CN102127582A - Method for promoting biosynthesis of 10-hydroxycamptothecine - Google Patents
Method for promoting biosynthesis of 10-hydroxycamptothecine Download PDFInfo
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- CN102127582A CN102127582A CN2010100101201A CN201010010120A CN102127582A CN 102127582 A CN102127582 A CN 102127582A CN 2010100101201 A CN2010100101201 A CN 2010100101201A CN 201010010120 A CN201010010120 A CN 201010010120A CN 102127582 A CN102127582 A CN 102127582A
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Abstract
The invention belongs to technical filed of biology, in particular to a method for promoting biosynthesis of 10-hydroxycamptothecine. The method comprises the following steps of: constructing a suspension culture system of camptotheca acuminata cells for an explant by using leaves of camptotheca acuminata plants; adding three kinds of exogenous substances such as cobalt nitrate, lanthanum nitrate and salicylic acid to regulate and control the secondary metabolism of the camptotheca acuminata cells; and inoculating two kinds of microorganisms such as Mucor spinosus and Aspergillus niger for biotransformation at the later stage of cell culture, placing the treated camptotheca acuminata cell culture into a pulp refiner, into which an ethanol solution is added, for homogenating, and filtering to obtain filtrate containing 10-hydroxycamptothecine. The method has the characteristics of short production period, low production cost and high yield of 10-hydroxycamptothecine, and is beneficial to the protection of the camptotheca acuminata plant resources.
Description
Technical field
The invention belongs to biological technical field.Be specifically related to the biosynthetic method of a kind of promotion 10-hydroxycamptothecine.
Background technology
10-hydroxycamptothecine is a kind of trace biology alkali that contains in the peculiar Nyssaceae plant camptotheca acuminata of China (Camptotheca acuminala Decne), it is an action target spot with the DNA topoisomerase I, by suppressing the synthetic antitumor action of bringing into play of organism DNA.It not only itself is a clinical effectiveness good antitumor medicine, but also is that the general important intermediate of replacing health (Topotecan) and irinotecan (Irinotecan) to wait other camptothecin derivative medicine is opened up in preparation; It demonstrates anti-tumor activity widely clinically, and toxic side effect is less relatively, and domestic clinical demand amount and export volume increase year by year.China enterprise directly extracts from the camplotheca acuminata plant for a long time, but because of its content low (about 0.002%), the production cost height is difficult to meet clinical needs.Therefore, much more more and more the preparation research of 10-hydroxycamptothecine comes into one's own, and it has become the second largest focus of the plant series antineoplastic medicament research except that taxanes.At present, to the preparation research of 10-hydroxycamptothecine comprise that chemistry is complete synthesis, chemistry is semi-synthetic, bio-transformation and in kindred plant, seek new medicine source etc., but because of various reasons, these methods are not used for industrialized production yet.
Summary of the invention
The inventor is in the research of carrying out suspension culture camplotheca acuminata cell preparation 10-hydroxycamptothecine, the camplotheca acuminata cell of finding suspension culture is undertaken after secondary metabolism regulation and control and inoculation certain micro-organisms carry out bio-transformation by adding some exogenous material, and the content of 10-hydroxycamptothecine is significantly promoted in the camplotheca acuminata cell culture.Therefore, carried out systematic research, promoted the biosynthetic means and the industrialization of 10-hydroxycamptothecine to lay a good foundation for setting up at aspects such as camplotheca acuminata cell secondary metabolism regulate and control method that improves the 10-hydroxycamptothecine yield and microbial conversion process.
The object of the present invention is to provide the biosynthetic method of a kind of promotion 10-hydroxycamptothecine, thereby the secondary metabolism of suspension culture camplotheca acuminata cell is regulated and control the production yield of the camplotheca acuminata cell of late stage of culture being carried out bio-transformation raising 10-hydroxycamptothecine with microbe inoculation by adding exogenous material.
The technical solution used in the present invention is: the biosynthetic method of a kind of promotion 10-hydroxycamptothecine is characterized in that this method is as follows:
(1) leaf of camplotheca acuminata plant is containing evoked callus on the MS substratum of hormone;
(2) callus is containing on the MS substratum of hormone succeeding transfer culture to obtain cell culture system;
(3) liquid nutrient medium of camplotheca acuminata cell suspension culture use is the MS substratum that contains hormone;
(4) in the camplotheca acuminata cell suspending culture solution, add Xiao Suangu 2～200 μ Mol/L, lanthanum nitrate 2～500 μ Mol/L and Whitfield's ointment 0.05～10mg/L;
(5) carry out bio-transformation at camplotheca acuminata cell cultures post incoulation mucor spinosus (Mucor spinosus) and aspergillus niger (Aspergillus.niger);
(6) camplotheca acuminata cell culture and ethanolic soln carry out homogenized and filter, and obtain containing the filtrate of 10-hydroxycamptothecine;
Wherein the hormone that uses in step (1), (2), (3) is 2,4 dichlorophenoxyacetic acid and 6-benzyladenine, and they unite use, and consumption is respectively 0.1～2.5mg/L.Particularly, its step is as follows:
1, the foundation of camplotheca acuminata cell culture system:
The acquisition of A, explant:
Get the leaf of camplotheca acuminata plant, use the clear water cleaning clean, immerse rinsing 2～6min in 60～85% ethanolic solns, use flushing with clean water then 2～6 times, be positioned over again among 0.1～2% chlorine bleach liquor who has dripped 2～10 tween-80s and soak 1～30min; Take out the back and use aseptic water washing, be cut into the long segment of 1～20mm to non-foam.
Inducing and succeeding transfer culture of B, camplotheca acuminata callus:
Induce and the succeeding transfer culture of camplotheca acuminata callus carry out in illumination box.The explant that A step is obtained inserts in the solid medium, illumination 11～20 hours/day, culture temperature is 20～35 ℃, cultivated 11～30 days, the camplotheca acuminata callus every 3～7 all subcultures once, subculture 2～6 times.
As induce, the solid medium of succeeding transfer culture camplotheca acuminata callus, its minimum medium is the MS substratum, wherein added 0.1～2.5mg/L 6-benzyladenine, 0.2～40g/L sucrose, 0.2～10g/L urea, 0.2～1.2mg/L 2,4 dichlorophenoxyacetic acid, peptizer is agar 0.1～6g/L, and pH is 4.5～7.5.
C, camplotheca acuminata cell cultures suspension system are set up:
B is gone on foot cultured camplotheca acuminata callus change in the gas lift type common loop reactor that air flow is 0.1～0.8L/min and cultivated 10～15 days, temperature is 20～35 ℃.
Carry out the liquid nutrient medium of suspension culture, its minimum medium is the MS substratum, wherein additional 0.1～2.5mg/L 6-benzyladenine, and 0.2～40g/L sucrose, 0.2～10g/L urea, 0.2～1.2mg/L 2,4 dichlorophenoxyacetic acid, pH are 4.5～7.5.
2, add exogenous material and carry out the regulation and control of suspension culture camplotheca acuminata cell secondary metabolism:
Adopt interpolation Xiao Suangu, lanthanum nitrate, three kinds of exogenous materials of Whitfield's ointment that the secondary metabolism of camplotheca acuminata cell is regulated and control.Cobalt nitrate solution adopts high-temperature sterilization, and adding concentration is 2～200 μ Mol/L, and the interpolation time is the 1st～20 day of cell cultures; Lanthanum nitrate hexahydrate adopts high-temperature sterilization, and adding concentration is 2～500 μ Mol/L, and the interpolation time is the 1st～20 day of cell cultures; Whitfield's ointment is sterilized with filtration method, and interpolation concentration is 0.05～10mg/L, and the interpolation time is the 1st～20 day of cell cultures.
Carry out the liquid nutrient medium of suspension culture, its minimum medium is the MS substratum, wherein additional 0.1～2.5mg/L 6-benzyladenine, and 0.2～40g/L sucrose, 0.2～10g/L urea, 0.2～1.2mg/L 2,4 dichlorophenoxyacetic acid, pH are 4.5～7.5.
The camplotheca acuminata cell culture period is 16～25 days.
3, microorganism is to the bio-transformation of suspension culture camplotheca acuminata cell:
A. the inoculation of camplotheca acuminata suspension culture of cells and microorganism:
The inoculation of mucor spinosus (Mucor spinosus), aspergillus niger (Aspergillus.niger): in the 5th～25 day inoculation 5～20% (W/V of camplotheca acuminata cell cultures phase, g/ml) mucor spinosus (Mucor spinosus), inoculate 1～20% (W/V, aspergillus niger g/ml) (Aspergillus.niger) after 1～100 hour.
B. the cultivation conversion condition of microorganism:
Initial p H value is 4.5～7.5, and air flow is 0.1～1.0L/min, and temperature is 25～35 ℃, cultivates after inoculated aspergillus niger (Aspergillus.niger) again and transforms 1～360 hour.
The results of C, camplotheca acuminata cell culture:
After the camplotheca acuminata cell culture results, clean with flushing with clean water, decompress filter is removed raffinate, and is standby.
4, the extraction of 10-hydroxycamptothecine:
With 3, the camplotheca acuminata cell culture that obtains of C is positioned in the refiner, 40～95% (V/V) ethanolic soln that adds 2～15 times of quality carries out homogenized and filters, and obtains containing the filtrate of 10-hydroxycamptothecine.
The invention has the beneficial effects as follows: the content of 10-hydroxycamptothecine is equivalent to 0.2～0.3% (g/g) of camplotheca acuminata dry cell weight in the gained extracting solution, and the content of 10-hydroxycamptothecine increases by 100～150 times than the camplotheca acuminata cell of handling; The microorganism of used substratum, the exogenous material of interpolation and inoculation is all cheap and easy to get, and production cost is low; With short production cycle, product production can increase along with production-scale expansion; The leaf that uses camplotheca acuminata does not influence the continued growth of former plant as explant, helps the protection of camplotheca acuminata plant resources.
Embodiment
The invention will be further described below in conjunction with embodiment, can make those skilled in the art more fully understand the present invention, but the protection domain that does not limit the present invention in any way.
1, the foundation of camplotheca acuminata cell culture system:
Inducing and succeeding transfer culture of A, camplotheca acuminata callus:
Evoked callus culture medium preparation: with one liter of distilled water preparation MS substratum.Add the 0.1mg/L 6-benzyladenine, 30g/L sucrose, 2g/L urea, 0.25mg/L 2,4 dichlorophenoxyacetic acid, peptizer are agar 6g/L, pH is 5.8.Average mark is loaded on 25 100mL triangle culturing bottles, after jumping a queue, places pressure kettle, under 121 ℃, 15 normal atmosphere, sterilizes 30 minutes.
Choosing leaf of Common Camptotheca is explant, cleans up with clear water earlier, immerses 70% ethanol rinsing 5min, takes out back flushing with clean water three times, soaks 5min again in 0.1% chlorine bleach liquor who has dripped two tween-80s, and aseptic water washing is to non-foam.Leaf is cut into the fritter of about 5mm * 5mm size under aseptic condition, inserts in the inducing culture for preparing.
In illumination box, cultivate, illumination 16 hours/day, culture temperature is 25 ℃, cultivates 15 days, will see has callus to produce.Every 21 days subcultures once (used subculture medium is identical with inducing culture).Through 3 succeeding transfer culture, it is frangible just can to obtain loosening, and growth conditions is good, is suitable for the camplotheca acuminata callus of suspension culture.
B, camplotheca acuminata cell cultures suspension system are set up:
The preparation of liquid nutrient medium: the composition of liquid nutrient medium is prepared 1 liter of MS substratum with distilled water.Add the 0.1mg/L6-benzyladenine, 30g/L sucrose, 2g/L urea, 0.5mg/L 2,4 dichlorophenoxyacetic acid, pH are 5.8.Be sub-packed in the 1L gas lift type common loop reactor each reaction unit 500ml nutrient solution behind the autoclaving.
To change in the liquid nutrient medium through above loose frangible, the eugonic camplotheca acuminata callus of 3 succeeding transfer culture, the inoculum size of each reaction unit (fresh weight) is 50 grams, is 10% of nutrient solution weight.Cultivate in air flow is the gas lift type common loop reactor of 0.45L/min, temperature is 25 ℃.Cultivated through 10 days, with the nutrient solution of 1/3 volume in the fresh changing liquid cultivation matrix bottle.Every 3 days, change the nutrient solution of 2/3 volume in the culturing bottle with fresh culture later on.After 15 days, callus has dispersed in nutrient solution and has been dispersed into unicellular or small-particle, is faint yellow, and it is vigorous to grow, and forms unicellular and the mixed uniformly camplotheca acuminata cell suspension culture of small cell cluster system.
2, add exogenous material suspension culture camplotheca acuminata cell carried out the secondary metabolism regulation and control:
The sterilization of 5L gas lift type common loop reactor: feed 0.1MPa steam sterilizing 40min.
Culture medium preparation: with 4 liters of distilled water preparation MS substratum.Add the 0.1mg/L6-benzyladenine, 30g/L sucrose, 2g/L urea, 0.5mg/L 2,4 dichlorophenoxyacetic acid, pH are 5.8.Adopt peristaltic pump to send in the sterilized 5L gas lift type common loop reactor behind the autoclaving.
The camplotheca acuminata cell seed of above-mentioned suspension culture is pressed in the reactor with sterile air, and inoculum size (fresh weight) is 400 grams, is 10% of nutrient solution weight; Air flow is 0.55L/min, and culture temperature is 25 ℃, cultivates 15 days.Added 0.016Mol/L lanthanum nitrate hexahydrate 5ml and 0.0008Mol/L cobalt nitrate solution 5ml at the 1st day that cultivates; Added 200mg/L salicylic acid solution 2ml on the 9th day.
3, microorganism is to the bio-transformation of suspension culture camplotheca acuminata cell:
A. the inoculation of camplotheca acuminata suspension culture of cells and microorganism:
Camplotheca acuminata cell suspension culture to 14 day under above-mentioned (2.) condition, (W/V, mucor spinosus g/ml) (Mucorspinosus) seed are inoculated 15% (W/V, aspergillus niger g/ml) (Aspergillus.niger) seed after 30 hours in inoculation 10%.
B. microbial transformation condition: initial p H value is 5.8, and air flow is 0.5L/min, and temperature is 28 ℃, transforms 72 hours again after inoculated aspergillus niger (Aspergillus.niger).
The results of C, camplotheca acuminata cell culture:
After the camplotheca acuminata cell culture results, clean with flushing with clean water, standby behind the decompress filter removal raffinate.
4,10-hydroxycamptothecine extraction and assay:
Take by weighing 100g camplotheca acuminata cell culture and pack in the refiner, 50% (V/V) ethanolic soln that adds 11 times of quality carries out homogenate, and homogenized temperature is 25 ℃, and the homogenate rotating speed is 12000 rev/mins, and the homogenate time is 8 minutes; Behind decompress filter, obtain containing the filtrate of 10-hydroxycamptothecine.Filtrate is got supernatant liquor through centrifugal, and through high-performance liquid chromatogram determination, the content of 10-hydroxycamptothecine is 132.07mg/L in the filtrate, and the content of 10-hydroxycamptothecine increases by 115 times than the camplotheca acuminata cell of handling.
Claims (5)
1. one kind promotes the biosynthetic method of 10-hydroxycamptothecine, it is characterized in that this method is as follows:
(1) leaf of camplotheca acuminata plant is containing evoked callus on the MS substratum of hormone;
(2) callus is containing on the MS substratum of hormone succeeding transfer culture to obtain cell culture system;
(3) liquid nutrient medium of camplotheca acuminata cell suspension culture use is the MS substratum that contains hormone;
(4) in the camplotheca acuminata cell suspending culture solution, add Xiao Suangu 2～200 μ Mol/L, lanthanum nitrate 2～500 μ Mol/L and Whitfield's ointment 0.05～10mg/L;
(5) carry out bio-transformation at camplotheca acuminata cell cultures post incoulation mucor spinosus (Mucor spinosus) and aspergillus niger (Aspergillus.niger);
(6) camplotheca acuminata cell culture and ethanolic soln carry out homogenized and filter, and obtain containing the filtrate of 10-hydroxycamptothecine;
Wherein the hormone that uses in step (1), (2), (3) is 6-benzyladenine and 2,4 dichlorophenoxyacetic acid, and they unite use, and consumption is respectively 0.1～2.5mg/L.
2. method according to claim 1, the condition of camplotheca acuminata cell suspension culture is: contain the hormone combinations of 0.1～2.5mg/L 6-benzyladenine and 0.2～1.2mg/L 2,4 dichlorophenoxyacetic acid in the MS substratum, the pH value is 4.5～7.5; Inoculum size be 8～15% (W/V, g/ml); Culture temperature is 20～35 ℃, and air flow is 0.1～0.8L/min, and incubation time is 16～25 days.
3. method according to claim 1, wherein in the step (4) the interpolation time of Xiao Suangu and lanthanum nitrate respectively the 1st～5 day of cell suspension culture; The salicylic interpolation time is the 5th～20 day of cell suspension culture.
4. method according to claim 1, wherein the condition of bio-transformation is in the step (5): at the 5th～25 day inoculation 5～20% (W/V of camplotheca acuminata cell cultures phase, g/ml) mucor spinosus (Mucor spinosus), inoculate 1～20% (W/V, aspergillus niger g/ml) (Aspergillus.niger) after 1～100 hour; Initial p H value is 4.5～7.5, and air flow is 0.1～1.0L/min, and culture temperature is 25～35 ℃, cultivates after inoculated aspergillus niger (Aspergillus.nigr) again and transforms 1～360 hour.
5. method according to claim 1, wherein the concentration of ethanolic soln is 40～95% (V/V) in the step (6), the add-on of ethanolic soln is 2～15 times of camplotheca acuminata cell culture fresh weight.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103493735A (en) * | 2013-09-30 | 2014-01-08 | 浙江农林大学 | Light regulation and control method for tissue culture and multiplication of camptotheca acuminata decaisne calluses |
| CN107043795A (en) * | 2017-02-04 | 2017-08-15 | 中国科学院成都生物研究所 | The method that camptothecine and 10 HCPTs are produced using camplotheca acuminata suspension cell |
-
2010
- 2010-01-18 CN CN2010100101201A patent/CN102127582A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103493735A (en) * | 2013-09-30 | 2014-01-08 | 浙江农林大学 | Light regulation and control method for tissue culture and multiplication of camptotheca acuminata decaisne calluses |
| CN103493735B (en) * | 2013-09-30 | 2016-05-04 | 浙江农林大学 | The light regulate and control method that a kind of camplotheca acuminata callus is cultivated and bred |
| CN107043795A (en) * | 2017-02-04 | 2017-08-15 | 中国科学院成都生物研究所 | The method that camptothecine and 10 HCPTs are produced using camplotheca acuminata suspension cell |
| CN107043795B (en) * | 2017-02-04 | 2020-10-09 | 中国科学院成都生物研究所 | Method for producing camptothecin and 10-hydroxycamptothecin by using camptotheca acuminata suspension cells |
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Application publication date: 20110720 |