CN101629202A - Method for improving content of phenethyl alcohol glucoside in cistanche salsa cultured cell - Google Patents
Method for improving content of phenethyl alcohol glucoside in cistanche salsa cultured cell Download PDFInfo
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- CN101629202A CN101629202A CN200910094853A CN200910094853A CN101629202A CN 101629202 A CN101629202 A CN 101629202A CN 200910094853 A CN200910094853 A CN 200910094853A CN 200910094853 A CN200910094853 A CN 200910094853A CN 101629202 A CN101629202 A CN 101629202A
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- culture
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- phenethyl alcohol
- cistanche salsa
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- MLRIJUWUQTVDQE-RKQHYHRCSA-N Phenylethyl beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=CC=C1 MLRIJUWUQTVDQE-RKQHYHRCSA-N 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 12
- 241000336315 Cistanche salsa Species 0.000 title claims abstract description 11
- 210000004748 cultured cell Anatomy 0.000 title claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 39
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 4
- 238000005286 illumination Methods 0.000 claims description 20
- 241000005787 Cistanche Species 0.000 claims description 15
- 230000000638 stimulation Effects 0.000 claims description 15
- 230000003203 everyday effect Effects 0.000 claims description 10
- 239000012879 subculture medium Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 229930000044 secondary metabolite Natural products 0.000 abstract description 4
- 238000004114 suspension culture Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 2
- 238000009630 liquid culture Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 108010079058 casein hydrolysate Proteins 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000002604 ultrasonography Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000036983 biotransformation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- -1 steroid glucoside Chemical class 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000336291 Cistanche deserticola Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000308150 Orobanchaceae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for improving the content of phenethyl alcohol glucoside in cistanche salsa cultured cells, which mainly comprises the following operation steps: carrying out the successive transfer culture of cistanche salsa cells; 2. carrying out the liquid culture and ultrasonic treatment of the cistanche salsa cells; and 3. measuring the content of the phenethyl alcohol glucoside. Using ultrasonic waves to stimulate the cistanche salsa cells in suspension culture is an effective means for improving the content of phenethyl alcohol glucoside in the cells. The method has simple operation, good effect and strong expansibility, can be applied to the mass culture process of a cistanche salsa cell bioreactor, and can improve the content of medicinal secondary metabolites of the cultured cells.
Description
Technical field
The present invention relates to a kind of method that improves the Yunnan content of phenethyl alcohol glucoside in cistanche salsa cultured cell with ultrasonic wave.
Background technology
Herba Cistanches (Cistanche deserticola Y.C.Ma) belongs to perennial high phytoparasite for the Orobanchaceae Herba Cistanches, little sad, the tepor of its sweet-salty, in " legendary god of farming hundred grass through ", classify as top gradely, have bowl spares, go into liver, effects such as benefit essence, nourshing kidney, establishing-Yang.Chemical ingredients in the Herba Cistanches is very complicated, kind surplus therefrom isolation identification has gone out 40 at present, main activeconstituents is phenethyl alcohol glucoside compounds (Phenylethanoid Glycosides, PeG), mainly comprise echinacosid, ergot steroid glucoside, acetyl ergot steroid glucoside and boschnialoside A, endocrine regulation is arranged, promote metabolism, remove free radical and the anti-ageing medicinal efficacy of waiting for a long time.Because long-term immoderate indiscriminate mining and serious waste, the wild resource of Herba Cistanches destroys serious, and imbalance between supply and demand highlights on the market.And utilize vegetable cell large scale culturing technology direct culturing cell in reactor to obtain medicinal secondary metabolite is to produce direct, the most effective means of plant amedica in the future.Carry out cell culture technology and produce medicinal secondary metabolite, at first will obtain a kind of method that can improve the effective pharmaceutical ingredient content of culturing cell fast,, shorten growth cycle, reduce production costs to guarantee production efficiency efficiently.
The application of ultrasonic wave in biology and medical diagnosis treatment had history decades, some results of study of forefathers show that low intensive ultrasonic stimulation can influence the growth of cell, the form of cytolemma and organoid is changed, and these variations can cause the change of cellular metabolism and membrane permeability.Existing many ultrasonic wave that studies show that can improve enzyme and the bio-transformation efficient of microorganism cells in reactor, but hyperacoustic in the bioprocesses to influence mechanism still not fully aware of, think that in most cases hyperacoustic mechanical stimulus strengthened mass transfer and mixing in the biotransformation, even improved the activity of enzyme, make the endocellular metabolism reaction more vigorous.Past, relevant ultrasonic wave Biological Effects concentrated on humans and animals cell and microorganism mostly, and the research that is applied to vegetable cell is less.
Summary of the invention
The purpose of this invention is to provide a kind of method that can effectively improve Herba Cistanches isolated culture cell content of phenethyl alcohol glucoside.
The operation steps of this method is as follows:
1) succeeding transfer culture of Herba Cistanches cell: subculture medium is: B5 basic medium+6-BA 0.5mg/L+GA
310mg/L+ caseinhydrolysate 800mg/L.Culture condition is a solid culture, pH value 5.6,25 ℃ of temperature, illumination every day 16h, intensity of illumination 3000Lux, culture cycle 30 days (purpose of Herba Cistanches cell succeeding transfer culture is in order to obtain the influence that enough cellular biomass are used to study the synthetic phenethyl alcohol glucoside ability of ultrasonic wave pair cell);
2) the Herba Cistanches cell supersonic wave is handled:
A. realize the ultrasound stimulation effect of pair cell with ultrasonic cleaner;
B. 15 days cell inoculation of succeeding transfer culture is cultivated in the triangular flask that 40mL liquid subculture medium is housed, culture temperature is 25 ℃, shaking speed 110r/min, and illumination every day 16h, intensity of illumination 3000Lux, cell culture period is 20d;
C. in 2-10 days of cell cultivation process, choose ultrasonic power 80-200W, ultrasonic treatment time 1-5min, cistanche salsa cultured cell is carried out ultrasonic stimulation;
3) continue behind the ultrasonic stimulation to cultivate, up to the culture cycle of finishing 18 days;
4) mensuration of content of phenethyl alcohol glucoside: the cell of healing of the Herba Cistanches after cultivate finishing is dried to constant weight under 60 ℃ condition, be ground into 40 orders then, and with methyl alcohol lixiviate 24h, filter the back concentrated extracting solution, dilute with water then, by macroporous adsorbent resin AB-8 post, successively water and methanol-eluted fractions are collected the content that methanol-eluted fractions partly is used to measure phenethyl alcohol glucoside.
Phenethyl alcohol glucoside has strong absorption peak at the 332nm place, but drawing standard curve thus obtains C=81.43X-0.69, R=0.9991, and wherein C is the phenethyl alcohol glucoside concentration (mg/L) in the methanol solution, and X is that this measures the solution absorbency value, and R is a relation conefficient.
S＝(CNV/W)×100％；P＝1000SW
Wherein, S is content of phenethyl alcohol glucoside (%), and P is phenethyl alcohol glucoside output (mg/L), and W is callus dry weight (g/L), and V is extraction liquid volume (L), and N is an extension rate.(this measuring method is a prior art)
The key point of content of the present invention is: 1. ultrasonic power chooses; 2. ultrasonic treatment time determines; 3. the ultrasonication moment chooses.Above key point if can be got hold of, and can significantly improve the content of cell phenethyl alcohol glucoside.
The present invention is easy and simple to handle, and is respond well, and extendability is strong, can be applicable to Herba Cistanches cell biological reactor large scale culturing process, improves the content of the medicinal secondary metabolite phenethyl alcohol glucoside of culturing cell compounds.
Embodiment
Following examples are used to illustrate the present invention, but are not limitations of the present invention.
Embodiment 1:
1) succeeding transfer culture of Herba Cistanches cell: subculture medium is: B5 basic medium+6-BA 0.5mg/L+GA
310mg/L+ caseinhydrolysate 800mg/L.Culture condition is a solid culture, pH value 5.6,25 ℃ of temperature, illumination every day 16h, intensity of illumination 3000Lux;
2) with 15 days cell inoculation of succeeding transfer culture suspension culture in the triangular flask that 40mL liquid subculture medium is housed, culture temperature is 25 ℃, shaking speed 110r/min, illumination every day 16h, intensity of illumination 3000Lux;
3) at the 2nd day that cultivates, it was that 80W, duration are the ultrasonic stimulation of 5min that suspended culture cell is applied power, and cell continues to cultivate, up to the culture cycle of finishing 18 days behind ultrasonic stimulation.
4) cultivating the content that finishes to measure the cell phenethyl alcohol glucoside in the back is 8.4%, has improved 18.3% than the control group without any ultrasound stimulation.
Embodiment 2:
1) succeeding transfer culture of Herba Cistanches cell: subculture medium is: B5 basic medium+6-BA 0.5mg/L+GA
310mg/L+ caseinhydrolysate 800mg/L.Culture condition is a solid culture, pH value 5.6,25 ℃ of temperature, illumination every day 16h, intensity of illumination 3000Lux;
2) with 15 days cell inoculation of succeeding transfer culture suspension culture in the triangular flask that 40mL liquid subculture medium is housed, culture temperature is 25 ℃, shaking speed 110r/min, illumination every day 16h, intensity of illumination 3000Lux;
3) at the 6th day that cultivates, it was that 140W, duration are the ultrasonic stimulation of 3min that suspended culture cell is applied power, and cell continues to cultivate, up to the culture cycle of finishing 18 days behind ultrasonic stimulation.
4) cultivating the content that finishes to measure the cell phenethyl alcohol glucoside in the back is 9.5%, has improved 33.8% than the control group without any ultrasound stimulation.
Embodiment 3:
1) succeeding transfer culture of Herba Cistanches cell: subculture medium is: B5 basic medium+6-BA 0.5mg/L+GA
310mg/L+ caseinhydrolysate 800mg/L.Culture condition is a solid culture, pH value 5.6,25 ℃ of temperature, illumination every day 16h, intensity of illumination 3000Lux;
2) with 15 days cell inoculation of succeeding transfer culture suspension culture in the triangular flask that 40mL liquid subculture medium is housed, culture temperature is 25 ℃, shaking speed 110r/min, illumination every day 16h, intensity of illumination 3000Lux;
3) at the 10th day that cultivates, it was that 200W, duration are the ultrasonic stimulation of 1min that suspended culture cell is applied power, and cell continues to cultivate, up to the culture cycle of finishing 18 days behind ultrasonic stimulation.
4) cultivating the content that finishes to measure the cell phenethyl alcohol glucoside in the back is 9.3%, has improved 31.0% than the control group without any ultrasound stimulation.
Claims (1)
1, a kind of method that improves content of phenethyl alcohol glucoside in cistanche salsa cultured cell is characterized in that carrying out according to the following steps:
1) succeeding transfer culture of Herba Cistanches cell: use subculture medium, culture condition is a solid culture, pH value 5.6,25 ℃ of temperature, illumination every day 16h, intensity of illumination 3000Lux;
2) 15 days Herba Cistanches cell inoculation of succeeding transfer culture is cultivated in the triangular flask that 40mL liquid subculture medium is housed, culture temperature is 25 ℃, shaking speed 110r/min, and illumination every day 16h, intensity of illumination 3000Lux;
3) in 2-10 days of cell cultivation process, choose ultrasonic power 80-200W, ultrasonic treatment time 1-5min, cistanche salsa cultured cell is carried out ultrasonic stimulation;
4) continue behind the ultrasonic stimulation to cultivate, up to the culture cycle of finishing 18 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910094853A CN101629202A (en) | 2009-08-20 | 2009-08-20 | Method for improving content of phenethyl alcohol glucoside in cistanche salsa cultured cell |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910094853A CN101629202A (en) | 2009-08-20 | 2009-08-20 | Method for improving content of phenethyl alcohol glucoside in cistanche salsa cultured cell |
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| CN101629202A true CN101629202A (en) | 2010-01-20 |
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| CN200910094853A Pending CN101629202A (en) | 2009-08-20 | 2009-08-20 | Method for improving content of phenethyl alcohol glucoside in cistanche salsa cultured cell |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113832167A (en) * | 2021-11-01 | 2021-12-24 | 昆明理工大学 | Gene and application thereof in improving yield of phenethyl alcohol and tryptophol |
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2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113832167A (en) * | 2021-11-01 | 2021-12-24 | 昆明理工大学 | Gene and application thereof in improving yield of phenethyl alcohol and tryptophol |
| CN113832167B (en) * | 2021-11-01 | 2023-04-21 | 昆明理工大学 | Gene and application thereof in increasing yield of phenethyl alcohol and tryptophane |
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Open date: 20100120 |