CN102080110A - Technical process for synthesizing camptothecin sugar derivative by artificially inducing nothapodytes nimmoniana (graham) mablerley endophyte - Google Patents
Technical process for synthesizing camptothecin sugar derivative by artificially inducing nothapodytes nimmoniana (graham) mablerley endophyte Download PDFInfo
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- CN102080110A CN102080110A CN2010105746231A CN201010574623A CN102080110A CN 102080110 A CN102080110 A CN 102080110A CN 2010105746231 A CN2010105746231 A CN 2010105746231A CN 201010574623 A CN201010574623 A CN 201010574623A CN 102080110 A CN102080110 A CN 102080110A
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Abstract
The invention relates to a technical process for synthesizing a camptothecin sugar derivative by artificially inducing nothapodytes nimmoniana (graham) mablerley endophyte. The process disclosed by the invention comprises the following steps of: charging immature cells of nothapodytes nimmoniana (graham) mablerley into an amplification culture medium according to the proportion of inoculation amount of 50-200g/L wet weight and culturing at 25-30 DEG C; adding 1-2,000 mummol/L of combined elicitor and 20-50g/L of sugar in the exponential growth initial period of the cells; adding a saccharified reagent and catalyst for carrying out saccharification reaction; and adding 1-2,000 mummol/L of combined elicitor for inducing in the exponential growth metaphase of the cells. The method disclosed by the invention realizes the synthesis of the camptothecin derivative in an artificial inducing way by utilizing the characteristics of the endophyte of the nothapodytes nimmoniana (graham) mablerley which is the camptothecin producing plant and cansolve the problems of lower conversion rate, high technical requirement on strain culture, more separating steps, and the like existing in the traditional biosynthesis methods and provide reference for the research of camptothecin as well as the anticancer effect and anticancer medicines thereof.
Description
Technical field
The present invention relates to a kind of technological method of synthetic camptothecin derivative, the technological method of the synthetic camptothecine sugar derivatives of especially a kind of artificial induction's form nothapodytes endophyte belongs to biological technical field.
Background technology
(camptothecin is to separate a kind of natural alkaloid that obtains from the Nyssaceae plant camptotheca acuminata CPT) to camptothecine.Camptothecine and derivative thereof have unique mechanism of action---and unique have selectivity inhibition DNA topoisomerase I (Topo I), so become one of focus of antitumor drug research over nearly more than 20 years.Because camptothecine is water-soluble and fat-soluble all relatively poor, the antitumour activity of its sodium salt is lower, and strict restriction has been received in clinical application.Thereafter people isolate 10-hydroxycamptothecine from camplotheca acuminata, have synthesized the camptothecin derivative that toxicity is little, antitumor action is stronger, have played significant role in treatment and prevention of tumour.Be applied at present clinical camptothecin derivative, for example be used for the treatment of the topotecan (Topotecan) of colorectum tumour irinotecan (Irinotecan), drugs approved by FDA listing in 1996 and health etc. is replaced in the shellfish promise of Korea S's approval listing in 2004 in Japan's approval listing in 1994, relevant pharmacological evaluation proves that some other camptothecin derivative has shown good antineoplastic activity clinically.
The natural content that is present in the camptothecine of camptothecin derivative is very low, therefore mainly is to be that raw material obtains through biotransformation method, the complete synthesis and chemical semisynthetic method of chemistry with the camptothecine at present.The method that chemistry is complete synthesis is because reactions steps is too much, and synthetic total recovery is low, the cost height, and can't realize industrialization.Shortcomings such as though and some biosynthetic methods have certain economic benefits, transformation efficiency is lower, strain culturing technical requirements height but still exist, and separating step is many, so its development is restricted.Because endophyte of plant distributes wide, kind is many, and easily cultivate, the easy to control and characteristics such as fast of growing, can also produce the meta-bolites of biologically active, therefore, by the artificial mode, the inducing plant endogenesis bacterium is synthesized camptothecin derivative, can be used as the route of synthesis of this class anticancer component of camptothecine and derivative thereof, for the research of camptothecine and antitumous effect thereof, cancer therapy drug provides reference.
Form nothapodytes this year is another main source of the camptothecine except camplotheca acuminata, be rich in camptothecine because of its full tree and caused concern, have and carry out correlative study, as patent " from the isolating camptothecine framework compound of Root-bark of Pittosporumlike Nothapodytes and as the purposes of the synthon of novel drugs and therapeutical agent " (ZL97194492.X), " from form nothapodytes, extract the method for camptothecine " (200810028740.0), " camplotheca acuminata and camptothecine development and use progress " Feng Jiancan etc., " forest-science ", 2000,36 rolled up for the 5th phases) etc., but rarely seen report by synthetic camptothecine of artificial induction's form nothapodytes endophyte and derivative thereof.
Summary of the invention
The technological method that the purpose of this invention is to provide the synthetic camptothecine sugar derivatives of a kind of artificial induction's form nothapodytes endophyte, this method is by with the form nothapodytes immature cell according to a certain percentage, place defined medium to cultivate, at the cell index early growth period, add combination elicitor and sugar, add saccharification reagent and catalyzer again, carry out saccharification react, grow mid-term at cell index at last, add the combination elicitor again the form nothapodytes endophyte is induced, realize the synthetic of camptothecine sugar derivatives.
The technical solution adopted in the present invention is:
(1) preparation amplification culture medium promptly adds naphthylacetic acid 50 ~ 80 μ mol/L, 6-benzyl aminopurine 5 ~ 10 μ mol/L, sucrose or glucose 30 ~ 50g/L, agar 1.5 ~ 2g/L on the basis of MS minimum medium; PH5.5 ~ 6; Is the ratio of 50 ~ 200g/L weight in wet base with the form nothapodytes immature cell according to inoculum size, places amplification culture medium, cultivates in 25 ~ 30 ℃;
(2) preparation combination elicitor: by 0.02 ~ 0.04mol form nothapodytes endophyte elicitor, 60 ~ 80g/L Whitfield's ointment, 20 ~ 60 hydrogen peroxide and 80 ~ 100 μ mol/L methyl jasmonate (mj)s are formed;
(3) inducing culture: at the cell index early growth period, adding concentration is the combination elicitor of 1 ~ 2000 μ mol/L and the sugar of 20 ~ 50g/L; Add saccharification reagent and catalyzer, carry out saccharification react; In cell index growth mid-term, the combination elicitor that adds concentration again and be 1 ~ 2000 μ mol/L is induced.
The present invention is with respect to the advantage of prior art: the mode that adopts the artificial induction, utilize the characteristic of producing camptothecin plant form nothapodytes endophyte, realize the synthetic of camptothecin derivative, can solve at present some biosynthetic methods and have still that transformation efficiency is lower, strain culturing technical requirements height, problems such as separating step is many, the research that can be camptothecine and antitumous effect thereof, cancer therapy drug provides reference.
Embodiment
Below by embodiment the present invention is described in further details, these embodiment only are used for illustrating the present invention, do not limit the scope of the invention.
Embodiment 1
(1) medium preparation: add naphthylacetic acid 50 μ mol/L, 6-benzyl aminopurine 5 μ mol/L, sucrose or glucose 30g/L, agar 1.5g/L in the MS minimum medium; PH5.5 ~ 6;
(2) inoculation culture: is the ratio of 100g/L weight in wet base with the form nothapodytes immature cell according to inoculum size, places 25 ℃ of shaking tables, rotating speed 80rpm, the dark cultivation;
(3) combination elicitor: by 0.02mol form nothapodytes endophyte elicitor, 60g/L Whitfield's ointment, 20 μ l/L hydrogen peroxide and 80 μ mol/L methyl jasmonate (mj)s are formed;
(4) combined induction, saccharification react: utilize inductor to cultivate in the culturing process, behind inoculation medium 5 days, adding concentration was the elicitor of 300 μ mol/L, and mends sugared 30g/L; Add the full acetyl bromide of 3g/L for semi-lactosi, 15 μ l/LBu
4NBr; Behind the inoculation medium 10 days, adding concentration again was the combination elicitor of 500 μ mol/L;
(5) extract: separated by the culture that obtains, extract, the productivity of acquisition is 34.38mg/ (Ld) the most, and production peak is 826.18mg/L.
Embodiment 2
(1) medium preparation: add naphthylacetic acid 50 μ mol/L, 6-benzyl aminopurine 5 μ mol/L, sucrose or glucose 30g/L, agar 1.5g/L in the MS minimum medium; PH5.5 ~ 6;
(2) inoculation culture: is the ratio of 100g/L weight in wet base with the form nothapodytes immature cell according to inoculum size, places 25 ℃ of shaking tables, rotating speed 100rpm, the dark cultivation;
(3) combination elicitor: by 0.02mol form nothapodytes endophyte elicitor, 60g/L Whitfield's ointment, 20 μ l/L hydrogen peroxide and 80 μ mol/L methyl jasmonate (mj)s are formed;
(4) combined induction, saccharification react: utilize inductor to cultivate in the culturing process, behind inoculation medium 7 days, adding concentration was the inductor of 600 μ mol/L, and mends sugared 30g/L; Add the full acetyl bromide of 6g/L for semi-lactosi, 15 μ l/LBu
4Behind the NBr inoculation medium 10 days, adding concentration again was the combination elicitor of 1000 μ mol/L;
(5) extract: separated by the culture that obtains, extract, the productivity of acquisition is 38.48mg/ (Ld) the most, and production peak is 958.20mg/L.
Embodiment 3
(1) medium preparation: add naphthylacetic acid 80 μ mol/L, 6-benzyl aminopurine 5 μ mol/L, sucrose or glucose 30g/L, agar 1.5g/L in the MS minimum medium; PH5.5 ~ 6;
(2) inoculation culture: is the ratio of 100g/L weight in wet base with the form nothapodytes immature cell according to inoculum size, places 25 ℃ of shaking tables, rotating speed 80rpm, the dark cultivation;
(3) combination elicitor: by 0.03mol form nothapodytes endophyte elicitor, 70g/L Whitfield's ointment, 20 μ l/L hydrogen peroxide and 80 μ mol/L methyl jasmonate (mj)s are formed;
(4) combined induction, saccharification react: utilize inductor to cultivate in the culturing process, behind inoculation medium 5 days, adding concentration was the inductor of 300 μ mol/L, and mends sugared 30g/L; Add the full acetyl bromide of 12g/L for semi-lactosi, 15 μ l/LBu
4NBr; Behind the inoculation medium 10 days, adding concentration again was the combination elicitor of 500 μ mol/L;
(5) extract: separate by the culture that obtains, extract the camptothecine of cell part and absorbed portion.The productivity that obtains reaches as high as 45.29mg/ (Ld), and production peak can reach 1015.0mg/L.
Claims (6)
1. the technological method of the synthetic camptothecine sugar derivatives of artificial induction's form nothapodytes endophyte, it is characterized in that being made up of following steps: with the form nothapodytes immature cell is the ratio of 50 ~ 200g/L weight in wet base according to inoculum size, place amplification culture medium, in 25 ~ 30 ℃, cultivate; At the cell index early growth period, adding concentration is the combination elicitor of 1 ~ 2000 μ mol/L and the sugar of 20 ~ 50g/L; Add saccharification reagent and catalyzer, carry out saccharification react; In cell index growth mid-term, the combination elicitor that adds concentration again and be 1 ~ 2000 μ mol/L is induced.
2. growth regulator according to claim 1 is characterized in that: described amplification culture medium is to have added naphthylacetic acid 50 ~ 80 μ mol/L, 6-benzyl aminopurine 5 ~ 10 μ mol/L, sucrose or glucose 30 ~ 50g/L, agar 1.5 ~ 2g/L on the basis of MS minimum medium; PH5.5 ~ 6.
3. method according to claim 1 is characterized in that described combination elicitor by 0.02 ~ 0.04mol form nothapodytes endophyte elicitor, 60 ~ 80g/L Whitfield's ointment, and 20 ~ 60 hydrogen peroxide and 80 ~ 100 μ mol/L methyl jasmonate (mj)s are formed.
4. method according to claim 1, it is characterized in that described saccharification reagent be the full acetyl bromide of 3 ~ 12g/L for semi-lactosi, described catalyzer is 15 ~ 30 μ l/LBu
4NBr.
5. method according to claim 1 is characterized in that described sugar is sucrose or glucose.
6. method according to claim 1 is characterized in that described cell index early growth period is 5 ~ 10 days behind the inoculation medium; The growth of described cell index is 10 ~ 20 days behind the inoculation medium mid-term.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948370A (en) * | 2012-12-13 | 2013-03-06 | 向华 | Rapid propagation method of nothapodytes pittosporoides |
| CN103202318A (en) * | 2013-04-08 | 2013-07-17 | 南京师范大学 | Accumulation promoter of atractylis lancea naphtha and preparation method thereof |
-
2010
- 2010-12-06 CN CN2010105746231A patent/CN102080110A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948370A (en) * | 2012-12-13 | 2013-03-06 | 向华 | Rapid propagation method of nothapodytes pittosporoides |
| CN103202318A (en) * | 2013-04-08 | 2013-07-17 | 南京师范大学 | Accumulation promoter of atractylis lancea naphtha and preparation method thereof |
| CN103202318B (en) * | 2013-04-08 | 2015-01-28 | 南京师范大学 | Accumulation promoter of atractylis lancea naphtha and preparation method thereof |
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Application publication date: 20110601 |