CN1055120C - Method for improving content and release of yew alcohol in Taxus chinensis cell - Google Patents
Method for improving content and release of yew alcohol in Taxus chinensis cell Download PDFInfo
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- CN1055120C CN1055120C CN96120776A CN96120776A CN1055120C CN 1055120 C CN1055120 C CN 1055120C CN 96120776 A CN96120776 A CN 96120776A CN 96120776 A CN96120776 A CN 96120776A CN 1055120 C CN1055120 C CN 1055120C
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- Prior art keywords
- taxol
- cell
- rare earth
- elicitor
- earth compound
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- 238000000034 method Methods 0.000 title claims abstract description 12
- 241001116500 Taxus Species 0.000 title description 11
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 title description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title 1
- 229930012538 Paclitaxel Natural products 0.000 claims abstract description 44
- 229960001592 paclitaxel Drugs 0.000 claims abstract description 44
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims abstract description 44
- -1 rare earth compound Chemical class 0.000 claims abstract description 17
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 17
- 239000005712 elicitor Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 230000010261 cell growth Effects 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 230000007423 decrease Effects 0.000 claims description 2
- 229910000333 cerium(III) sulfate Inorganic materials 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 abstract description 2
- 230000001939 inductive effect Effects 0.000 abstract 2
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 28
- 239000000243 solution Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- PGJHGXFYDZHMAV-UHFFFAOYSA-K azanium;cerium(3+);disulfate Chemical compound [NH4+].[Ce+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O PGJHGXFYDZHMAV-UHFFFAOYSA-K 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 244000050510 Cunninghamia lanceolata Species 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 241001116498 Taxus baccata Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- HSJPMRKMPBAUAU-UHFFFAOYSA-N cerium(3+);trinitrate Chemical compound [Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O HSJPMRKMPBAUAU-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- GJKFIJKSBFYMQK-UHFFFAOYSA-N lanthanum(3+);trinitrate;hexahydrate Chemical compound O.O.O.O.O.O.[La+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O GJKFIJKSBFYMQK-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention discloses a method for enhancing the content and release of taxol in yew cells. Yew cells are inoculated in a B5 culture medium. When the cell production exists in a log phase stage, rare earth compound inducing elements are added, the taxol content in the cells can be enhanced and can reach 2.5 to 6 times, and the release quantity can reach 1.2 to 5 times. The rare earth compound inducing elements adopted by the present invention have easy raw material obtainment and low price. The operation running process is reliable, and the three waste pollution can not be generated.
Description
The present invention relates to content of taxol and method for releasing in a kind of raising yew cell.It belongs to plant cell culture technology.
Taxol has good antitumous effect, especially to advanced ovarian cancer, metastatic breast cancer and nonsmall-cell lung cancer all have significant curative effect, for this reason, development research for it is subject to people's attention, and the huge fund that has dropped into 100,015,000 dollars as U.S. BMS company is used for the exploitation of taxol and the research of application.
Taxol is by separating in the stem of yewtree, the skin the earliest.A kind of secondary metabolite that yew cell produces is taxol.This product is few at the cell intensive amount, causes industrial production cost very expensive.Therefore, the content that how to improve taxol in the yew cell then becomes the problem that people extremely pay close attention to, and also is a problem that difficulty is bigger simultaneously.One of effective means is to add elicitor in the cytotropic nutrient solution at present.The elicitor that uses has two classes: a class is biotic induce, as the extract of the fungi of high pressure deactivation, bacterium, yeast cells wall etc.This type of elicitor is difficult to quantitative control because they self extraction and purification technology is all very complicated in addition in the industrial production, it is big therefore to use difficulty.Another kind of is the heavy metallic salt elicitor, as vanadate, lead, mercury etc.This type of elicitor is because pair cell has big toxicity and injury effect, and easy again contaminate environment is so should not adopt.
Purpose of the present invention is to provide a kind of content of taxol in the yew cell and method of release of improving.This method not only can obviously improve the content of taxol in the yew cell, and helps taxol and discharge from cell.
In order to achieve the above object, the present invention is realized by following technical proposals.Yew cell is inoculated in B
5In the liquid nutrient medium, adopt shake flask fermentation, with 120～130rpm and temperature culturing cell under 24～26 ℃ and dark place condition, can obviously present lag period, logarithmic phase, the stage of stable development, decline phase four-stage the vegetative period of cell at shaking table.For improving the content and the release of taxol in the cell, to B
5Add elicitor in the liquid nutrient medium, its technical characterictic is: when the cell growth is in logarithmic phase, to B
5Add ammonium cerous sulfate rare earth compound elicitor in the nutrient solution, perhaps add lanthanum nitrate rare earth compound elicitor, perhaps add cerous nitrate rare earth compound elicitor.The amount of above-mentioned added rare earth compound is 0.01～0.4mg/ml
Fermented liquidThe time best results.
Below the present invention is described in detail.At first prepare B
5Liquid nutrient medium and rare earth compound solution.Inoculate yew cell in prepared culture medium, the growth conditions of culturing cell is: adopt and shake bottle, shaking speed is 120～130rpm, and temperature keeps 24～26 ℃, and the place is dark place.The process of growth of observation of cell, when cell growth is in logarithmic phase, with prepare and through the rare earth compound solution of sterilising treatment according to 0.01～0.4mg/ml
Fermented liquidRatio add in the nutrient solution, continue the culturing cell growth then and reach 10～15 days, behind the cell maturation, fermented liquid is carried out centrifugation, processing such as supernatant liquor that separation is obtained and throw out extract again obtain containing the medicinal extract of taxol, adopt the content of high pressure (efficiently) liquid chromatography test taxol, and the inside and outside content of cell how much as can be known, simultaneously also can pass through the contrast experiment, find out effect of the present invention.
Effect of the present invention is very remarkable and heartening.Experimentize with taxus chinensis in northeast, consequently: the content of taxol in the yew cell, the content that adds the rare earth compound elicitor is 2.5～6 times of content when not adding rare earth compound, reach as high as 7 times, the burst size of taxol, the burst size that adds rare earth compound are 1.2～5.0 times of burst size when not adding rare earth compound.The present invention be advantageous in that the rare earth compound that is adopted, raw material is easy to get inexpensive, operates reliablely, and the process three wastes are free from environmental pollution.
The invention will be further described with embodiment below.
Example one:
1. compound concentration is the aseptic ammonium cerous sulfate solution of 10mg/ml, and is standby.
2. the bottle that shakes at 250ml claims dress 30mlB
5Liquid nutrient medium.
3. in inoculation of medium 20ml concentration the taxus chinensis in northeast cell seed liquor of 50g (fresh weight)/l logarithmic phase.
4. add the aseptic ammonium cerous sulfate solution of 1ml, and at 25 ℃, shaking table 120～135rpm, stuck fermentation after 14 days is cultivated at dark place.
5. with 4000rpm whizzer isolated cell (throw out) and supernatant liquor.
6. extract the taxol in cell and the supernatant liquor respectively.
7. adopt the content of external standard method taxol with high performance liquid chromatography, its result as shown in Table 1.
Table one
| Taxol mg/g (dry weight) in the born of the same parents | The outer taxol mg/l of born of the same parents | Dry cell weight g/l | Taxol total amount mg/l in one liter | The outer taxol of born of the same parents/total taxol | |
| Control group | 0.036 | 0.069 | 12.01 | 0.489 | 12％ |
| Experimental group | 0.251 | 1.509 | 5.42 | 2.867 | 53％ |
By table one data as can be known: content of taxol improves 5.9 times, and burst size improves 4.4 times.
Example two:
1. compound concentration is the aseptic ammonium cerous sulfate solution of 0.5mg/ml, 10mg/ml, 20mg/ml respectively, and is standby.
The 2nd～7 step is identical with example one, shown in the results list two.
Table two
| Ultimate density in the fermented liquid | 0.01mg/ml | 0.2mg/ml | 0.4mg/ml |
| Dry cell weight mg/l | 14.12 | 5.42 | 3.86 |
| Total purple pure China fir mg/l | 0.629 | 2.867 | 0.872 |
| The outer taxol of born of the same parents/total taxol | 16％ | 53％ | 31％ |
Example three:
1. compound concentration is the aseptic lanthanum nitrate hexahydrate of 10mg/ml, and is standby.
The 2nd～7 step is with example one.Its result is with shown in the table three.
Table three
| Taxol mg/g (dry weight) in the born of the same parents | The outer taxol mg/l of born of the same parents | Dry cell weight g/l | Total purple pure China fir mg/l | The outer taxol of born of the same parents/total taxol | |
| Control group | 0.036 | 0.059 | 12.01 | 0.489 | 12％ |
| Experimental group | 0.231 | 0.490 | 4.12 | 1.438 | 34％ |
Improve 2.9 times by table three visible cell content of taxol, the taxol burst size improves 2.8 times.
Example four:
1. compound concentration is the cerous nitrate sterile solution of 10mg/ml, and is standby.
The 2nd～7 step is identical with example one.Its result is with shown in the table four.
Table four
| Taxol mg/g (dry weight) in the born of the same parents | The outer taxol mg/l of born of the same parents | Dry cell weight g/l | Total purple pure China fir mg/l | The outer taxol of born of the same parents/total taxol | |
| Control group | 0.036 | 0.059 | 12.01 | 0.489 | 12％ |
| Experimental group | 0.352 | 0.629 | 4.04 | 2.044 | 31％ |
By table four as seen, the content of taxol improves 4.2 times in the yew cell, and the taxol burst size improves 2.6 times.
Example five:
The 1st～2 step is with example one.
The 3rd step added 1ml in lag period, logarithmic phase, the stage of stable development of cell growth respectively in substratum, concentration is the aseptic ammonium cerous sulfate solution of 10mg/ml.
The 4th～7 step is with example one, and its result is with shown in the table five.
Table five
| Lag period | Logarithmic phase | The stage of stable development | |
| Taxol mg/g in the born of the same parents | 0.08 | 0.06 | 0.03 |
| The outer taxol mg/l of born of the same parents | 1.02 | 1.79 | 0.87 |
| Dry cell weight g/l | 4.02 | 5.87 | 6.41 |
| Total taxol mg/l | 1.34 | 2.14 | 1.06 |
Claims (2)
1. a method that improves content of taxol and release in the yew cell is that yew cell is inoculated in B
5In the liquid nutrient medium, adopt shake flask fermentation, with 120～130rpm and temperature culturing cell under the condition at 24 ℃～26 ℃ and dark place, the process of growth of cell is lag period, logarithmic phase, the stage of stable development, decline phase four-stage at shaking table, and to B
5Add elicitor in the liquid nutrient medium, it is characterized in that: when the cell growth is in logarithmic phase, to B
5Add cerous sulfate rare earth compound elicitor in the nutrient solution, perhaps add lanthanum nitrate rare earth compound elicitor, perhaps add cerous nitrate rare earth compound elicitor.
2. by the method for content of taxol in the described raising yew cell of claim 1 and release, it is characterized in that: the amount of the rare earth compound elicitor that is added is 0.01～0.4mg/ml
Fermented liquid
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96120776A CN1055120C (en) | 1996-11-27 | 1996-11-27 | Method for improving content and release of yew alcohol in Taxus chinensis cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96120776A CN1055120C (en) | 1996-11-27 | 1996-11-27 | Method for improving content and release of yew alcohol in Taxus chinensis cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1158356A CN1158356A (en) | 1997-09-03 |
| CN1055120C true CN1055120C (en) | 2000-08-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96120776A Expired - Fee Related CN1055120C (en) | 1996-11-27 | 1996-11-27 | Method for improving content and release of yew alcohol in Taxus chinensis cell |
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| Country | Link |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304561C (en) * | 2004-11-13 | 2007-03-14 | 西南师范大学 | Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material |
| CN113528601B (en) * | 2020-04-13 | 2023-08-04 | 菲鹏生物股份有限公司 | Cell culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1079000A (en) * | 1992-04-01 | 1993-12-01 | 友联坎普公司 | What the taxus somatic embryo took place induces, and produces the alkaloid that contains taxane-ring by it |
| CN1096820A (en) * | 1993-06-25 | 1994-12-28 | 华中理工大学 | The method of producing taxol by means of cell culture |
| CN1111911A (en) * | 1993-07-06 | 1995-11-15 | 大韩民国山林厅林木育种研究所 | A method for producing taxol and taxanes from embryo cultures of taxus species |
| CN1132792A (en) * | 1994-11-25 | 1996-10-09 | 三井石油化学工业株式会社 | Method of producing taxane-type diterpene |
-
1996
- 1996-11-27 CN CN96120776A patent/CN1055120C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1079000A (en) * | 1992-04-01 | 1993-12-01 | 友联坎普公司 | What the taxus somatic embryo took place induces, and produces the alkaloid that contains taxane-ring by it |
| CN1096820A (en) * | 1993-06-25 | 1994-12-28 | 华中理工大学 | The method of producing taxol by means of cell culture |
| CN1111911A (en) * | 1993-07-06 | 1995-11-15 | 大韩民国山林厅林木育种研究所 | A method for producing taxol and taxanes from embryo cultures of taxus species |
| CN1132792A (en) * | 1994-11-25 | 1996-10-09 | 三井石油化学工业株式会社 | Method of producing taxane-type diterpene |
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| Publication number | Publication date |
|---|---|
| CN1158356A (en) | 1997-09-03 |
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