CN101338298B - Saussurea involucrata cell lines - Google Patents
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Abstract
The invention provides a purple cell system of a Saussurea Involucrata Sau-1 CGMCC No.2506 which stably products flavone with high yield. The content of a flavone compound can achieve 8 to 12 percent of the dry weight of a culture which is 7 to 10 times of a natural Involucrata. The invention also provides a culture technology of the cell system and a method for fast producing the culture of the cell system in a large scale. The method adopts the combination of liquid-solid culture, manufactures the seeds by liquid and obtains the culture by solid culture, selects the seeds with better comprehensive situations for liquid culture and repeats in the way. The method can be used for large scale production. Compared with a wild type, the production period is greatly shortened; besides, the quality is stable and the yield is high. Besides, the method does not occupy the farming land, does not damage the wild resource of the Involucrata., protects the environment and solves the problem of lack of the source of Saussurea Involucrata.
Description
Technical field
The present invention relates to a kind of saussurea involucrata cell line, the invention still further relates to the preparation method of this clone and the large scale production method of culture thereof.
Background technology
Saussurea involucrata is the geographic medicinal plant among the people of high mountain, be used for dispelling cold and removing dampness, promoting blood circulation to restore menstrual flow, relieving inflammation and relaxing pain etc., be used for rheumatic arthritis, women's cold and pain in the lower abdomen, amenorrhoea among the people more, the treatment of diseases such as retention of placenta, paralysis are not saturating, lung cold cough, impotence, high mountain inadaptation.In recent years, saussurea involucrata receives much attention in the effect aspect relieving inflammation and relaxing pain, antiearly pregnancy, the anti-ageing and anticancer hyperplasia as ethnic drug.But China saussurea involucrata mainly is distributed in the above plateau refrigerant latitudes of 4000m, as Xinjiang, Gansu, Sichuan, Yunnan and Tibet.These local climates are changeable, cold and hot impermanence, the highest monthly mean temperature 3-10 ℃, minimum monthly mean temperature is then spent to tens degree subzero tens, growing environment is very abominable, have only cold-resistant sedge genus, wormwood to belong to and the association with it of minority high mountain per nnial herb, general saussurea involucrata plant under this environment mostly is perennial greatly, poor growth, the artificial culture difficulty, predatoriness is excavated and is made the saussurea involucrata resource seriously deficient for a long time, has made that saussurea involucrata becomes endangered species, and natural resources is difficult to satisfy clinical growing needs.Therefore the exploitation of carrying out saussurea involucrata of application cell cultured method promptly can be satisfied clinical demand to the saussurea involucrata medicine, also can protect national resource, and safeguards ecotope.
Application number is that the Chinese patent of 00123739.X and 00123740.3 discloses two kinds of Saussurea medusa high yield clones respectively, and flavones content accounts for 6.9% and 6.23% of dry cell weight respectively.But also there is not the relevant report that further improves flavones content.In addition, saussurea involucrata cell line is unstable in amplification process, when amplification culture content of effective after the certain scale can reduce the effective means that does not also address the above problem at present.
Summary of the invention
First purpose of the present invention is to provide a kind of saussurea involucrata cell line of high yield flavones.
The present invention is with Herba Saussureae Involueratae (its embryo, root, stem, leaf, perhaps by the aseptic seedling of seed germination) as explant, evoked callus, and carry out succeeding transfer culture repeatedly, obtain four kinds of Sinkiang saussurea involucrata cell lines: white color system, yellow system, green system and purplish red colour system; 4 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 total flavones compounds content is the highest by detection to its speed of growth, total flavones compounds content.From isolated cells system, select bright-colored red-purple, compact structure, the group's granulous 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, integrated survey content of effective and growth characteristics, the stable high yield saussurea involucrata cell line Sau-1 that has finally obtained infinitely to go down to posterity and cultivated, this clone has been deposited in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on May 20th, 2008, Institute of Microorganism, Academia Sinica, 100101), preserving number is CGMMCC No.2506, classification called after Herba Saussureae Involueratae (Saussura involucrate) (Herba Saussureae Involueratae is Herba Saussureae Involueratae).This saussurea involucrata cell line has following feature: grow vigorous, bright-colored, be red-purple, compact structure, be a shape, quality evenly, slightly partially firmly, chromocor compound content is the 8-12% of culture dry weight, culture cycle 10-14 days.Specifically, can prepare above-mentioned clone by the following method:
(1) cultivation of Herba Saussureae Involueratae aseptic seedling
Get the seed of natural Herba Saussureae Involueratae, 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 on the solid medium that adds 0.5-3mg/L GA (Plant hormones regulators,gibberellins), 10-30g/L sucrose, 0.6g/L agar among the 1/2MS (prescription sees attached list 1) with aseptic nipper, 22-27 ℃ of illumination cultivation, 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/L6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose among the MS (prescription sees attached list 1), 22-27 ℃ of dark the cultivation induced callus in 7-10 days.
(3) subculture of callus screening
The present invention by to the saussurea involucrata callus induce and cultivation has obtained four kinds of Sinkiang saussurea involucrata cell lines: white color system, yellow system, green system and purplish red colour system; 4 kinds of clones have been carried out succeeding transfer culture respectively,, determined that the red-purple cell is that growing way is better, the clone that the total flavones compounds content is the highest by detection to its speed of growth, total flavones compounds content.
Choosing the red-purple cell is initiating cell system, selects color red-purple callus dark, that be in bright colors with appearance method from solid medium and continues succeeding transfer culture at the solid medium that adds 0.2mg/L-0.5mg/L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose in MS (prescription sees attached list 1).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 saussurea involucrata cell line Sau-1 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 saussurea involucrata cell line, its used substratum is to add 0.2mg/L-0.5mg/L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose in the MS substratum (prescription sees attached list 1), pH value 5.2-5.8, the inoculation piece is 4-6mm, culture condition: temperature 22-27 ℃, illumination 10-12 hour, incubation time 15～20 days.Select during subculture growth vigorous, bright-colored, be red-purple, compact structure, be a shape, quality evenly, hard slightly partially be the culture subculture of feature.In addition flavones, chlorogenic acid, isochlorogenic acid and the Syringa oblata Lindl. saponin content of culture are investigated, selected 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) 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.
Specifically, can adopt following method to carry out scale operation:
Sau-1 does seed with the high yield saussurea involucrata cell line, suspension culture in the 500mL culturing bottle that the 150mL liquid nutrient medium is housed, liquid nutrient medium is that MS substratum (prescription sees attached list 1) adds 0.2mg/L-0.5mg/L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthylacetic acid) and 30g/L sucrose, pH value 5.2-5.8, inoculum size 3-6% (W/V, g.fw/ml), culture condition: temperature is 22-27 ℃, shaking speed 100-120rpm, incubation time 8-10 days, illumination 10-12 hour, 2000lax.
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 that MS substratum (prescription sees attached list 1) adds 0.2mg/L-0.5mg/L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose, pH value 5.2-5.8; Culture condition: temperature 22-27 ℃, illumination 10-12 hour, 20001ax, incubation time 15-20 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 a shape, 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 generally is no more than 15% of gross production, all the other are collected and low-temperature vacuum drying promptly gets saussurea involucrata 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.Can also investigate the content of flavones, chlorogenic acid, isochlorogenic acid and the Syringa oblata Lindl. saponin of culture in process of production, 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, protective foods and medicine.
Clone of the present invention can be used for the scale operation of saussurea involucrata cell culture, has not only satisfied clinical demand, and has protected the wild saussurea involucrata of national rare plant.
Advantage of the present invention mainly embodies:
(1) clone flavones content height provided by the invention, Saussurea involucrate general flavone content accounts for the 8-12% of culture dry weight, and high-efficiency liquid-phase fingerprint shows that ingredient and wild saussurea involucrata are basic identical in the culture.(2) growth is vigorous, and culture cycle is short, 15-20 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 saussurea involucrata wild resource, has protected ecotope, has solved the problem of Herba Saussureae Involueratae shortage of resources.
Description of drawings
Fig. 1 tenuigenin spectrum analysis of the present invention HPLC spectrogram.
Embodiment
Further specify the present invention below in conjunction with 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 Herba Saussureae Involueratae red-purple clone
Get the seed of natural Herba Saussureae Involueratae, 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 on the solid medium that adds 2.5mg/L GA (Plant hormones regulators,gibberellins), 30g/L sucrose, 0.6g/L agar among the 1/2MS (prescription sees attached list 1) with aseptic nipper, 25 ℃ of illumination cultivation, seed can be sprouted and grow aseptic seedling in about 10 days.
Get the aseptic seedling plumule and do explant, under aseptic condition, with aseptic operation scissors and aseptic nipper the aseptic seedling plumule is cut into the long segment of 2-5mm, be seeded on the solid medium that adds 0.4mg/L 6-BA (6-benzyl purine), 3mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose among the MS (prescription sees attached list 1), 25 ℃ of dark cultivations induced callus in 8 days.
The present invention by to the saussurea involucrata callus induce and cultivation has obtained four kinds of Sinkiang saussurea involucrata cell lines: white color system, yellow system, green system and purplish red colour system; 4 kinds of clones have been carried out succeeding transfer culture respectively,, determined that the red-purple cell is that growing way is better, the clone that the total flavones compounds content is the highest by detection to its speed of growth, total flavones compounds content.Root, stem, leaf and cotyledon with aseptic seedling also all can obtain above-mentioned red-purple clone, preferably adopt stem.
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 0.5mg/L 6-BA (6-benzyl purine), 4mg/L NAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose in MS (prescription sees attached list 1) solid medium with appearance method and continue succeeding transfer culture.Culture condition is 25 ℃, illumination 10-12 hour, 20001ax.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 saussurea involucrata cell line Sau-1 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 a shape, quality homogeneous, separate easily; Its high-efficiency liquid-phase fingerprint (Fig. 1, qualification result sees Table 1) and wild saussurea involucrata are identical substantially, and the content of chlorogenic acid, isochlorogenic acid all is higher than wild saussurea involucrata; Saussurea involucrate general flavone content is the 8-12% of dry cell weight.
Table 1HPLC qualification result table
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 10-14 days, growing way is better, and is bright-colored, is red-purple, uniform particles, and the quality homogeneous, segregative clone Sau-1 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 to add 0.4mg/L 6-BA (6-benzyl purine), 3mg/L NAA (naphthylacetic acid) and 30g/L sucrose among the MS (prescription sees attached list 1), and the pH value is 5.5; 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 5% (W/V, g.fw/ml).
1.4 shaking table is cultivated
Culture condition: culture temperature is 25 ℃, shaking speed 110rpm, incubation time 9d, illumination 11h, 20001ax.
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 0.3mg/L 6-BA (6-benzyl purine), 3mg/LNAA (naphthylacetic acid), 0.6g/L agar and 30g/L sucrose among the MS (prescription sees attached list 1).
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: 25 ℃, illumination 11h, 20001ax; Culture cycle: 15 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 a shape, 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 is advisable to be no more than 15% of gross production.
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 flavones and other activeconstituentss.
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; General flavone content height in the dry product culture reaches the 8-12% 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.
Claims (9)
1. the succeeding transfer culture method of Herba Saussureae Involueratae red-purple clone, it is characterized in that, used substratum is the MS substratum that contains 0.1-0.5mg/L 6-BA and 1-5mg/L NAA, culture temperature is 22～27 ℃, 15～20 days subcultures once, described Herba Saussureae Involueratae red-purple cell is that Herba Saussureae Involueratae (Saussurea involucrate) Sau-1 preserving number is CGMCCNo.2506.
2. succeeding transfer culture method as claimed in claim 1 is characterized in that, selects vigorous, bright-colored red-purple, compact structure, the tangible culture subculture of granular structure of being of growth during subculture.
3. succeeding transfer culture method as claimed in claim 2 is characterized in that, also comprises during subculture flavones, chlorogenic acid, isochlorogenic acid and Syringa oblata Lindl. saponin content in the culture are investigated, and selects the good culture subculture of comprehensive proterties.
4. the large scale production method of the culture of Herba Saussureae Involueratae red-purple clone, its step comprises:
1) Herba Saussureae Involueratae red-purple clone is adopted 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,
Described Herba Saussureae Involueratae red-purple cell is that Herba Saussureae Involueratae (Saussurea involucrate) Sau-1 preserving number is CGMCC No.2506.
5. method as claimed in claim 4, it is characterized in that, step 1) liquid shaking table is cultivated used substratum for adding the MS liquid nutrient medium of 0.1-0.5mg/L 6-BA and 1-5mg/L NAA, inoculum size 3-6%, culture condition: the pH value is 5.2-5.8, and culture temperature is 22-27 ℃, shaking speed 100-120rpm, cultivated illumination 10-12 every day hour, 2000lax 8-10 days.
6. method as claimed in claim 5, 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 on the MS solid medium that contains 0.1-0.5mg/L 6-BA and 1-5mg/L NAA, the inoculation piece is 4-6mm, culture condition: temperature 22-27 ℃, illumination 10-12 every day hour was cultivated 15～20 days.
7. as each described method of claim 4-6, it is characterized in that comprise that also flavones, chlorogenic acid, isochlorogenic acid and the Syringa oblata Lindl. saponin content to culture investigated, good culture is reserved seed for planting to select comprehensive proterties.
8. the application of Herba Saussureae Involueratae red-purple clone in preparation food, healthcare products and medicine, described Herba Saussureae Involueratae red-purple cell is that Herba Saussureae Involueratae (Saussurea involucrate) Sau-1 preserving number is CGMCC No.2506.
9. application as claimed in claim 8, wherein said food are protective foods.
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| CN102450647B (en) * | 2010-10-26 | 2013-06-19 | 天津天狮生物发展有限公司 | Snow lotus culture composition for preventing and relieving arthritis |
| CN103305454A (en) * | 2012-03-14 | 2013-09-18 | 中国医学科学院药物研究所 | Method for producing Syringin, chlorogenic acid and 1, 5-dicaffeoylquinic acid by cell culture of herba saussureae involucratae |
| CN107646685A (en) * | 2017-10-30 | 2018-02-02 | 江苏凤谷生物有限公司 | A kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones |
| CN108504623A (en) * | 2018-04-28 | 2018-09-07 | 大连普瑞康生物技术有限公司 | A kind of medusa-snow lotus cell line for generating flavone with high output and preparation method thereof |
| CN115786232B (en) * | 2022-12-19 | 2023-10-24 | 中国科学院青岛生物能源与过程研究所 | Quick suspension culture and genetic transformation method for saussurea involucrata cells |
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