ice.Ethyl acetate measures its suction in 551nm after being settled to 5ml
Light value (matched group is 200 μ l5% vanillin -+800 microlitres of glacial acetic acid perchloric acid+4ml ethyl acetate).
The standard curve of total triterpene is drawn with oleanolic acid for standard substance, and its regression equation is y=43.044x-
0.6438, correlation coefficient r2=0.997, oleanolic acid has good linear relationship in 4～32mg/l content range.
5) extraction of oleanolic acid and assay
With reference to the method for Tan Chaoyang [80] etc., concrete grammar is as follows for the extraction of oleanolic acid: precision weighs 0.5g cell and does
Sample, adds 25ml hydrochloric acid-ethanol solution (2: 8), is heated to reflux 3h, lets cool, shake up and filter, precision measures subsequent filtrate 15ml,
Plus distilled water 15ml, it is placed in and boils off ethanol in 80 DEG C of water-baths, then extracted 3 times with ether, each 20ml, merge ether extraction liquid
And be evaporated in 40 DEG C of low temperature, plus 1ml methanol dissolved residue, and used 0.45 μm of organic filter membrane filtration, this is sample detection liquid,
Then detected using high performance liquid chromatography.
High performance liquid chromatography (hplc) testing conditions: with waters company 600-717-2487 chromatographic system, chromatographic column hiq
sil c18v4.6mm×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: accurate draw the oleanolic acid standard solution 0.2 that concentration is 0.5mg/ml,
0.5th, 1,2,3,4,5ml, is respectively placed in 5ml volumetric flask, plus 95% ethanol dilution, to scale, shakes up, the accurate 20 μ l that draw enter
Sample, measures its integrating peak areas value.With concentration as abscissa, integrating peak areas value is vertical coordinate, draws standard curve.Olive
Sour regression equation is: y=107x+33446, r2=0.9992.Result shows, oleanolic acid has in 0.2-5.0 μ g range
Good linear relationship.
The effect of the present invention: the present invention promotes ability, the Betula platyphylla Suk. total triterpene of acquisition and the Qi Dun that cell produces using snp
Tartaric acid content greatly improves.By technique scheme, tested with Betula platyphylla Suk. flower pesticide suspension cell for material, its result is: snp
After process, in Betula platyphylla Suk. flower pesticide suspension cell, content of oleanolic acid is 1.3089mg/gdw, is the 569% of matched group.Intracellular total
Triterpene content reaches 23.86mg/gdw, is the 143.20% of matched group.Therefore, this is that a kind of have very much the white of prospects for commercial application
Birch total triterpene and the production method of oleanolic acid.
Claims (1)
1. a kind of method improving total triterpene and content of oleanolic acid in Betula platyphylla Suk. cell using snp (sodium nitroprusside): with Betula platyphylla Suk. flower pesticide
Suspension cell is experiment material, is inoculated in 100ml fluid medium by 50g/l inoculum concentration, and this liquid culture based component is b5
Minimal medium adds 0.1～0.3mg/l 6-ba, 0.5～1.0mg/l tdz, sucrose 20g/l and acid hydrolyzed casein 1g/l,
Ph5.5～6.0, use after 121 DEG C of autoclave sterilization 20min, condition of culture is: 120rpm, 25 DEG C, intensity of illumination
2000lx, photoperiod 16h, humidity 40%～50%, add within the 8th day the donor snp (sodium nitroprusside) of no in cell suspension cultures, make
Its final concentration of 1mmol/l, culture 12h or 48h harvests, and harvests, intracellular content of oleanolic acid reaches after culture 12h
1.31mg/gdw, is the 569.00% of matched group；Culture 48h harvests, and intracellular total triterpene contentses reach 23.86mg/gdw, are
The 143.20% of matched group.
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| CN201410260611.XA CN104031970B (en) | 2013-12-22 | 2014-06-12 | Method for increasing content of total triterpenes and oleanolic acid in white birch cells by use of SNP (sodium nitroprusside) |
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| CN201310711853 | 2013-12-22 | ||
| CN201310711853.1 | 2013-12-22 | ||
| CN2013107118531 | 2013-12-22 | ||
| CN201410260611.XA CN104031970B (en) | 2013-12-22 | 2014-06-12 | Method for increasing content of total triterpenes and oleanolic acid in white birch cells by use of SNP (sodium nitroprusside) |
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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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| 任春林.诱导子组合对白桦三萜合成调控及MeJA抑制性消减文库分析.《中国优秀硕士学位论文全文数据库》.2013,第10页2.1.2和2.1.3，第11页2.1.4.3以及摘要部分. * |
| 肖强等.外源NO供体硝普钠（SNP）对盐胁迫下水稻幼苗中叶绿素和游离脯氨酸含量以及抗氧化酶的影响.《作物学报》.2008,第34卷(第10期),第1849-1853页. * |
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