CN101289651A - Sinkiang alkanna tinctoria cell lines and process for producing sinkiang alkanna tinctoria alkannin by cell culture - Google Patents
Sinkiang alkanna tinctoria cell lines and process for producing sinkiang alkanna tinctoria alkannin by cell culture Download PDFInfo
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Abstract
The invention provides an Arnebia euchroma mauve cell line for stable and high yield of alkannin, wherein, the content of alkannin compounds can reach 6 to 8 percent of the dry weight of culture and is 5 to 9 times of that of wild and natural Arnebia euchroma. The invention also provides a culture technique for the cell line and a method for mass and quick production of the culture of the cell line. The method breaks the routine lithospermum cell two-step culture method, adopts one-step culture method to cultivate the lithospermum cell culture and then realizes production of Arnebia euchroma cells through the method of combination of cell suspension culture and culture of solid culture medium and quick production of effective compositions. The method adopts combination of liquid culture and solid culture, produces seeds through liquid culture, obtains the culture through solid culture and selects and uses reserved seeds with good comprehensive properties for liquid culture, and the steps are repeated. Moreover, the production of the Arnebia euchroma cells by adoption of the method does not occupy arable lands and destroy wild resources of Arnebia euchroma, thereby the environment is protected and the problem of shortage of Arnebia euchroma resources is solved.
Description
Technical field
The present invention relates to a kind of lithospermum euchromum Royle clone, the invention still further relates to the preparation method of this clone and the production method of culture thereof.
Background technology
Asian puccoon is a kind of perennial herb medicinal plant that is used as medicine with root of Lamiales Boraginaceae.China's Asian puccoon resource can be divided into substantially: lithospermum euchromum Royle (Arnebia euchroma (Royle) Johnst.), gromwell root (Lithospermum erythrorhizonS.et Z.), Yunnan Asian puccoon (Onosma paniculata Bur.Et Fr.), arnebia guttata Bunge (Arnebia guttata Bunge.) and Tibet Asian puccoon classifications such as (Onosma hookeri Clarke var.Longiflorum Duthie.).Wherein, lithospermum euchromum Royle is because of pigment content height and the good main source that becomes the commodity Asian puccoon of pharmacological action.
Lithospermum euchromum Royle is practised the title Radix Arnebiae, grows in the high hill grove of height above sea level 2500-4200 rice or the hillside fields that faces south, and mainly is distributed in ground such as Xinjiang, Tibet.Plant height 15-35cm, the appearance red-purple, root is sturdy.Medicinal part is its root, medicinal ingredients is Shikonin and derivative thereof, mainly comprise: Shikonin, β, β '-dimethyl allene acyl A Kaning, deoxyshikonin, racemization acetyl A Kaning, acetyl A Kaning, β-acetoxyl group isovaleryl A Kaning, Shikalkin, beta-hydroxy isovaleryl A Kaning, 1-methoxyl group acetylshikonin, acetylshikonin etc., all belong to 2,4-naphthoquinones class organic compound.
Shikonin and derivative thereof contained in the lithospermum euchromum Royle have important pharmaceutical use.Studies show that: Shikonin and derivative thereof have antitumor, anti-AIDS, antiviral, antibiotic, anti-inflammatory and promote the effect of recycle system round-robin.External application can be treated skin carcinoma, lupus erythematosus, eczema, Acyclovir and burn and scald; The assisting therapy that can be used for chorioepithelioma, viral hepatitis, lung cancer and liver cancer chemicotherapy for oral administration.Simultaneously, Shikonin and derivative thereof become the ideal natural red colouring matter because of having look valency height, strong adhesion and absorbing the strong advantage of uv-radiation function, are described as the king of haematochrome in the world.
Though China's Asian puccoon resource is than horn of plenty, because of people excessively gather, the wild alkanna resource is near exhausted.But the demand to contained Shikonin of lithospermum euchromum Royle and derivative thereof in medicinal industry is bigger again, thereby serious imbalance between supply and demand occurred.Search out the propagation method of suitable lithospermum euchromum Royle, produce Shikonin and derivative thereof fast to meet the need of market, significant.
At present, the research of lithospermum euchromum Royle tissue culture aspect is mainly concentrated both ways: on the one hand, from callus, filter out good clone, in order to produce useful secondary metabolite; On the other hand, utilize tissue culture technique, induce and filter out excellent strain, and further it is bred into whole plant fast.The research for lithospermum euchromum Royle at present mainly concentrates on the former, but because of it has working condition requirement height, complex process, and long flow path, the shortcoming that production cost is high, thereby applying of this technology be subjected to greatly restriction, awaits further research.
Summary of the invention
First purpose of the present invention is to provide a kind of lithospermum euchromum Royle clone of high yield Shikonin.As explant, evoked callus by succeeding transfer culture repeatedly, obtains three kinds of lithospermum euchromum Royle clones: white color system, yellow system, purplish red colour system with lithospermum euchromum Royle (its root, stem, leaf are perhaps by the aseptic seedling of seed germination) in the present invention; 3 kinds of clones are carried out succeeding transfer culture respectively,, determine that the red-purple cell is that growing way is better, the clone that the Shikonin compounds content is the highest by detection to its speed of growth, Shikonin compounds content.From isolated cells system, select bright-colored red-purple, compact structure, the crumby culture of being, be clone of the present invention.
The present invention further carries out succeeding transfer culture and screening to the red-purple clone that obtains, and integrated survey content of effective and growth characteristics have finally obtained the stable high yield lithospermum euchromum Royle clone that can infinitely go down to posterity and cultivate.This clone has following feature: grow vigorous, bright-colored, be red-purple, compact structure, be agglomerate and open, quality evenly, slightly partially firmly, Shikonin and derivative content thereof are the 6-8% of culture dry weight, culture cycle 30-35 days.Specifically, can prepare above-mentioned clone by the following method:
1) cultivation of lithospermum euchromum Royle aseptic seedling
Get the seed of natural lithospermum euchromum Royle, identify by institute of Chinese Academy of Sciences sample shop (national herbarium shop), earlier with 75% alcohol-pickled 10-30s, then with 1% mercuric chloride solution sterilization 8-12min, wash 4-5 time repeatedly with sterilized water again, be placed at last and blot seed-coat moisture on the aseptic filter paper, be seeded in the solid medium that adds 10-30g/L sucrose, 0.6g/L agar in the basic MS (prescription is built subordinate list 1) with aseptic nipper, 23-25 ℃, unglazed according to cultivating, seed can be sprouted and grow aseptic seedling in about 10 days.
2) callus inducing and cultivating
Get aseptic shoot root, stem, leaf and cotyledon, plumule is done explant, under aseptic condition, with aseptic operation scissors and aseptic nipper aseptic shoot root, stem, leaf and cotyledon, plumule are cut into the long segment of 2-5mm, be seeded on the solid medium that adds 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/LKT, 0.6g/L agar and 30g/L sucrose among the N6 (prescription sees attached list 2), 23-25 ℃ of dark the cultivation induced callus in 7-10 days.
3) subculture of callus screening
The present invention by to the lithospermum euchromum Royle callus induce and cultivation has obtained three kinds of Sinkiang saussurea involucrata cell lines: white color system, yellow system and purplish red colour system; 3 kinds of clones have been carried out succeeding transfer culture respectively,, determined that the red-purple cell is that growing way is better, Shikonin and the highest clone of derivative content thereof by detection to its speed of growth, Shikonin and derivative content thereof.
Choose the red-purple cell and be initiating cell system, from solid medium, select color red-purple callus dark, that be in bright colors adds 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose in N6 (prescription sees attached list 2) solid medium with appearance method and continue succeeding transfer culture.Be better optimize seed, purifying cells system, the inoculation piece size is controlled at below the 3mm during subculture.So repeatedly, selected clone is progressively tamed, the present invention has obtained the stable high yield lithospermum euchromum Royle clone that can infinitely go down to posterity and cultivate through the screening of nearly 100 generations.
Second purpose of the present invention is to provide the succeeding transfer culture method of above-mentioned lithospermum euchromum Royle cloned culture, its used substratum is to add 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose among the N6 (prescription sees attached list 2), pH value 5.2-5.8, the inoculation piece is 4-6mm, culture condition: temperature 23-25 ℃, unglazed photograph, incubation time 30-35 days.Select during subculture growth vigorous, bright-colored, be red-purple, compact structure, be lumps, quality evenly, hard slightly partially be the culture subculture of feature.In addition to culture Shikonin and derivative (β thereof, β '-dimethyl allene acyl A Kaning, deoxyshikonin, racemization acetyl A Kaning, acetyl A Kaning, β-acetoxyl group isovaleryl A Kaning, Shikalkin, beta-hydroxy isovaleryl A Kaning, 1-methoxyl group acetylshikonin, acetylshikonin etc., all belong to 2,4-naphthoquinones class organic compound) content is investigated, and selects the good culture subculture of comprehensive proterties.
The 3rd purpose of the present invention is to provide the method for the above-mentioned cloned culture of a kind of scale operation.The inventive method is cultivated the liquid shaking table and is combined with solid culture, comprises the steps: 1) production of hybrid seeds of employing liquid shaking table cultured method; 2) growth culture vigorous, bright-colored, the red-purple feature is reserved seed for planting in the culture, and the shaking table that is used for step 1) is cultivated, and all the other results get culture.
Specifically, can adopt following method to carry out scale operation:
Do seed with high yield lithospermum euchromum Royle clone, suspension culture in the 500mL culturing bottle that the 150mL liquid nutrient medium is housed, liquid nutrient medium is interpolation 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT among the N6 (prescription sees attached list 2), reaches 30g/L sucrose, pH value 5.2-5.8, and inoculum size 3-6% (W/V, g.fw/ml), culture condition: temperature is 23-25 ℃, shaking speed 100-120rpm, incubation time 8-10 days, unglazed according to cultivating.
From liquid nutrient medium, select growth vigorous, bright-colored, be red-purple, good dispersity, uniform particles, free of contamination cell mass are done to produce with planting, it is transferred on the solid medium cultivate, note picking out the cell mass that is a bit darkish in color, turns white, the cell mass size of inoculation is 4-6mm, solid medium is to add 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose, pH value 5.2-5.8 among the N6 (prescription sees attached list 2); Culture condition: temperature 23-25 ℃, unglazed according to cultivating incubation time 30-35 days.
When utilizing production to carry out scale operation with kind, according to the turnout size, can therefrom select to grow vigorous, bright-colored, be red-purple, compact structure, be lumps, quality is even, summary is reserved seed for planting for the callus of feature partially firmly, carries out the liquid shaking table again and cultivates the production of hybrid seeds, and the seed selection amount can not surpass 15% of gross production, all the other are collected and low-temperature vacuum drying promptly gets lithospermum euchromum Royle cell culture dry product, so repeatedly.
But liquid culture and solid culture are carried out the same period in the aforesaid method in addition, can be shortened to 15-20 days the production cycle like this.In process of production can also be to the careless element and the derivative (β thereof of culture, β '-dimethyl allene acyl A Kaning, deoxyshikonin, racemization acetyl A Kaning, acetyl A Kaning, β-acetoxyl group isovaleryl A Kaning, Shikalkin, beta-hydroxy isovaleryl A Kaning, 1-methoxyl group acetylshikonin, acetylshikonin etc., all belong to 2,4-naphthoquinones class organic compound) content is investigated, and good culture is reserved seed for planting to select comprehensive proterties.
Clone of the present invention has high-load effective constituent, can be used for preparing food, makeup, health care food and medicine.
Clone of the present invention can be used for the scale operation of lithospermum euchromum Royle cell culture, has not only satisfied clinical demand, and has protected the wild lithospermum euchromum Royle of national rare plant.
Advantage of the present invention mainly embodies:
(1) clone Shikonin provided by the invention and derivative content height thereof, Shikonin and derivative content thereof account for the 8-10% of culture dry weight, and high-efficiency liquid-phase fingerprint shows that ingredient and wild lithospermum euchromum Royle are basic identical in the culture.(2) growth is vigorous, and cell growth rate can reach 12-18g dry weight/liter/month, and culture cycle is short, 30-35 days.(3) stability is high, can infinitely go down to posterity.(4) can carry out scale operation by the inventive method, compare the production cycle shortens greatly with wild-type, and the culture steady quality output height of being produced.(5) the inventive method does not account for the arable land, does not destroy the lithospermum euchromum Royle wild resource, has protected ecotope, has solved the problem of lithospermum euchromum Royle shortage of resources.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
The acquisition of embodiment 1 lithospermum euchromum Royle clone
Get the seed of natural lithospermum euchromum Royle, earlier with 75% alcohol-pickled 10-30s, then with 1% mercuric chloride solution sterilization 8-12min, wash 4-5 time repeatedly with sterilized water again, be placed at last and blot seed-coat moisture on the aseptic filter paper, be seeded in the basic MS (prescription sees attached list 1) with aseptic nipper and add, on the solid medium of 10-30g/L sucrose, 0.6g/L agar, 23-25 ℃ unglazed according to cultivating, and seed can be sprouted and grow aseptic seedling in about 10 days.
Get aseptic plumule and do explant, under aseptic condition, with aseptic operation scissors and aseptic nipper aseptic shoot root, stem, leaf and cotyledon, plumule are cut into the long segment of 2-5mm, be seeded on the solid medium that adds 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose among the N6 (prescription sees attached list 2), 25 ℃ of dark cultivations induced callus in 10 days.
The present invention by to the saussurea involucrata callus induce and cultivation has obtained three kinds of Sinkiang saussurea involucrata cell lines: white color system, yellow system and purplish red colour system; 3 kinds of clones have been carried out succeeding transfer culture respectively,, determined that the red-purple cell is that growing way is better, Shikonin and the highest clone of derivative content thereof by detection to its speed of growth, total flavones compounds content.
Choose the red-purple cell and be initiating cell system, from solid medium, select color red-purple callus dark, that be in bright colors adds 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose in N6 (prescription sees attached list 2) solid medium with appearance method and continue succeeding transfer culture.Culture condition is 23-25 ℃, and is unglazed according to cultivating.Be better optimize seed, purifying cells system, the inoculation piece size is controlled at below the 3mm during subculture.So repeatedly, selected clone is progressively tamed, and the present invention has obtained the stable high yield lithospermum euchromum Royle clone that can infinitely go down to posterity and cultivate through the screening of nearly 100 generations, and this cell line growth is vigorous, bright-colored, be red-purple, compact structure, be lumps, quality homogeneous, separate easily; Its high-efficiency liquid-phase fingerprint and wild alkanna are identical substantially, and the content of Shikonin and derivative thereof all is higher than wild lithospermum euchromum Royle; The content of Shikonin and derivative thereof is the 6-8% of dry cell weight.
Embodiment 2 liquid-solid alternately production methods
1, the liquid shaking table is cultivated the production of hybrid seeds
1.1 seed selection
Choose and cultivated 20-25 days, growing way is better, and is bright-colored, is red-purple, uniform particles, and the quality homogeneous, segregative cloned culture of the present invention is made seed.
1.2 substratum preparation
Adopt the 500ml culturing bottle, every bottled 120-150ml nutrient solution, nutrient solution composition are interpolation 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT among the N6 (prescription sees attached list 2), reach 30g/L sucrose, and the pH value is 5.2-5.8; High-temperature sterilization: 121 ℃, 20min.
1.3 inoculation
Under the aseptic condition, culture is inoculated in the 500ml culturing bottle that the 120-150ml nutrient solution is housed after broken with tweezers folders, and inoculum size 3-6% (W/V, g.fw/ml).
1.4 shaking table is cultivated
Culture condition: culture temperature is 22-27 ℃, shaking speed 100-120rpm, and incubation time 8-10d is as illumination cultivation.In the culturing process, observe more, the discovery microbiological contamination is chosen immediately.
2, solid culture
2.1 choosing of liquid seed
Detect with microscopic examination, get colors bright-coloured, be red-purple, growing way better, good dispersity, the free of contamination culture of uniform particles do to produce with kind.
2.2 substratum
Add the solid medium of 0.2mg/L-0.5mg/L IAA, 1mg/L-5mg/L KT, 0.6g/L agar and 30g/L sucrose among the N6 (prescription sees attached list 2)
2.3 inoculation
Under the aseptic condition, with the nutrient solution filtering in the culturing bottle, culture is transferred in the sterile petri dish that is lined with aseptic filter paper, picking growth is vigorous, bright-colored, be red-purple, good dispersity, uniform particles, free of contamination cell mass are forwarded on the solid medium, and the inoculation piece size is 4-6mm.
2.4 solid culture
Culture temperature: 23-25 ℃, unglazed according to cultivating culture cycle: 30-35 days.
The collection of 3 cultures and cyclic production
3.1 reserve seed for planting
In the time of scale operation, can therefrom select to grow vigorous, bright-colored, be red-purple, compact structure, be lumps, quality evenly, reserve seed for planting for the callus of feature firmly slightly partially, the liquid shaking table that is used for step 1 is cultivated, and the seed selection amount can not surpass 15% of gross production and be advisable.
3.2 the collection of culture
Collect the residue culture, low-temperature vacuum drying gets final product.As required, the exsiccant culture can be worn into meal or fine powder, perhaps be used for extracting Shikonin and derivatives active composition thereof.
After measured, the scale operation cost that carries out with this method is lower, and the culture growth velocity is fast, and output can reach 20-25g dry weight/moon/rise substratum; Shikonin and derivative content height thereof in the dry product culture reach the 6-8% of culture dry weight and culture steady quality.
More than provided preferred implementation of the present invention, those skilled in the art are easy to expect adopting similar method that condition of the present invention is replaced, for example that succeeding transfer culture is used hormone replaces to the hormone of similar functions, used substratum is replaced to the substratum of similar functions, culture condition is made an amendment, yet these changes do not change essence of the present invention, thereby all belong within the scope of the present invention.
Subordinate list 1MS culture medium prescription
Subordinate list 2N6 culture medium prescription
Claims (12)
1, a kind of lithospermum euchromum Royle red-purple clone, it prepares by the following method: adopting the embryo of lithospermum euchromum Royle or the aseptic seedling of seed germination is explant, through evoked callus and succeeding transfer culture repeatedly, from isolated cells system, select bright-colored red-purple, the compact structure of being, crumby culture, promptly.
2, clone as claimed in claim 1, it is a lithospermum euchromum Royle red-purple clone.
3, by claim 1 or 2 cultures of telling the clone acquisition.
4, claim 1 or 2 succeeding transfer culture methods of telling clone is characterized in that used substratum is the N6 substratum that contains 0.1-0.5mg/LIAA and 1-5mg/L KT, and culture temperature is 23～25 ℃, and unglazed according to cultivating, 30～35 days subcultures once.
5, as claim 4 described succeeding transfer culture methods, it is characterized in that, select vigorous, bright-colored red-purple, compact structure, the tangible culture subculture of crumb structure of being of growth during subculture.
6, succeeding transfer culture method as claimed in claim 5, it is characterized in that, also comprise during subculture Shikonin in the culture and derivative (β thereof, β '-dimethyl allene acyl A Kaning, deoxyshikonin, racemization acetyl A Kaning, acetyl A Kaning, β-acetoxyl group isovaleryl A Kaning, Shikalkin, beta-hydroxy isovaleryl A Kaning, 1-methoxyl group acetylshikonin, acetylshikonin etc., all belong to 2,4-naphthoquinones class organic compound) content is investigated, and selects the good culture subculture of comprehensive proterties.
7, as the large scale production method of culture as described in the claim 3, its step comprises:
1) claim 1 or 2 are told clone and adopt the production of hybrid seeds of liquid shaking table cultured method;
2) seed that step 1) is obtained is used for solid culture;
3) selection step 2) growth culture vigorous, bright-colored, the red-purple feature is reserved seed for planting in the culture, and the shaking table that is used for step 1) is cultivated, and all the other results get culture.
8, method as claimed in claim 7, it is characterized in that, step 1) liquid shaking table is cultivated used substratum for adding the N6 liquid nutrient medium of 0.1-0.5mg/L IAA and 1-5mg/L KT, inoculum size 3-6%, culture condition: pH value is 5.2-5.8, and culture temperature is 23-25 ℃, shaking speed 100-120rpm, unglazed according to cultivating incubation time 8-10 days.
9, method as claimed in claim 8, it is characterized in that, step 2) solid culture select bright-colored, be red-purple, grow fine, good dispersity, uniform particles, free of contamination culture carry out solid-state cultivation production as seed, be inoculated into the N6 solid medium that contains 0.1-0.5mg/L IAA and 1-5mg/L KT, inoculation is 4-6mm soon, culture condition: 23～25 ℃ of temperature, unglazed according to cultivating, cultivated 30～35 days.
10, as each described method of claim 7-9, it is characterized in that, also comprise Shikonin and derivative (β thereof to culture, β '-dimethyl allene acyl A Kaning, deoxyshikonin, racemization acetyl A Kaning, acetyl A Kaning, β-acetoxyl group isovaleryl A Kaning, Shikalkin, beta-hydroxy isovaleryl A Kaning, 1-methoxyl group acetylshikonin, acetylshikonin etc., all belong to 2,4-naphthoquinones class organic compound) content is investigated, and good culture is reserved seed for planting to select comprehensive proterties.
11, claim 1 or 2 described clones are used in preparation food, protective foods, makeup, healthcare products and medicine.
12, claim 3 described cultures are used in preparation food, protective foods, makeup, healthcare products and medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745482A (en) * | 2014-09-12 | 2015-07-01 | 中国人民解放军第二军医大学 | Endophytic fungi of amebia enchroma and application of endophytic fungi |
| CN104771384A (en) * | 2014-01-15 | 2015-07-15 | 中国科学院上海生命科学研究院湖州营养与健康产业创新中心 | Pharmaceutical use of alkannin |
| CN110269852A (en) * | 2019-08-07 | 2019-09-24 | 中美（河南）荷美尔肿瘤研究院 | β, beta-dimethyl-acry-lalkannin application in preparation of anti-tumor drugs |
-
2008
- 2008-04-01 CN CNA2008100108788A patent/CN101289651A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104771384A (en) * | 2014-01-15 | 2015-07-15 | 中国科学院上海生命科学研究院湖州营养与健康产业创新中心 | Pharmaceutical use of alkannin |
| CN104745482A (en) * | 2014-09-12 | 2015-07-01 | 中国人民解放军第二军医大学 | Endophytic fungi of amebia enchroma and application of endophytic fungi |
| CN110269852A (en) * | 2019-08-07 | 2019-09-24 | 中美（河南）荷美尔肿瘤研究院 | β, beta-dimethyl-acry-lalkannin application in preparation of anti-tumor drugs |
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Open date: 20081022 |