CN104087644A - Method for utilizing hydrogen to improve content of total triterpenes and betulin in betula platyphylla cells - Google Patents
Method for utilizing hydrogen to improve content of total triterpenes and betulin in betula platyphylla cells Download PDFInfo
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Abstract
The invention belongs to the field of biological engineering, and concretely relates to a method for utilizing hydrogen to improve the content of total triterpenes and betulin in betula platyphylla cells. The method comprises: taking betula platyphylla suspension cells as a material, inoculating to 100 mL of B5 liquid culture medium according to an inoculation amount of 50 g/L, additionally adding 0.1-0.3 mg/L of 6-BA and 0.5-4.0 mg/L of TDZ, 20 g/L of cane sugar and 1 g/L of acid-hydrolyzed casein, and controlling pH to be 5.5-6.0; introducing hydrogen to enable the final concentration to be 0.5-1.5 mmol/L on the eighth day of cell suspension culture; and culturing for 12 h and harvesting, so as to obtain betulin with the highest content of 1.11 mg/gDW which is 185.1% of that of a control group, or culturing for 24 h and harvesting, so as to obtain intracellular total triterpenes with the content of 24.37 mg/gDW which is 181.32% of that the a control group. Hydrogen has the advantages of nontoxicity and innocuousness, and is convenient for separation and purification of a metabolite. The method provides a new approach for solving the shortage problem of natural plant medicine sources and industrialized large-scale production of betula platyphylla triterpenes and betulin medicines.
Description
Technical field:
The present invention relates to hydrogen and promote the synthetic of secondary metabolite, it belongs to technical field of bioengineering.
Background technology:
Hydrogen is that nature extensively exists and the simplest element of molecular structure, and in universe, 90% composition is made up of hydrogen.Hydrogen is diatomic gas colourless, odorless, tasteless, that have certain reductibility.In submarine medicine field, hydrogen is widely used in high deep hydrogen-oxygen mixing diving process.The solubleness of hydrogen is lower, and can not be absorbed in a large number by body, so people never pay attention to the effect of hydrogen in higher organism body.The reductibility of hydrogen is the most important chemical property of hydrogen, and we just learnt in middle school, and it is copper that hydrogen can reduce heated oxide copper.But many people also do not know, hydrogen also has unique biological property, and past field of biology thinks that hydrogen belongs to physiological rare gas element always, thinks that hydrogen is the gas without any biological effect.But from internationally famous magazine " Nature Medicine " report in 2007, hydrogen had magical selective antioxidation, and it is 2% hydrogen that patient only need to breathe 35min concentration, just can very effectively treat cerebral ischemia re-pouring injured.Breathe hydrogen and also can treat newborn baby Following Cerebral Hypoxia-ischemia disease.Subsequently, hydrogen is becoming new study hotspot aspect phytology, preclinical medicine and clinical Study on Transformation.Think that the huge applications potentiality of hydrogen are not also really familiar with.
Up to the present, approach 500 sections about the biomedical research paper of hydrogen in the world, within average 2 days, just increased by 1 section.Because hydrogen is in the field that does not medically have to use for reference, as a kind of therapeutic gas, efficient manner is by drinking hydrogen saturation water at present, and clinical application can be by breathing hydrogen and oxygen mixed gas and injection hydrogen saturated solution.
Experiment shows hydrogen water, H
2suck, and the disease that intraperitoneal, the saturated brine of intravenous injection hydrogen cause active oxygen all has provide protection.The evidence accumulating in these researchs shows, H
2can protect various cells, tissue and organ are avoided oxidative damage.H in bacterium and algae
2metabolism carried out many investigation.Some early stage researchs show H in some higher plants
2develop and absorb the hydrogenase of luminous leaf and hypothesis existence.But, in higher plant, seldom there are the physiological action of research examination hydrogen manufacturing and the mechanism of this process of support.
In recent years, the research that utilizes Vitro Plant culture technique to produce useful secondary metabolites makes great progress, as paniculatum cell is produced Shikonin, ginseng-cell is cultivated production saponin, purple foxglove cell cultures production alkaloid etc. and all reached industrialized industrial scale, cultivate production anti-cancer alkaloid with Catharanthus Roseus Cell and reached pilot scale level (Liu Chunchao etc., 1997; Hu Liyong, 2004).But the low yield phenomenon of secondary metabolite is one of key problem of restriction cell cultures natural plant product technology industrialization application, understanding and grasping vegetable cell Secondary Metabolic Regulation of Callus rule is the basis addressing this problem.Although, investigator is studied for secondary metabolite low yield problem in plant cell culture both at home and abroad, optimization, the culture technique that comprises seed selection, the culture condition of high-quality clone improved and (Guo Xiaohong etc., 2005 such as the clone of the synthetic key gene of active substance; Xu Maojun, 2009; Qian Dandan etc., 2011).But up to the present, in vegetable cell, the low yield problem of secondary metabolite is well solved not yet.
In white birch (Betula platyphylla suk) leaf and bark, contain important secondary metabolite---white birch triterpene (TBP), main Types comprises lupinane type, dammarane type, oleanane type triterpene material (Zhang, 2003), be great exploitation potential for its anticancer, Cardiovarscular, protect pioneer's medicine (Li Wei, 2000 of liver; Ye Yinying, 2000,2001).Compared with nascent metabolism, Secondary Metabolism of Plant has very strong Modulatory character.For the low yield problem of natural active matter in plant cell culture, investigator has carried out a large amount of research and probes both at home and abroad.Mechanism synthetic relevant with secondary metabolite in research and inquirement vegetable cell not only contributes to grasp the regulation rule of Secondary Metabolism of Plant but also significant to solving plant cell culture secondary metabolite low yield problem in production practice.For the effect that can promote that white birch triterpene is synthetic, study the synthetic molecular mechanism of triterpene in its regulation and control white birch suspension culturing cell, emphasis is inquired into H
2induction on the synthetic impact of triterpene etc., is set up the synthetic technical system of hydrogen regulation and control white birch triterpene, for the problem that solves low yield in Production of Secondary Metabolites By Plant Cell Cultures provides reliable and stable technology.
Content of the present invention:
For protection white birch natural resources; set up the resource production sequence of Sustainable Development and Utilization; utilize callus and the suspended culture cell of nontoxic hydrogen induction from white birch induction; carry out the synthetic of total triterpene and trochol and produce, utilize high performance liquid chromatography to carry out detection by quantitative simultaneously.For the novel method that provides of the total triterpene of industrialization method scale operation and trochol class medicine is provided.
Embodiment:
(1) cultivation of suspension cell
On Bechtop, cut axillalry bud, with 70% alcohol-pickled 5min, again with 5% the chlorine bleach liquor 10-20min that sterilizes, then be put in sterilized culture dish, be inoculated into (NT+1.0mg/L caseinhydrolysate+3% sucrose+0.5～4.0mg/LBA, 0.2～4.0mg/L NAA) on the substratum of the various combinations of passing through in advance autoclave sterilization and carry out the induction of callus.The about 3-5 of an every bottle graft kind axillalry bud.First after one week, carry out light cultivation with dark cultivation.Cultivate after approximately 7 weeks, obtain callus.Callus, after succeeding transfer culture, is bred rapidly.
Select high cell growth speed, the high callus of loose and total triterpenoid content, carries out subculture and suspension culture.And culturing cell is in the B5+0.1～0.3mg/L6-BA+0.5～4.0mg/LTDZ suspension medium that is conducive to white birch suspension Growth of Cells and secondary metabolite accumulation.Obtain high yield white birch triterpene and trochol suspension cell line, after filtration, dry, pulverize, organic solvent (95% ethanol) extraction step, utilizes liquid-phase chromatography method to detect and quantitative analysis betulin and trochol.
(2) abstraction and quantification of betulin
The abstraction and quantification employing model cassia twig of betulin etc. 2007, the method for Zhang Ze etc. 2004.Extract reagent: 95% ethanol; Extracting method: ultrasonic extraction; Measuring method: high performance liquid chromatography, detect wavelength 210nm, moving phase is acetonitrile and water (V:V=8:2), flow velocity 1.0mL/min.
This patent has following characteristics:
1) hydrogen treat has nontoxic advantage, and production environment is not subject to the impact of the physical environments such as region, season, water quality, disease and pest, weather simultaneously.
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) ability that the white birch suspension cell of cultivating has many kinds of substances such as producing white birch triterpene and trochol, has realized the exploitation of white birch natural plant resource and medicinal ingredients thereof.
4) utilize efficient liquid-phase chromatography method to carry out trochol and detect, method is simple, extracts solvent cleaned nontoxic, easy to operate.
5) be that solution anti-AIDS and antitumor natural drug source are in short supply, accelerate clinical large-scale application, a kind of new way is provided, there is very high economic benefit and social benefit.
Embodiment
1) vegetable material source
White birch is taken from the select tree in Northeast Forestry University's white birch strengthening breeding garden.Select axillalry bud, after sterilization, be inoculated into (NT+1.0mg/L caseinhydrolysate+3% sucrose+0.5～4.0mg/LBA, 0.2～4.0mg/L NAA) on the substratum of the various combinations of passing through in advance autoclave sterilization and carry out the induction of callus.Callus, after succeeding transfer culture, is bred rapidly.Select the callus that growth conditions is good it is repeatedly transferred to liquid nutrient medium suspension culture after subculture.Through switchings more than 6 generations, set up stable suspended culture cell system, suspension culture subculture cycle is 15d, inoculum size is that 5g cell is in 100mL liquid nutrient medium.2) culture condition
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 0.0.1-0.3mg/L6-BA and 0.5-4mg/L TDZ, sucrose 20g/L, acid hydrolyzed casein 1g/L, pH5.5～6.0,121 DEG C of autoclave sterilization 20min.
3) preparation of rich hydrogen water is added
The hydrogen delivery port of SHC type hydrogen generator (purchased from Shandong Sai Kesaisi hydrogen energy source company limited) is inserted to half hour in sterile purified water, distilled water become 99.99% rich hydrogen water, in the time of white birch suspension cell cultures to the 8 days, be added in substratum, final concentration is respectively 0.5-1.5mmol/L, 12 to 24h harvested cells after adding.
4) extraction and the assay of total triterpene
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=45.036x+0.0417,, coefficient R 2=0.9983, Oleanolic Acid has good linear relationship in 0.005～0.025mg/mL content range.
5) extraction of trochol and assay
The extraction of trochol is with reference to model cassia twig etc. 2007, the method method of Zhang Ze etc. 2004, concrete grammar 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 subsequent filtrate 15mL, adding distil water 15mL, be placed in 80 DEG C of water-baths and boil off ethanol, then use extracted with diethyl ether 3 times, each 20mL, merge ether extraction liquid in 40 DEG C of low temperature evaporates to dryness, add 1ml dissolve with methanol residue, and used the organic filter membrane of 0.45 μ m to filter, this is sample detection liquid, then utilize 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.
Trochol linear relationship is investigated: accurate trochol standard solution that concentration is 0.5mg/mL 0.2,0.4,0.6,0.8, the 1mL 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.Trochol regression equation is: Y=3E+06x-128674.Result shows, trochol has good linear relationship within the scope of 0.2～1mg/ml.
Effect of the present invention: the present invention utilizes hydrogen to promote the ability of cells produce triterpene, and the total triterpene of white birch and the trochol content of acquisition improve greatly.By technique scheme, test taking white birch suspension cell as material, consequently: after hydrogen treat, trochol content is up to 1.11mg/gDW, be control group 185.1%.After cultivating 12-24h, gather in the crops, in cell, total triterpene contents reaches 24.37mg/gDW, is 181.32% of control group.Therefore, this is a kind of have the very much total triterpene of white birch of prospects for commercial application and production method of trochol.
Accompanying drawing 1: white birch solid callus
Accompanying drawing 2: white birch suspension cell
Accompanying drawing 3: trochol standard substance go out peak figure
Accompanying drawing 4:H
2process trochol and go out peak figure
Accompanying drawing 5: the typical curve (taking Oleanolic Acid as standard substance) that total triterpene contents is measured
Accompanying drawing 6: the typical curve of trochol assay.
Claims (6)
1. patent proposition hydrogen of the present invention promotes white birch triterpene synthetic technology to be: 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 0.1～0.3mg/L6-BA and 0.5～4.0mg/L TDZ, sucrose 20g/L, acid hydrolyzed casein 1g/L, pH5.5～6.0,121 DEG C of autoclave sterilization 20min.Within the 8th day, adding rich hydrogen water to make its final concentration in cell suspension culture is 0.5-1.5mmol/L, after cultivating 12 to 24h, gather in the crops, trochol content is up to 1.11mg/gDW, is control group 185.1%, in cell, total triterpene contents reaches 24.37mg/gDW, is 181.32% of control group.
2. according to the method described in claims 1, liquid nutrient medium is wherein for being B5 minimum medium, and additional hormone and concentration are 0.1～0.3mg/L6-BA and 0.5～4.0mg/L TDZ, sucrose 20g/L, acid hydrolyzed casein 1g/L.
3. according to the method described in claims 1, white birch cell inoculum size is wherein that 5g fresh cell contains in the 100ml triangular flask of 50ml nutrient solution, shaking speed 120r/min.
4. according to the method described in claims 1, wherein rich hydrogen water obtains by the preparation of SHC type hydrogen generator, within the 8th day, adds in suspension culture, and culture cycle is 10 days, and the final concentration of rich hydrogen water is 0.5-1.5mmol/L.
5. according to the method described in claims 1, harvest time is wherein to gather in the crops suspension cell after rich hydrogen water adds 12-24h.
6. according to the method described in claims 1, wherein after hydrogen treat, trochol content is up to 1.11mg/gDW, is control group 185.1%.In cell, total triterpene contents reaches 24.37mg/gDW, is 181.32% of control group.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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) |
| 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 |
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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 |
| 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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| JIQING ZENG ET AL.: "Molecular Hydrogen Is Involved in Phytohormone Signaling and Stress Responses in Plants", 《PLOS ONE》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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