CN1158356A

CN1158356A - Method for improving content and release of yew alcohol in Taxus chinensis cell

Info

Publication number: CN1158356A
Application number: CN 96120776
Authority: CN
Inventors: 元英进; 胡国武; 那平; 王传贵; 周永洽; 申泮文
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 1996-11-27
Filing date: 1996-11-27
Publication date: 1997-09-03
Anticipated expiration: 2016-11-27
Also published as: CN1055120C

Abstract

A process for increasing taxusol content in enqlish yew cells and the taxusol amount released from them features that the enqlish yew cells are inculated into B5 culture medium and inductor of rare-earth compound is added to at logarithnic phase of cell production. The taxusol content in the cess is increased by 2.5-6 times and the taxusol amount released from the cell by 1.2-5 times. Said inductor of rare-earth compound features easily available raw material, reliable operation and no environmental pollution.

Description

The method of content of taxol and release in the raising yew cell

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, and 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, and 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 liquid}The 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 the cell growth is in logarithmic phase, with prepare and add in the nutrient solution through the rare earth compound solution of sterilising treatment ratio according to 0.01～0.4mg/ml fermented liquid, continue the culturing cell growth then and reach 10～15 days, behind the cell maturation, fermented liquid is carried out centrifugation, supernatant liquor that separation is obtained and throw out such as extract again at the medicinal extract that processing obtains containing taxol, adopt the content of high pressure (efficiently) liquid chromatography test taxol, content inside and outside the 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)/1 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
	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％	Control group	???0.036	???0.069	???12.01	??0.489	????12％

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
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	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
	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％	Control group	????0.036	????0.059	??12.01	???0.489	????12％

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
	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％	Control group	????0.036	????0.059	???12.01	???0.489	????12％

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
	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	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	Dry cell weight g/l	????4.02	????5.87	????6.41

Claims

1. the method for content of taxol and release in the sharp raising 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, 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.

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 added is 0.01～0.4mg/ml _{Fermented liquid}The time best results.