CN1095498C - Saffron polyploid cell culture process to produce crosin and similar active matter - Google Patents
Saffron polyploid cell culture process to produce crosin and similar active matter Download PDFInfo
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- CN1095498C CN1095498C CN00102919A CN00102919A CN1095498C CN 1095498 C CN1095498 C CN 1095498C CN 00102919 A CN00102919 A CN 00102919A CN 00102919 A CN00102919 A CN 00102919A CN 1095498 C CN1095498 C CN 1095498C
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Abstract
The present invention relates to a method for producing crocin active substances by culturing saffron polyploid cells. Firstly, cell auxin and cytokinin are added to a culture medium, and then sucrose is added; a pH value is adjusted by acid or alkali, and agar is added so that the culture medium becomes a basic culture medium; secondly, the leaf sheaths of the saffron is disinfected to serve as an explant which is inoculated on the basic culture medium, and a cell division inhibitor is added to the culture medium so as to culture a suspended single-cell line; finally, amino acid, elicitors, a metabolism precursor and a coenzyme are added to obtain the product of the present invention, which can be made into a novel anticancer drug, and can also be made into health products for preventing tumors.
Description
The present invention relates to a kind of method of utilizing Stigma Croci to cultivate the polyploid cell that to produce crocin, belong to the bio-pharmaceuticals field of engineering technology.
Stigma Croci (Crocus sativus L) is a kind of herbal medicine of preciousness, originates in ground such as all states in southern Europe and Iran.Because it is had relatively high expectations to procreation enviroment, has only ground such as Xinjiang, Tibet that a small amount of cultivation is arranged in China.Since ancient times, use mainly in China that Stigma Croci is cured wound, typhoid fever, the Yu Kaijie that looses, promoting blood circulation and removing blood stasis, hemoptysis is spitted blood and disease such as the cerebrovascular.In recent years find that the crocetin (Crocitin), safranal (safranal), crocin (Crocin) and the Picrocrocin materials such as (Protocrocin) that contain in the Stigma Croci column cap have stronger antitumour activity.Especially leukemia, papillary carcinoma, pinacocyte knurl and soft tissue sarcoma etc. had stronger restraining effect.Its anticancer mechanism is the expression from DNA and rna level inhibition cell protein kinase activity and proto-oncogene, suppresses the genetoxic of benzopyrene and 12-0-14 acyl group phosphorus ketone-13 acetate (TPA).The Lethal Dose 50 of its cancer cells is 0.8～2.0nmol/L, and side effect is far smaller than vitamin A acid, and therefore, materials such as crocin might become one of ideal cancer therapy drug fully.
But owing to croceous medicinal ingredients mainly exists in the column cap, and the output of dry column cap extremely low (6kg/hm2) if carry out mass production, not only needs big area to plough, and also will spend a large amount of labours.In addition, because the molecular configuration of crocin (Crocin) and Picrocrocin (Picrocrocin) is very complicated, be difficult to use the chemical method synthetic.Therefore, utilizing the culture plant cell method to produce crocin kind anti-cancer drugs thing is one of effective ways that solve the Stigma Croci supply and demand.
The objective of the invention is to propose a kind of method of utilizing Stigma Croci to cultivate the polyploid cell that to produce crocin, utilize the production of culture plant cell method to have the medicine of antitumour activity or treatment cardiovascular and cerebrovascular diseases.
The Stigma Croci that utilizes that the present invention proposes is cultivated the method for the polyploid cell that can produce crocin, and this method may further comprise the steps:
1. in plant tissue culture media commonly used, add 0～10mg/L archusia or 0～10mg/L phytokinin by every liter of nutrient solution, add 20～60g/L sucrose again, with acid or alkali its pH value is adjusted to 5～7, add 7～10g/L agar, after the dissolving of heating, be divided in the test tube, through 105～125 ℃ of high temperature, 0.1～0.15MPa pressure makes minimum medium through cooling after sterilizing down;
With croceous leaf sheath with the clorox sterilization back of 70% ethanol and 3%～10% as growing body outward.Be seeded on the above-mentioned minimum medium, at 20～25 ℃, dark condition was cultivated 20～40 days down, formed callus;
3. prepare above-mentioned minimum medium again, add cell division inhibitor in substratum, add-on is 10-100mg/L, and the callus that the second above-mentioned one-step inducing is gone out is seeded on this substratum, at 20～25 ℃, cultivates under the lucifuge condition 10～20 days;
4. the callus cell with the 3rd step is placed in the minimum medium that the above-mentioned the first step do not add agar, and at 20～25 ℃, under the lucifuge condition, rotating and culturing is 10～20 days on the 90rpm bottle swingging machine, turns out the suspension monoclonal;
5. go on foot unicellular evenly being seeded on the dull and stereotyped minimum medium of suspension of turning out with the 4th, at 20～25 ℃, cultivated under the lucifuge condition 20～30 days, form new cell mass, then with after the staining agent dyeing, observe its chromosome number with powerful microscope (1600 times), pick out polyploid cell system;
6. configuration minimum medium, and add amino acid/11～100mg/L, elicitor 1～100ml/L, metabolic precursor thereof 10～200mg/L, coenzyme 0.1～10mg/L respectively, the polyploid cell that the 5th step was obtained is seeded in this substratum, at 20～25 ℃, cultivated under the lucifuge condition 20～30 days, and promptly can from harvested cell, be separated to crocin class active substance of the present invention.
Utilize the crocin of method preparation of the present invention, can solve problem such as Stigma Croci resource critical shortage and Stigma Croci selling at exorbitant prices for a long time.Particularly chemical substances such as isolated crocin, Picrocrocin, safranal or crocetin have good anti-cancer activity from the Stigma Croci cell, have the bright prospect that is developed to the new type anticancer medicine, also can be developed to the healthcare products that prophylaxis of tumours takes place.Can also develop treatment cardiovascular and cerebrovascular diseases and hemoptysis, haematemesis and good medicine promoting blood circulation and removing blood stasis.
Introduce embodiments of the invention below.
Example one
(1) at Murashige ﹠amp; In Skoog (MS) minimum medium, add 1.0mg/L archusia naa and 4.0mg/L phytokinin Bian aminopurine (BA) by every liter of nutrient solution, add 20g/L sucrose again, with sodium hydroxide or hydrochloric acid its pH value is adjusted to 6, add 7g/L agar, after the dissolving of heating, be divided in the test tube, through 125 ℃ of high temperature, 0.1MPa pressure is sterilized down after cooling makes minimum medium;
(2) with croceous leaf sheath with 70% ethanol sterilization 1 minute, with 3% clorox sterilization 10 minutes, behind aseptic water washing three times, be seeded on the above-mentioned minimum medium again, at 25 ℃, dark condition was cultivated 40 days down, formed callus;
(3) prepare above-mentioned minimum medium again, add 30mg/L cell division inhibitor colchicine in substratum, the callus that the second above-mentioned one-step inducing is gone out is seeded on this substratum, at 25 ℃, cultivates 20 days under the lucifuge condition;
(4) callus cell with the 3rd step is placed in the liquid-based basal culture medium that does not add agar, and at 25 ℃, under the lucifuge condition, rotating and culturing is 20 days on the 90rpm bottle swingging machine, turns out the suspension monoclonal.
(5) unicellular evenly being seeded on the dull and stereotyped minimum medium of suspension that the 4th step was turned out, at 25 ℃, cultivated 30 days under the lucifuge condition, form new cell mass, after using cell observation cell Giemsa stain agent (GiemsaStain) dyeing then, observe its chromosome number with powerful microscope (1600 times), pick out polyploid cell system;
(6) configuration minimum medium, and add amino acid, ornithine 10mg/L, elicitor staphylococcus respectively and cultivate bacterium liquid 50ml/L, metabolic precursor thereof lycopin 30mg/L, coenzyme NAD P10mg/L (buying) from U.S. SIGMA company, the polyploid cell system that the 5th step was obtained is seeded in this substratum, at 25 ℃, cultivated 30 days under the lucifuge condition, promptly can from harvested cell, be separated to crocin, Picrocrocin, safranal and Stigma Croci acids active substance.
Example two
(1) in Gamborg (B5) minimum medium, add 1.0mg/L2 by every liter of nutrient solution, 4-D and 4.0mg/L Bian aminopurine (BA), add 20g/L sucrose again, its pH value is adjusted to 5.8, add 7g/L agar with acid or alkali, after the dissolving of heating, be divided in the test tube, through 125 ℃ of high temperature, 0.1MPa pressure is sterilized down after cooling makes minimum medium;
(2) with croceous leaf sheath with 70% ethanol sterilization 1 minute, again with 3% clorox sterilization 10 minutes, behind aseptic water washing three times, by kind on above-mentioned minimum medium, at 25 ℃, dark condition was cultivated 40 days down, formed callus;
(3) prepare above-mentioned minimum medium again, add the 50mg/L colchicine in substratum, the callus that the second above-mentioned one-step inducing is gone out is seeded on this substratum, at 25 ℃, cultivates 20 days under the lucifuge condition;
(4) callus cell with the 3rd step is placed on (liquid) in the minimum medium that does not add agar, and at 25 ℃, under the lucifuge condition, rotating and culturing is 20 days on the 90rpm bottle swingging machine, turns out the suspension monoclonal.
(5) unicellular evenly being seeded on the dull and stereotyped minimum medium of suspension that the 4th step was turned out at 25 ℃, cultivated 30 days under the lucifuge condition, form new cell mass, with after the staining agent dyeing, observe its chromosome number then, pick out polyploid cell system with powerful microscope (1600 times);
(6) configuration minimum medium, and add phenylalanine 10mg/L tooth branch cladosporium respectively and cultivate bacterium liquid 50ml/L, gentiobiose 30mg/L, NAD10mg/L, the polyploid cell that the 5th step was obtained is seeded in this substratum, at 25 ℃, cultivated 30 days under the lucifuge condition, promptly can from harvested cell, be separated to crocin, Picrocrocin, safranal and Stigma Croci acids active substance.
Example three
(1) in White (W) minimum medium, add 1.0mg/L Yin tremble acetate and 4.0mg/L Bian aminopurine (BA) by every liter of nutrient solution, add 20g/L sucrose again, with acid or alkali its pH value is adjusted to 5.8, add 7g/L agar, after the dissolving of heating, be divided in the test tube, through 125 ℃ of high temperature, 0.1MPa pressure is sterilized down after cooling makes minimum medium;
(2) with croceous leaf sheath with 70% ethanol sterilization 1 minute, with 3% clorox sterilization 10 minutes, behind aseptic water washing three times, be seeded on the above-mentioned minimum medium again, at 25 ℃, dark condition was cultivated 40 days down, formed callus;
(3) prepare above-mentioned minimum medium again, add the 100mg/L colchicine in substratum, the callus that the second above-mentioned one-step inducing is gone out is seeded on this substratum, at 25 ℃, cultivates 20 days under the lucifuge condition;
(4) callus cell with the 3rd step is placed on (liquid) in the minimum medium that does not add agar, and at 25 ℃, under the lucifuge condition, rotating and culturing is 20 days on the 90rpm bottle swingging machine, turns out the suspension monoclonal.
(5) unicellular evenly being seeded on the dull and stereotyped minimum medium of suspension that the 4th step was turned out at 25 ℃, cultivated 30 days under the lucifuge condition, form new cell mass, with after the staining agent dyeing, observe its chromosome number then, pick out polyploid cell system with powerful microscope (1600 times);
(6) configuration minimum medium, and add tryptophane 10m/L, paecilomycerol respectively and cultivate bacterium liquid 50ml/L, lycopin 50mg/L, NADP20mg/L, the polyploid cell that the 5th step was obtained is seeded in this substratum, at 25 ℃, cultivated 30 days under the lucifuge condition, promptly can from harvested cell, be separated to crocin, Picrocrocin, safranal and Stigma Croci acids active substance.
Claims (1)
1, a kind of method of utilizing Stigma Croci to cultivate the polyploid cell that can produce crocin is characterized in that this method may further comprise the steps:
(1) in plant tissue culture media commonly used, add 0～10mg/L archusia or 0～10mg/L phytokinin by every liter of nutrient solution, add 20～60g/L sucrose again, with acid or alkali its pH value is adjusted to 5～7, add 7～10g/L agar, after the dissolving of heating, be divided in the test tube, through 105～125 ℃ of high temperature, 0.1～0.15MPa pressure makes minimum medium through cooling after sterilizing down;
(2) with croceous leaf sheath with the clorox sterilization back of 70% ethanol and 3%～10% as growing body outward.Be seeded on the above-mentioned minimum medium, at 20～25 ℃, dark condition was cultivated 20～40 days down, formed callus;
(3) prepare above-mentioned minimum medium again, add cell division inhibitor in substratum, add-on is 10-100mg/L, and the callus that the second above-mentioned one-step inducing is gone out is seeded on this substratum, at 20～25 ℃, cultivates under the lucifuge condition 10～20 days;
(4) callus cell with the 3rd step is placed on the above-mentioned the first step but does not add in the minimum medium of agar, and at 20～25 ℃, under the lucifuge condition, rotating and culturing is 10～20 days on the 90rpm bottle swingging machine, turns out the suspension monoclonal;
(5) unicellular evenly being seeded on the dull and stereotyped minimum medium of suspension that the 4th step was turned out at 20～25 ℃, cultivated under the lucifuge condition 20～30 days, form new cell mass, with after the staining agent dyeing,, pick out polyploid cell system then with its chromosome number of ultramicroscopic observation;
(6) configuration minimum medium, and add amino acid/11～100mg/L, elicitor 1～100ml/L, metabolic precursor thereof 10～200mg/L, coenzyme 0.1～10mg/L respectively, the polyploid cell that the 5th step was obtained is seeded in this substratum, at 20～25 ℃, cultivated under the lucifuge condition 20～30 days, and promptly can from harvested cell, be separated to crocin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343655B (en) * | 2008-08-29 | 2010-05-12 | 北京林业大学 | Method for identifying poplar polyploid with cell interphase nucleus chromocenter number |
| CN101695281B (en) * | 2009-10-26 | 2011-06-01 | 中国科学院新疆理化技术研究所 | Method for tissue culture and rapid propagation of thornless safflower |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303203C (en) * | 2004-01-09 | 2007-03-07 | 清华大学 | Method of obtaining saline cistanche phenylethanol glycoside kind compound using bio-conversion technique |
| CN104054576B (en) * | 2014-06-23 | 2015-09-09 | 上海交通大学 | The method for creating of hexaploid safflower germ plasm resource |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0233040A2 (en) * | 1986-02-04 | 1987-08-19 | Ajinomoto Co., Inc. | Stigma of crocus sativus L. and method for the production thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0233040A2 (en) * | 1986-02-04 | 1987-08-19 | Ajinomoto Co., Inc. | Stigma of crocus sativus L. and method for the production thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343655B (en) * | 2008-08-29 | 2010-05-12 | 北京林业大学 | Method for identifying poplar polyploid with cell interphase nucleus chromocenter number |
| CN101695281B (en) * | 2009-10-26 | 2011-06-01 | 中国科学院新疆理化技术研究所 | Method for tissue culture and rapid propagation of thornless safflower |
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