[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2021103863A1 - 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法 - Google Patents

一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法 Download PDF

Info

Publication number
WO2021103863A1
WO2021103863A1 PCT/CN2020/122753 CN2020122753W WO2021103863A1 WO 2021103863 A1 WO2021103863 A1 WO 2021103863A1 CN 2020122753 W CN2020122753 W CN 2020122753W WO 2021103863 A1 WO2021103863 A1 WO 2021103863A1
Authority
WO
WIPO (PCT)
Prior art keywords
culture
medium
content
inducer
saussurea
Prior art date
Application number
PCT/CN2020/122753
Other languages
English (en)
French (fr)
Inventor
刘汉石
金银兵
刘雅萍
Original Assignee
大连普瑞康生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 大连普瑞康生物技术有限公司 filed Critical 大连普瑞康生物技术有限公司
Priority to EP20894710.1A priority Critical patent/EP4067479A4/en
Priority to US17/756,713 priority patent/US20230287336A1/en
Publication of WO2021103863A1 publication Critical patent/WO2021103863A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/02—Flowers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids

Definitions

  • the invention belongs to the field of plant biotechnology engineering, and relates to a tissue culture of medicinal plants and a culture method thereof, in particular to a method for improving the flavonoid phenylpropanoid compound of the Saussurea cell culture.
  • Saussurea is a folk medicinal plant in high mountains. It is used to dispel cold and dehumidify, promote blood circulation and relieve menstruation, anti-inflammatory and analgesic, etc. It is mostly used for rheumatoid arthritis, cold pain in the lower abdomen of women, amenorrhea, fetal clothing, numbness and coldness Treatment of cough, impotence, mountain maladaptation and other symptoms. In recent years, as a national medicine, Saussurea has attracted much attention in anti-inflammatory and analgesic, anti-early pregnancy, anti-aging and inhibiting cancer cell proliferation.
  • the flavonoids and their glycosides, sesquiterpene lactones, coumarins, lignans, steroids, polysaccharides and other ingredients contained in Saussurea lotus have anti-rheumatism, analgesia, regulation of the cardiovascular system, anti-cancer, anti-aging, family planning , Anti-radiation and other biological activities, have great development and utilization value, and have good application prospects.
  • syringin has anti-inflammatory, analgesic, anti-hyperglycemic, anti-depressant and anti-cancer activities; chlorogenic acid has anti-inflammatory, anti-hyperglycemic and anti-hepatitis B virus effects; 1,5-dicaffeoylquine Acid has the effect of anti-oxidant treatment of diabetes and anti-AIDS, as a new anti-AIDS drug has entered the stage of clinical research.
  • syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid have a variety of medicinal activities and have important application values.
  • Syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid are all active ingredients possessed by the wild Saussurea serrata, but the content is low. The production of these three components by cell culture of Saussurea serrata has a good prospect.
  • the obtained culture has a single composition, mostly with a single total flavonoids as an indicator, and the content is low, which makes it difficult to realize the true value of Saussurea; 2. In view of the problem of low content of certain components, no effective means to obtain high-yielding cell lines has been found Etc.; 3. The inducer of the culture is only to obtain higher total flavonoids, and other components of the snow lotus itself are ignored; 4. The metabolic regulation only pays attention to the addition of the inducer, and does not block the flow of branch metabolic pathways. The synthesis of metabolites is not required in order to achieve a method of increasing product yield; 5.
  • the culture process is mostly a common basic medium, and no basic medium for snow lotus is established according to the characteristics of the snow lotus and the quality of the culture; 6. Most of the cultures Staying in the laboratory, it is difficult to cultivate on a large scale, and it is unstable during the scale-up process. When the scale-up culture reaches a certain scale, the content of active ingredients will decrease.
  • domestic and foreign researchers have cultivated the callus and cell suspension of Saussurea A lot of work has been done in cultivation and fermentation culture.
  • the current culturing effect of Saussurea cellulosae and the medicinal ingredients of Saussurea culture are single, and the improvement of the content of main medicinal ingredients needs to be explored.
  • the lack of optimization of the basic culture environment and the lack of regulation of metabolic pathways have resulted in the lack of a stable, high-yield, and rapid regulation process for large-scale cultivation of Saussurea cells.
  • the present invention provides a method for improving the flavonoid phenylpropanoid compound of the Saussurea cell culture.
  • a medium for improving the flavonoid phenylpropanoid compound of Saussurea cell culture in the medium, the concentration of phosphorus element is 0.5-3 mmol/L, the concentration of NO 3 - ion is 20-35 mmol/L, and the NH 4 +- ion concentration of 0-30mmol / L, Ca 2+ ion concentration 0.5-3mmol / L, Mg 2+ ion concentration of 0.2-1.5mmol / L, the concentration of boron ions 0.02-0.1mmol /.
  • the phosphorus element concentration is 2.7 mmol/L
  • the NO 3 - ion concentration is 30 mmol/L
  • the NH 4 + -ion concentration is 18 mmol/L
  • the Ca 2+ ion concentration is 0.6 mmol/L
  • Mg 2 +The ion concentration is 0.3mmol/L
  • the boron ion concentration is 0.03mmol/L.
  • the medium is (mg/L): potassium nitrate 3000, ammonium sulfate 793, diammonium hydrogen phosphate 340, magnesium sulfate heptahydrate 74, calcium chloride dihydrate 88, potassium iodide 0.83, boric acid 2, sulfuric acid tetrahydrate Manganese 22.3, ferrous sulfate heptahydrate 27.8, zinc sulfate heptahydrate 8.6, inositol 100, sodium molybdate dihydrate 0.25, thiamine hydrochloride 0.1, copper sulfate pentahydrate 0.025, pyridoxine hydrochloride 0.5, cobalt chloride hexahydrate 0.025, niacin 0.5, disodium edetate 37.3, glycine 2.
  • an inducer for improving the flavonoid phenylpropanoid compound of Saussurea cell culture is one or more of SNP inducer, MeJ inducer, SA inducer, silver ion and its complex inducer Among them, the concentration of SNP inducer is 1-50mmol/L, the concentration of MeJ inducer is 1-50mg/L, the concentration of SA inducer is 1-50mg/L, silver ion and its complex inducer The added concentration is 1-50mg/L.
  • the concentration of the SNP inducer is 10 mmol/L
  • the concentration of the MeJ inducer is 20 mg/L
  • the concentration of the SA inducer is 30 mg/L
  • the concentration of silver ion and its complex inducer is 20 mg. /L.
  • a precursor for improving the flavonoid phenylpropanoid compound of Saussurea cell culture wherein the precursor is one or a combination of cinnamic acid, phenylalanine, sodium acetate, tyrosine, and oxalic acid; wherein, Add 1-20mg/L of cinnamic acid, 1-20mg/L of phenylalanine, 1-20mg/L of sodium acetate, 1-50mg/L of tyrosine, and 1-20mgl/L of oxalic acid.
  • cinnamic acid was added 10mg/L
  • phenylalanine was added 10mg/L
  • sodium acetate was added 10mg/L
  • tyrosine was added 20mg/L
  • oxalic acid was added 10mgl/L.
  • the bypass metabolism inhibitor is arginine, trisodium citrate, sodium fluoride, cyclohexylalanine, One or several combinations of 5-MT (5-methoxytryptophan); among them, arginine 0-100mg/L, trisodium citrate 0-50nmg/L, sodium fluoride 0-100mg/L , Cyclohexylalanine 0-100mg/L, 5-MT (5-methoxytryptophan) 0-100mg/L.
  • arginine added 10mg/L
  • trisodium citrate added 15mg/L
  • sodium fluoride added 15mg/L
  • cyclohexylalanine added 10mg/L
  • 5-MT(5-methoxytryptophan) Acid Add 10mg/L.
  • a method for improving the flavonoid phenylpropanoid compound of Saussurea cell culture comprising the following steps:
  • Saussurea praecox Using seed embryos of Saussurea praecox or plants germinated from seeds as explants, inducing callus in an induction medium and performing repeated subcultures to obtain Saussurea cell lines;
  • the Saussurea cell line obtained in step (1) is inoculated into the culture medium, the inoculation amount is 1 ⁇ 50g cell fresh weight per liter of culture medium, illumination 1000-3000lxu, illumination time 12-24h/day, 100-130 revolutions 1/min shaker, subculture once every 7 to 20 days, after culturing for 3 to 4 generations, a stable and high-yield suspension cell culture line of Saussurea tianshanensis can be established;
  • step (3) Large-scale cultivation: select the culture in step (2), culture it in a medium, inoculate 10 to 80 g of fresh cell weight per liter of medium, and after suspension culture for 14 to 20 days, filter and harvest the cells;
  • the medium of step (2) and step (3) is the aforementioned medium; or the medium is a medium supplemented with the aforementioned precursor; or the medium is a medium supplemented with the aforementioned inducer; or The medium is a medium to which the aforementioned bypass metabolism inhibitor is added.
  • the precursor when the precursor is cinnamic acid, phenylalanine, or sodium acetate, it is added at the time of inoculation; when the precursor is tyrosine or oxalic acid, it is added at the middle of the culture; the inducer is added at the middle of the culture Add at the time; the bypass metabolism inhibitor is added at the later stage of the culture.
  • step (3) large-scale culture, the cells are inserted into a culture flask containing 5-20 glass beads with a diameter of 1-10 mm in the culture flask for culture.
  • the culture conditions are the same as the original cells, and the cells are eliminated during the subculture process.
  • a shear-resistant, stable, high-yielding suspension cell culture line of Saussurea tianshanensis is established after 10 generations.
  • the method of the present invention uses the culture medium, inducer, precursor, and bypass metabolism inhibitor of the present invention to significantly increase the total flavonoid content and the content of the Saussurea cell culture.
  • the seeds of natural Xinjiang Saussurea have been identified by the Herbarium of the Institute of Chinese Academy of Sciences (National Herbarium), first soaked in 75% alcohol for 10-30s, then sterilized with 1% mercuric chloride solution for 8-12 minutes, and then sterilized Rinse 4-5 times with water, and finally put it on sterile filter paper to absorb the moisture on the surface of the seeds, and inoculate with sterile tweezers in 1/2MS (see attached table 3 for the formula) and add 0.5-3mg/L GA (gibberellin) , 10-30g/L sucrose, 0.6g/L agar solid medium, 22-27 °C light culture, about 10 days after the seeds can germinate and grow sterile seedlings.
  • GA gibberellin
  • the saussurea cells expanded and cultured as described in Example 3 were inoculated into liquid synthetic medium (see Table 1) +0.2mg/L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/ L NAA (naphthalene acetic acid), 30g/L sucrose, in shake flask suspension culture, specifically: use the culture flask as the culture container, use the shaker to rotate and shake the culture, the inoculation amount is 1-50g cell fresh weight per liter of culture medium, At 25 ⁇ 2°C, light 1000-3000lxu, light time 12-24h/day, 100-130 rpm shaker, subculture once every 7d ⁇ 20d, stable and high yield can be established after 3 ⁇ 4 generations The Tianshan Saussurea suspension cell culture system.
  • liquid synthetic medium see Table 1 +0.2mg/L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/ L NAA (naphthalene acetic acid), 30g
  • the Saussurea cells in Example 3 or 4 were connected to a culture flask containing 5-20 glass beads with a diameter of 1-10 mm in the culture flask for culture.
  • the culture medium was the same as that used for the original cells.
  • the culture conditions were: 25 ⁇ 2°C, light 1000-3000lxu, light time 12-24h/day, 100-130 rpm shaker, subculture 1 time every 7d ⁇ 20d, weed out culture flasks with poor growth during subculture, 10
  • a stable, high-yielding suspension cell culture system of Saussurea tianshanensis with shear resistance was established.
  • the optimized cell line was continuously subcultured and optimized under the same shear force in the culture flask, and the culture could be used for the next step of fermentation tank scale-up culture. .
  • Example 3 The cultures of Example 3 were selected from the cultures of Example 3 and cultured for 8-12 days. The cultures were well grown, brightly colored, purple-red, uniform particles, uniform texture, and easily separated cultures as seeds.
  • each bottle contains 500ml culture medium, and add 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g on the basis of MS medium (see Table 2).
  • 6-BA 6-benzylpurine
  • NAA naphthalene acetic acid
  • MS medium see Table 2.
  • /L sucrose, pH value is 5.5; high temperature sterilization: 121°C, 20min.
  • the culture was inoculated into a culture flask containing a culture solution, and the inoculum amount was 5% (W/V, g.fw/ml).
  • Culture conditions culture temperature is 25 ⁇ 2°C, shaker speed is 110rpm, culture time is 9d, illumination is 11h, 2000lux.
  • the dried culture can be ground into coarse powder or fine powder, or used to extract flavonoids and other active ingredients.
  • the growth is good, the color is bright, the purple-red, the particles are uniform, the texture is uniform, the high-yield Tianshan Saussurea suspension cell culture seeds are expanded and cultured, and the culture barrel equipment is used ( Patent publication number: CN208857314U), equipped with 20-100L synthetic medium (see Table 1), air 0-1m 3 /h, specifically: the medium is synthetic medium + 0.2mg/L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 1-20g dry cell weight per liter of culture medium, at 25 ⁇ 2°C, light 1000-3000lxu, illumination time 12h, suspension culture for 14 days, filter and harvest the cells.
  • the medium is synthetic medium + 0.2mg/L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 15% of the dry cell weight, the chlorogenic acid content is more than 1.03%, the syringin content is more than 0.85%, the 1,5-dicaffeoylquinic acid content is more than 1.6%, and the rutin content is more than 0.06%.
  • Preparation of the reference solution accurately weigh 10 mg of the rutin reference substance dried at 120°C to a constant weight, put it in a 50mL volumetric flask, add an appropriate amount of methanol, heat in a water bath to dissolve, let cool, add methanol to the mark, and shake well. Get it immediately (each mL contains 0.2mg of anhydrous rutin);
  • Preparation of standard curve Precisely measure 1mL, 2mL, 3mL, 4mL, 5mL, 6mL of the reference solution, put them into 25mL volumetric flasks, add water to 6mL, add 1mL of 5% sodium nitrite solution, shake well, and place for 6min. Add 1mL of 10% aluminum nitrate, shake well, place for 6min, add 10mL of sodium hydroxide test solution, add water to the mark, shake well, place for 15min, use the corresponding reagent as a blank, measure the absorbance at 500nm, take the absorbance as the ordinate, The concentration is the abscissa, and the standard curve is drawn;
  • Determination Take about 0.10g of the crude powder of this product, accurately weigh it, put it in a Sox-type extractor, add about 90mL of methanol, and heat to reflux until the extract is colorless (the number of reflux for each sample shall not be less than 4 times and 2 hours). Transfer to a 50mL volumetric flask, add methanol to the mark, and shake well. Accurately measure 5mL and place it in a 25mL volumetric flask. According to the method under the standard curve preparation item, start from "adding water to 6mL", measure the absorbance in accordance with the law, and read the weight of the anhydrous rutin in the test solution from the standard curve (mg), calculate and get.
  • Example 8 Precursor cinnamic acid was added to the medium to produce a large-scale Saussurea culture rich in medicinal ingredients
  • Example 4 Select the cell line in Example 4 as seeds and transfer it to a synthetic medium containing cinnamic acid.
  • the specific formula is: synthetic medium (see Table 1) + different concentrations of cinnamic acid + 0.2mg/L-0.5mg/ L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, use a shaker to culture for more than 3 generations, and use seeds that are multiplied and stabilized for subsequent generations.
  • Plant cell culture barrel equipment Patent Publication No. CN208857314U
  • 20-100L culture medium equipped with 20-100L culture medium, inoculation amount of fresh weight 10g/L, culture time 14 days, light intensity about 1500lux, light time 12h/day
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours.
  • the yield of the dry product is determined to be 15g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 22% of the dry cell weight, the chlorogenic acid content is more than 1.66%, the syringin content is more than 1.45%, the 1,5-dicaffeoylquinic acid content is more than 2.81%, and the rutin content is more than 0.066%.
  • Example 9 Precursor phenylalanine was added to the medium to produce a large-scale Saussurea culture rich in medicinal ingredients
  • the specific formula is: synthetic medium (see Table 1) + different concentrations of phenylalanine + 0.2mg/L -0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose specific formula see the attached table, use a shaker to culture for more than 3 generations, expand The seeds that have been multiplied and stabilized for subsequent generations are transferred to the production culture device.
  • the culture device uses the equipment in the publication number CN208857314U.
  • the device is equipped with 10-50L of culture medium, air 0-1m 3 /h, and the inoculum amount Fresh weight is 40g/L, culture time is 15 days, light intensity is about 1500lux, light time is 12h/day
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours.
  • the yield of the dry product is determined to be 15g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the content of flavonoids accounts for more than 22.7% of the dry cell weight, the content of chlorogenic acid is more than 1.28%, the content of syringin is more than 2.07%, the content of 1,5-dicaffeoylquinic acid is more than 2.48%, and the content of rutin is more than 0.078%.
  • Example 10 Adding sodium acetate to the culture medium to produce a large-scale Saussurea culture rich in medicinal ingredients
  • Example 4 Select the cell line in Example 4 as seeds and transfer it to a synthetic medium containing sodium acetate.
  • the specific formula is: synthetic medium (see Table 1) + different concentrations of sodium acetate + 0.2mg/L-0.5mg /L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, see the attached table for the specific formula.
  • the culture device is CN208857314U medium culture equipment, equipped with 10-50L medium, air 0-1m 3 /h, inoculum weight 50g/L, culture temperature 25 ⁇ 2°C, cultivation time 12 days, light intensity about 1500lux, light time 12h/day.
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours.
  • the yield of the dry product is determined to be 15g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the content of flavonoids accounts for more than 21.8% of the dry cell weight, the content of chlorogenic acid is more than 1.79%, the content of syringin is more than 1.22%, the content of 1,5-dicaffeoylquinic acid is more than 2.35%, and the content of rutin is more than 0.064%.
  • composition of the culture solution is a synthetic medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH 6.0; high temperature sterilization Bacteria: 121°C, 20min.
  • the culture device is a culture barrel in the patent CN208857315U, equipped with 40L culture medium, air 0-1m 3 /h, inoculation volume is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light intensity 1000-2000lux, The light time is 12h/day.
  • the culture reaches the 10th day, add 10-200ml of inducer to the culture bag, and continue the culture for 6 days to collect the culture.
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 18.2 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 19.4% of the dry cell weight, the chlorogenic acid content is more than 0.62%, the syringin content is more than 1.49%, the 1,5-dicaffeoylquinic acid content is more than 2.75%, and the rutin content is more than 0.062%.
  • Example 12 The medium-term addition of oxalic acid to the medium produces a large-scale Saussurea culture rich in medicinal ingredients
  • composition of the culture solution is a synthetic medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH 6.0; high temperature sterilization Bacteria: 121°C, 20min.
  • the culture device is the culture equipment in the patent publication number CN208857314U, equipped with 16L culture medium, air 0-1m 3 /h, and the culture medium is synthetic medium (see attached table) + 0.2mg/L-0.5mg/ L 6-BA (6-benzyl purine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light intensity 1000- 2000lux, illumination time 12h/day, when the culture reaches the 10th day, add the inducer to the culture tank at the ratio of 0.2-4ml/L medium, and collect the culture after culturing for 6 days
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 19.6% of the dry cell weight, the chlorogenic acid content is more than 1.27%, the syringin content is more than 0.95%, the 1,5-dicaffeoylquinic acid content is more than 1.72%, and the rutin content is more than 0.071%.
  • Example 13 Adding the inducer methyl jasmonate to the medium to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is a culture device in the patent publication number CN208857314U, equipped with 10-50L culture medium, air 0-1m 3 /h, and the culture medium is synthetic medium (see Table 1)+0.2mg/L- 0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light The intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 10th day, the inducer is added to the culture tank at the ratio of 0.2-6ml/L medium, and the culture is collected after 6 days of continuous culture.
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 20.4 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the content of flavonoids accounts for more than 24.1% of the dry cell weight, the content of chlorogenic acid is more than 2.44%, the content of syringin is more than 1.89%, the content of 1,5-dicaffeoylquinic acid is more than 4.12%, and the content of rutin is more than 0.081%.
  • Example 14 Adding the inducer silver nitrate to the medium in the mid-term improves the phenylpropanoids in the Saussurea culture
  • the composition of the culture medium is a synthetic medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH 6.0; high temperature sterilization Bacteria: 121°C, 20min.
  • the culture device is a culture device in the patent publication number CN208857314U, equipped with 10-50L culture medium, air 0-1m 3 /h, and the culture medium is synthetic medium (see Table 1)+0.2mg/L- 0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light The intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 10th day, the inducer is added to the culture barrel according to the ratio of 0.2-5ml/L medium, and the culture is collected after 6 days of continuous culture
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 19.5% of the dry cell weight, the chlorogenic acid content is more than 1.47%, the syringin content is more than 1.38%, the 1,5-dicaffeoylquinic acid content is more than 2.97%, and the rutin content is more than 0.065%.
  • Example 15 Adding the inducer salicylic acid to the medium in the mid-term improves the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min;
  • the culture device is the culture device in the patent number CN208577726U, equipped with 10-100L synthetic medium, air 0-1m 3 /h, and the medium is synthetic medium (see Table 1)+0.2mg/L- 0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light The intensity is 1000-2000lux, the illumination time is 12h/day, when the culture reaches the 10th day, the inducer is added to the culture bag according to the ratio of 0.2-8ml/L medium, and the culture is collected after 6 days of continuous culture;
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16.8 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 21.2% of the dry cell weight, the chlorogenic acid content is more than 1.86%, the syringin content is more than 1.09%, the 1,5-dicaffeoylquinic acid content is more than 3.55%, and the rutin content is more than 0.072%.
  • Example 16 Adding a combination inducer to the medium to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is MS medium (see Table 2) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is the culture equipment in the patent number CN208577726U, equipped with 300L synthetic raw medium, air 0-50m 3 /h, and the medium is synthetic medium (see Table 1) + 0.2mg/L-0.5mg /L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light intensity 1000 -2000lux, illumination time 12h/day, when the culture reaches the 10th day, add the inducer to the culture tank according to the ratio of 0.2-5ml/L medium, and collect the culture after 6 days of continuous culture
  • the culture solution is filtered, the upper layer of cells are collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the content of flavonoids accounts for more than 18.1% of the dry cell weight, the content of chlorogenic acid is more than 0.68%, the content of syringin is more than 0.77%, the content of 1,5-dicaffeoylquinic acid is more than 1.85%, and the content of rutin is more than 0.028%.
  • Example 17 Adding a combination inducer to the medium to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is the culture device in the patent number CN208577726U, equipped with 300L synthetic medium, air 0-50m 3 /h, and the medium is synthetic medium (see Table 1) + 0.2mg/ L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation volume of fresh weight 30g/L, culture temperature 25 ⁇ 2°C , Light intensity 1000-2000lux, light time 12h/day, when the culture reaches the 10th day, add the inducer to the culture tank according to the ratio of 0.2-4ml/L medium, and continue the culture for 6 days and then collect the culture
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16.9 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 18.27% of the dry cell weight, the chlorogenic acid content is more than 1.41%, the syringin content is more than 1.04%, the 1,5-dicaffeoylquinic acid content is more than 2.57%, and the rutin content is more than 0.066%.
  • Example 18 Adding a combination inducer to the medium in the mid-term to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is the culture device in the patent number CN208577726U, equipped with 300L synthetic medium, air 0-50m 3 /h, and the medium is synthetic medium (see Table 1) + 0.2mg/ L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation volume of fresh weight 30g/L, culture temperature 25 ⁇ 2°C , The light intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 10th day, the inducer is added to the culture tank at the ratio of 0.2-6ml/L medium, and the culture is collected after 6 days of continuous culture.
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 16.8 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 24.9% of the dry cell weight, the chlorogenic acid content is more than 2.58%, the syringin content is more than 2.07%, the 1,5-dicaffeoylquinic acid content is more than 4.37%, and the rutin content is more than 0.095%.
  • Example 19 Adding a combination inducer to the medium in the mid-term improves the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is the culture device in the patent number CN208577726U, equipped with 300L synthetic medium, air 0-50m 3 /h, and the medium is synthetic medium (see Table 1) + 0.2mg/ L-0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation volume of fresh weight 30g/L, culture temperature 25 ⁇ 2°C , The light intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 10th day, the inducer is added to the culture tank at the ratio of 0.2-6ml/L medium, and the culture is collected after 6 days of continuous culture.
  • the culture solution was filtered, the upper layer of cells was collected, and dried at 50°C for 24 hours, and the weight of the dried culture was determined to be 16.3 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the content of flavonoids accounts for more than 23.5% of the dry cell weight, the content of chlorogenic acid is more than 2.27%, the content of syringin is more than 1.33%, the content of 1,5-dicaffeoylquinic acid is more than 3.64%, and the content of rutin is more than 0.067%.
  • Example 20 Adding bypass metabolism inhibitors in the late stage of the culture medium to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is a culture device in the patent publication number CN208857314U, equipped with 10-50L culture medium, air 0-1m 3 /h, and the culture medium is synthetic medium (see Table 1)+0.2mg/L- 0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light The intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 13th day, the metabolic inhibitor is added to the culture tank at the ratio of 0.2-5ml/L medium, and the culture is collected after the culture is continued for 2 days.
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 14.8 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 18.5% of the dry cell weight, the chlorogenic acid content is more than 1.87%, the syringin content is more than 1.1%, the 1,5-dicaffeoylquinic acid content is more than 2.3%, and the rutin content is more than 0.042%.
  • Example 21 Adding bypass metabolism inhibitors in the late stage of the culture medium to increase the phenylpropanoids in the Saussurea culture
  • composition of the culture medium is a special medium (see Table 1) with 0.4mg/L 6-BA (6-benzylpurine), 2mg/L NAA (naphthalene acetic acid) and 30g/L sucrose, pH value is 6.0; Bacteria: 121°C, 20min.
  • the culture device is a culture device in the patent publication number CN208857314U, equipped with 10-50L culture medium, air 0-1m 3 /h, and the culture medium is synthetic medium (see Table 1)+0.2mg/L- 0.5mg/L 6-BA (6-benzylpurine), 2mg/L-4mg/L NAA (naphthalene acetic acid), 30g/L sucrose, inoculation amount is 30g/L fresh weight, culture temperature 25 ⁇ 2°C, light The intensity is 1000-2000lux, the light time is 12h/day, when the culture reaches the 13th day, the metabolic inhibitor is added to the culture tank at the ratio of 0.2-5ml/L medium, and the culture is collected after the culture is continued for 2 days.
  • the culture solution is filtered, the upper layer of cells is collected, and dried at 50°C for 24 hours, and the weight of the dried culture is determined to be 15.0 g/L.
  • the total flavonoid content in the cells was measured with a spectrophotometer, and the secondary metabolites of synthetic snow lotus, rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid were detected by HPLC.
  • the results showed that the snow lotus
  • the flavonoid content accounts for more than 20% of the dry cell weight, the chlorogenic acid content is more than 2.0%, the syringin content is more than 1.2%, the 1,5-dicaffeoylquinic acid content is more than 2.2%, and the rutin content is more than 0.052%.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Physiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法。该方法使用的培养基中，磷元素浓度0.5-3mmol/L，NO3-离子浓度20-35mmol/L，NH4+-离子浓度0-30mmol/L，Ca2+离子浓度0.5-3mmol/L，Mg2+离子浓度为0.2-1.5mmol/L，硼离子浓度0.02-0.1mmol/；在培养基中可添加诱导剂或前体或旁路代谢抑制剂。

Description

一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法 技术领域
本发明属于植物生物技术工程领域，涉及药用植物的组织培养物及其培养方法，具体是一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法。
背景技术
雪莲是高山地区的民间药用植物，用于散寒除湿、活血通经、抗炎镇痛等，民间多用于风湿性关节炎、妇女小腹冷痛、闭经，胎衣不下、麻痹不透、肺寒咳嗽、阳痿、高山不适应症等症的治疗。近年来，雪莲作为民族药在抗炎镇痛、抗早孕、抗衰老及抑制癌细胞增生方面的作用倍受关注。但我国雪莲主要分布在4000m以上的高原寒带地区，如新疆、甘肃、四川、云南和西藏。这些地区气候多变，冷热无常，最高月平均气温3～10℃，最低月平均气温则在零下十几度到几十度，生长环境十分恶劣，只有耐寒的苔草属、蒿草属以及少数高山多年生草本植物与之伴生，一般在此环境下的雪莲植物大多为多年生，生长缓慢，人工栽培困难，长期以来掠夺性采挖已使雪莲资源严重匮乏，已使得雪莲成为濒危物种，自然资源难以满足临床日益增长的需要。
雪莲中含有的黄酮及其苷、倍半萜内酯、香豆素、木脂素、甾体、多糖等成分，具有抗风湿、镇痛、调节心血管系统、抗癌、延缓衰老、计划生育、防辐射等生物活性，有很大的开发利用价值，应用前景良好。研究证明：紫丁香苷具有抗炎镇痛、抗高血糖、抗抑郁及抗癌活性；绿原酸具有抗炎、抗高血糖和抗乙型肝炎病毒作用；1，5-二咖啡酰奎尼酸具有抗氧化治疗糖尿病和抗艾滋病作用，作为新型抗艾滋病药物已进入临床研究阶段。综上所述，紫丁香苷、绿原酸和1，5-二咖啡酰奎尼酸具有多种药用活性，具有重要应用价值。紫丁香苷、绿原酸和1，5-二咖啡酰奎尼酸均为野生天山雪莲所具有的活性成分，但含量低，利用天山雪莲细胞培养生产该三种成分具有良好的前景。
应用细胞培养的方法进行雪莲的开发即可以满足社会对雪莲资源的需求，也可以保护自然资源，维护生态环境。但在有关雪莲组织培养的研究报道中，因存在许多问题极大地限制了其在实际生产中的应用，存在的问题有：
1.获得的培养物成分单一，多以单一的总黄酮为指标，而且含量低，难以实现雪莲真正的价值；2.针对某些成分含量低的问题，没有找到有效的获得高产细胞系的手段等；3.培养物的诱导剂仅仅为了获得更高的总黄酮，而忽略的雪莲本身的其他成分；4.代谢调控中只注重诱导剂的添加，没有通过改变分支代谢途径的流向，阻断不需要代谢产物的合成，以达 到提高产物产量的方法；5.培养工艺多为常用基本培养基，而没有根据雪莲的特点以及培养物的质量建立雪莲专用的基本培养基；6.培养物大多停留在实验室当中，难以大规模培养，在放大过程中不稳定，当放大培养到一定规模后有效成分的含量会降低；综上所述，国内外研究者在雪莲愈伤组织培养、细胞悬浮培养、发酵培养等方面做了大量工作，然而目前雪莲细胞的培养效果以及雪莲培养物药用成分单一，主要药用成分的含量量的提升有待挖掘。基本培养环境的没有优化，代谢路径的调控少等，致使现在缺少一种稳定高产且快速的大规模培养天山雪莲细胞的调控工艺。
发明内容
为解决现有技术缺陷，本发明提供一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法。
为实现上述发明目的，本发明采用以下技术方案：
一种提高雪莲细胞培养物黄酮苯丙素类化合物的培养基，所述培养基中，磷元素浓度为0.5-3mmol/L，NO 3 -离子浓度为20-35mmol/L，NH 4 +-离子浓度0-30mmol/L，Ca 2+离子浓度0.5-3mmol/L，Mg 2+离子浓度为0.2-1.5mmol/L，硼离子浓度为0.02-0.1mmol/。
进一步地，所述养基中，磷元素浓度为2.7mmol/L，NO 3 -离子浓度为30mmol/L，NH 4 +-离子浓度18mmol/L，Ca 2+离子浓度0.6mmol/L，Mg 2+离子浓度为0.3mmol/L，硼离子浓度为0.03mmol/L。
进一步地，所述培养基为(mg/L)：硝酸钾3000，硫酸铵793，磷酸氢二铵340，七水硫酸镁74，二水氯化钙88，碘化钾0.83，硼酸2，四水硫酸锰22.3，七水硫酸亚铁27.8，七水硫酸锌8.6，肌醇100，二水钼酸钠0.25，盐酸硫胺素0.1，五水硫酸铜0.025，盐酸吡哆醇0.5，六水氯化钴0.025，烟酸0.5，乙二胺四乙酸二钠37.3，甘氨酸2。
一种提高雪莲细胞培养物黄酮苯丙素类化合物的诱导剂，所述诱导剂为SNP诱导剂、MeJ诱导剂、SA诱导剂、银离子以及其络合物诱导剂中的一种或几种的组合；其中，SNP诱导剂添加浓度为1-50mmol/L，MeJ诱导剂添加浓度为1-50mg/L，SA诱导剂添加浓度为1-50mg/L，银离子以及其络合物诱导剂添加浓度为1-50mg/L。
进一步地，所述SNP诱导剂添加浓度为10mmol/L，所述MeJ诱导剂添加浓度为20mg/L，SA诱导剂添加浓度为30mg/L，银离子以及其络合物诱导剂添加浓度为20mg/L。
一种提高雪莲细胞培养物黄酮苯丙素类化合物的前体，所述前体为肉桂酸、苯丙氨酸、乙酸钠、酪氨酸、草酸中的一种或几种的组合；其中，肉桂酸添加1-20mg/L，苯丙氨酸添加1-20mg/L，乙酸钠添加1-20mg/L，酪氨酸添加1-50mg/L，草酸添加1-20mgl/L。
进一步地，所述肉桂酸添加10mg/L，苯丙氨酸添加10mg/L，乙酸钠添加10mg/L，酪氨 酸添加20mg/L，草酸添加10mgl/L。
一种抑制旁路代谢提高雪莲细胞培养物黄酮苯丙素类化合物合成的代谢抑制剂，所述旁路代谢抑制剂为精氨酸、柠檬酸三钠、氟化钠、环己丙氨酸、5-MT(5-甲氧基色氨酸)的一种或几种的组合；其中，精氨酸0-100mg/L、柠檬酸三钠0-50nmg/L、氟化钠0-100mg/L、环己丙氨酸0-100mg/L、5-MT(5-甲氧基色氨酸)0-100mg/L。
进一步地，所述精氨酸添加10mg/L、柠檬酸三钠添加15mg/L、氟化钠添加15mg/L、环己丙氨酸添加10mg/L、5-MT(5-甲氧基色氨酸)添加10mg/L。
一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法，所述方法包括以下步骤：
(1)原始细胞系的获得：
采用雪莲的种胚或由种子萌发的植株为外植体，在诱导培养基下诱导愈伤组织并进行反复继代培养，获得雪莲细胞系；
(2)步骤(1)得到的雪莲细胞系接种到培养基中培养，接种量为1～50g细胞鲜重每升培养基，光照1000-3000lxu，光照时间12-24h/天，100-130转/分的摇床，每7d～20d继代1次，培养3～4代后即可建立起稳定、高产的天山雪莲悬浮细胞培养系；
(3)大规模培养：选取步骤(2)中的培养物，在培养基中培养，接种量为10～80g细胞鲜重每升培养基，悬浮培养14～20天后，过滤收获细胞；
其中，所述步骤(2)和步骤(3)的培养基为前述培养基；或所述培养基为添加前述前体的培养基；或所述培养基为添加前述诱导剂的培养基；或所述培养基为添加前述旁路代谢抑制剂的培养基。
进一步地，所述前体为肉桂酸、苯丙氨酸、乙酸钠时，在接种时添加；所述前体为酪氨酸、草酸时，在培养中期时添加；所述诱导剂在培养中期时添加；所述旁路代谢抑制剂在培养后期时添加。
进一步地，步骤(3)大规模培养前，将细胞接入到培养瓶中含量1-10mm直径的玻璃珠5-20枚的培养瓶中进行培养，培养条件同原细胞，继代过程中淘汰生长不好的培养瓶，10代后建立起耐剪切的稳定、高产的天山雪莲悬浮细胞培养系。
本发明有益效果：本发明方法在制备雪莲细胞培养物的过程中，使用本发明的培养基、诱导剂、前体、旁路代谢抑制剂，显著提高了雪莲细胞培养物的总黄酮含量及芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量。
具体实施方式
以下结合具体实施例对本发明作进一步说明，但不以任何方式限制本发明。
实施例1 新疆雪莲无菌苗的培育
取天然新疆雪莲的种子，已通过中国科学院研究所标本馆(国家植物标本馆)鉴定，先用75％酒精浸泡10-30s，然后用1％氯化汞溶液消毒8-12min，再用无菌水反复冲洗4-5次，最后放在无菌滤纸上吸干种子表面水分，用无菌镊子接种在1/2MS(配方见附表3)中添加0.5-3mg/L GA(赤霉素)、10-30g/L蔗糖、0.6g/L琼脂的固体培养基上，22-27℃光照培养，约10天种子即可萌发长出无菌苗。
实施例2 愈伤组织的诱导和培养
取无菌苗根、茎、叶及子叶、胚芽做外植体，在无菌条件下，用无菌手术剪刀和无菌镊子将无菌苗根、茎、叶及子叶、胚芽切成2-5mm长的小段，接种在MS(配方见附表2)中添加0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)、0.6g/L琼脂及30g/L蔗糖的固体培养基上，22-27℃暗培养，7-10天诱导出愈伤组织。
实施例3 愈伤组织的继代筛选
通过对雪莲愈伤组织的诱导和培养得到了四种新疆雪莲细胞系：白色系、黄色系、绿色系和紫红色系；对4种细胞系分别进行了继代培养，通过对其生长速度、总黄酮化合物含量的检测以，确定紫红色细胞系为长势较好，总黄酮化合物含量最高的细胞系。
选取紫红色细胞系为原始细胞系，用目测法从固体培养基中挑选颜色较深、色彩鲜亮的紫红色愈伤组织在MS(配方见附表2)中添加0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)、0.6g/L琼脂及30g/L蔗糖的固体培养基上继续继代培养。为更好的优化种子、纯化细胞系，继代时接种块大小控制在3mm以下。如此反复，对所选择细胞系逐步驯化，经过近100代筛选获得了可以无限传代培养的稳定高产雪莲细胞系。
实施例4 液态悬浮培养种子培养系的建立
将实施例3所述扩增培养的雪莲细胞接种到液体合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，中进行摇瓶悬浮培养，具体为：以培养瓶为培养容器，采用摇床旋转振荡培养，接种量为1～50g细胞鲜重每升培养基，在25±2℃，光照1000-3000lxu，光照时间12-24h/天，100-130转/分的摇床，每7d～20d继代1次，3～4代后即可建立起稳定、高产的天山雪莲悬浮细胞培养系。
实施例5 搅拌发酵罐用种的建立
将实施例3或4中的雪莲细胞，接入到培养瓶中含量1-10mm直径的玻璃珠5-20枚的培养瓶中进行培养，培养基同原细胞所使用的培养基，培养条件：25±2℃，光照1000-3000lxu，光照时间12-24h/天，100-130转/分的摇床，每7d～20d继代1次，继代过程中淘汰生长不好的培养瓶，10代后建立起耐剪切的稳定、高产的天山雪莲悬浮细胞培养系，优化好的细胞系 在同样剪切力的培养瓶下不断继代培养优化，培养物可用于下一步的发酵罐放大培养。
实施例6 雪莲细胞的大规模培养
1.1选种
从实施例3的培养物中选取培养8-12天，长势较好，颜色鲜艳，呈紫红色，颗粒均匀，质地均一，容易分离的培养物作种子。
1.2培养基配制
采用1000ml培养瓶，每瓶装500ml培养液，培养液MS培养基(见表2)为基础内添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为5.5；高温灭菌：121℃，20min。
1.3接种
无菌条件下，将培养物接种到装有培养液的培养瓶中，接种量5％(W/V，g.fw/ml)。
1.4摇床培养
培养条件：培养温度为25±2℃，摇床转速110rpm，培养时间9d，光照11h、2000lux。
培养过程中，多观察，发现染菌立即挑出。
3培养物的收集及循环生产
3.1留种
大规模生产的同时，可以从中选择生长旺盛、颜色鲜艳、呈紫红色，结构致密、呈团粒状，质地均匀、略偏硬为特征的愈伤组织留种，选种量以不超过总生产量的15％为宜。
3.2培养物的收集
收集剩余培养物，低温真空干燥，即可。根据需要，可以将干燥的培养物磨成粗粉或细粉，或者用来提取黄酮及其他活性成分。
经测定，用此方法进行的大规模生产成本较低，培养物生长速率快，产量可达20-25g干重/月/升培养基；干品培养物中总黄酮含量高，达培养物干重的8-12％。HPLC检测细胞中芦丁0.0003％，1.5-O-二咖啡酰奎尼酸0.8％，紫丁香苷0.31％，绿原酸0.35％。
实施例7 使用合成培养基大规模生产高含量细胞培养物
从实施例4的培养物中选取培养8-12天，长势较好，颜色鲜艳，呈紫红色，颗粒均匀，质地均一，扩增培养的高产的天山雪莲悬浮细胞培养种子，采用培养桶设备(专利公开号：CN208857314U)，装有20-100L合成生培养基(见表1)，通空气0-1m 3/h，具体为：培养基为合成培养基+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为1～20g细胞干重每升培养基，在25±2℃，光照1000-3000lxu，光照时间12h，悬浮培养14天后，过滤收获细胞。
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的15％以上，绿原酸含量1.03％以上，紫丁香苷含量0.85％以上，1，5-二咖啡酰奎尼酸含量1.6％以上，芦丁0.06％以上。
含量测定
(1)测量干细胞重
将细胞培养液倒入120目不锈钢筛网中，过滤，收获细胞，蒸馏水清洗3次，用吸水纸充分吸干细胞表面的水份，称鲜重，将湿细胞置于60℃烘箱烘至恒重，得干细胞重。
(2)测定雪莲总黄酮的方法：
对照品溶液的制备：精密称取在120℃干燥至恒重的芦丁对照品10mg，置50mL容量瓶中，加甲醇适量，水浴加热使溶解，放冷，加甲醇至刻度，摇匀。即得(每mL中含无水芦丁0.2mg)；
标准曲线的制备：精密量取对照品溶液1mL、2mL、3mL、4mL、5mL、6mL，分别置25mL容量瓶中，各加水至6mL，加5％亚硝酸钠溶液1mL，摇匀，放置6min，加10％硝酸铝1mL，摇匀，放置6min，加氢氧化钠试液10mL，再加水至刻度，摇匀，放置15min，以相应试剂为空白，在500nm处测定吸光度，以吸光度为纵坐标，浓度为横坐标，绘制标准曲线；
测定：取本品粗粉约0.10g，精密称定，置索式提取器中，加甲醇约90mL，加热回流至提取液无色(每个样品回流次数不得少于4次和2小时)，转移至50mL容量瓶中，加甲醇至刻度，摇匀。精密量取5mL，置25mL容量瓶中，照标准曲线制备项下的方法，自“加水至6mL”起，依法测定吸光度，从标准曲线上读出供试品溶液中含无水芦丁的重量(mg)，计算，即得。
(3)苯丙素类含量测定
取干燥至恒重的天山雪莲培养细胞，研细，精密称取细粉约1.000mg，置于具塞试管中，用20％甲醇50mL超声提取1h，用甲醇补足失重，15000r/min离心20min，取上清过0.45μm滤膜过滤，通过HPLC同时测定芦丁、紫丁香苷、绿原酸和1，5-二咖啡酰奎尼酸的含量。Shiseido capcell pak C18 MGⅡ色谱柱(4.6mm×250mm，5μm)；
流动相A为0.2％磷酸缓冲盐溶液(含50mmo/L-1KH 2PO 4)，流动相B为乙腈，梯度洗脱。梯度条件：0min(VA∶VB＝92∶8)～20min(VA∶VB＝90∶10)～50min(VA∶VB＝70∶30)～60min(VA∶VB＝50∶50)；流速1.0mL/min -1；柱温30℃；检测波长265nm(紫丁香苷)；进样量20μ。
实施例8 培养基中加入前体肉桂酸大批量生产富含药用成分的雪莲培养物
选取实例4中的细胞系为种子，将其转接到含有肉桂酸合成培养基中，具体配方为：合成培养基(见表1)+不同浓度的肉桂酸+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，使用摇床进行培养3代以上，将扩繁并稳定继代的种子，采用植物细胞培养桶设备(专利公开号CN208857314U)，装有20-100L培养基，接种量为鲜重10g/L，培养时间14天，光照强度1500lux左右，光照时间12h/天
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，的干品测定其产量为15g/L。
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的22％以上，绿原酸含量1.66％以上，紫丁香苷含量1.45％以上，1，5-二咖啡酰奎尼酸含量2.81％以上，芦丁0.066％以上。
实施例9 培养基中加入前体苯丙氨酸大批量生产富含药用成分的雪莲培养物
选取实例4中的细胞系为种子，将其转接到含有苯丙氨酸合成培养基中，具体配方为：合成培养基(见表1)+不同浓度的苯丙氨酸+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖具体配方见附表，使用摇床进行培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置使用的是公开号为CN208857314U中的设备，装置中装有10-50L培养基，通空气0-1m 3/h，接种量为鲜重40g/L，培养时间15天，光照强度1500lux左右，光照时间12h/天
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，的干品测定其产量为15g/L。
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的22.7％以上，绿原酸含量1.28％以上，紫丁香苷含量2.07％以上，1，5-二咖啡酰奎尼酸含量2.48％以上，芦丁0.078％以上。
实施例10 培养基中加入乙酸钠大批量生产富含药用成分的雪莲培养物
选取实例4中的细胞系为种子，将其转接到含有乙酸钠的合成培养基中，具体配方为：合成培养基(见表1)+不同浓度的乙酸钠+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖具体配方见附表，使用摇床进行培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为CN208857314U中培养设备，装有10-50L培养基，通空气0-1m 3/h，接种量为鲜重50g/L，培养温度25±2℃，培养时间12天，光照强度 1500lux左右，光照时间12h/天。
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，的干品测定其产量为15g/L。
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的21.8％以上，绿原酸含量1.79％以上，紫丁香苷含量1.22％以上，1，5-二咖啡酰奎尼酸含量2.35％以上，芦丁0.064％以上
实施例11 培养基中期添加酪氨酸大批量生产富含药用成分的雪莲培养物
培养基配制：
培养液成分为合成培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取酪氨酸0.04g，溶解到100mL纯净水中，121℃灭菌20min备用
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利CN208857315U中的培养桶，装有40L培养基，通空气0-1m 3/h，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂10-200ml加入到培养袋内，继续培养6天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量18.2g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的19.4％以上，绿原酸含量0.62％以上，紫丁香苷含量1.49％以上，1，5-二咖啡酰奎尼酸含量2.75％以上，芦丁0.062％以上。
实施例12 培养基中期添加草酸大批量生产富含药用成分的雪莲培养物
培养基配制：
培养液成分为合成培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取草酸0.45g，溶解到1000mL纯净水中，配置成5mmol/L的诱导剂，使用NaOH调整pH至5.8，然后121℃灭菌20min备用
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利公开号CN208857314U中的培养设备，装有16L培养基，通空气0-1m 3/h，培养基为合成培养基(见附表)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-4ml/L培养基的比例加入到培养桶内，继续培养6天后对培养物进行收集
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的19.6％以上，绿原酸含量1.27％以上，紫丁香苷含量0.95％以上，1，5-二咖啡酰奎尼酸含量1.72％以上，芦丁0.071％以上。
实施例13 培养基中期添加诱导剂茉莉酸甲酯提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取茉莉酸甲酯0.5g，溶解到100mL纯净水中，121℃灭菌20min备用
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利公开号为CN208857314U中的培养设备，装有10-50L培养基，通空气0-1m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天， 培养到第10天时，将诱导剂按照0.2-6ml/L培养基的比例加入到培养桶内，继续培养6天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量20.4g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的24.1％以上，绿原酸含量2.44％以上，紫丁香苷含量1.89％以上，1，5-二咖啡酰奎尼酸含量4.12％以上，芦丁0.081％以上。
实施例14 培养基中期添加诱导剂硝酸银提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为合成培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取硝酸银0.5g，溶解到100mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用；
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利公开号为CN208857314U中的培养设备，装有10-50L培养基，通空气0-1m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-5ml/L培养基的比例加入到培养桶内，继续培养6天后对培养物进行收集
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的 19.5％以上，绿原酸含量1.47％以上，紫丁香苷含量1.38％以上，1，5-二咖啡酰奎尼酸含量2.97％以上，芦丁0.065％以上
实施例15 培养基中期添加诱导剂水杨酸提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min；
诱导剂配置：
称取水杨酸0.5g，溶解到100mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用；
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利号CN208577726U中的培养设备，装有10-100L合成生培养基，通空气0-1m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-8ml/L培养基的比例加入到培养袋内，继续培养6天后对培养物进行收集；
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16.8g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的21.2％以上，绿原酸含量1.86％以上，紫丁香苷含量1.09％以上，1，5-二咖啡酰奎尼酸含量3.55％以上，芦丁0.072％以上。
实施例16 培养基中期添加组合诱导剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为MS培养基(见表2)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取水杨酸0.5g，硝酸银0.5g，茉莉酸甲酯0.5g，溶解到100mL纯净水中，装入到黑色 培养瓶中，121℃灭菌20min备用；
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利号CN208577726U中的培养设备，装有300L合成生培养基，通空气0-50m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-5ml/L培养基的比例加入到培养罐内，继续培养6天后对培养物进行收集
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的18.1％以上，绿原酸含量0.68％以上，紫丁香苷含量0.77％以上，1，5-二咖啡酰奎尼酸含量1.85％以上，芦丁0.028％以上。
实施例17 培养基中期添加组合诱导剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取水杨酸10.0g，硝酸银10.0g溶解到1000mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用
培养物的生产：
选取实例6中的耐剪切的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利号CN208577726U中的培养设备，装有300L合成生培养基，通空气0-50m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-4ml/L培养基的比例加入到培养罐内，继续培养6 天后对培养物进行收集
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16.9g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的18.27％以上，绿原酸含量1.41％以上，紫丁香苷含量1.04％以上，1，5-二咖啡酰奎尼酸含量2.57％以上，芦丁0.066％以上。
实施例18 培养基中期添加组合诱导剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取茉莉酸甲酯10.0g，水杨酸10.0g，硝酸银10.0g溶解到1000mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用
培养物的生产：
选取实例6中的耐剪切的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利号CN208577726U中的培养设备，装有300L合成生培养基，通空气0-50m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-6ml/L培养基的比例加入到培养罐内，继续培养6天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16.8g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的24.9％以上，绿原酸含量2.58％以上，紫丁香苷含量2.07％以上，1，5-二咖啡酰奎尼酸含 量4.37％以上，芦丁0.095％以上。
实施例19 培养基中期添加组合诱导剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
诱导剂配置：
称取水杨酸10.0g，硝酸银10.0g，茉莉酸甲酯10.0g，硝普钠10.0g溶解到1000mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用
培养物的生产：
选取实例6中的耐剪切的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利号CN208577726U中的培养设备，装有300L合成生培养基，通空气0-50m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第10天时，将诱导剂按照0.2-6ml/L培养基的比例加入到培养罐内，继续培养6天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量16.3g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的23.5％以上，绿原酸含量2.27％以上，紫丁香苷含量1.33％以上，1，5-二咖啡酰奎尼酸含量3.64％以上，芦丁0.067％以上。
实施例20 培养基后期添加旁路代谢抑制剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
抑制剂配置：
称取精氨酸10.0g，D-丙氨酸10.0g，柠檬酸钠15.0g溶解到1000mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用。
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利公开号为CN208857314U中的培养设备，装有10-50L培养基，通空气0-1m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第13天时，将代谢抑剂按照0.2-5ml/L培养基的比例加入到培养桶内，继续培养2天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量14.8g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的18.5％以上，绿原酸含量1.87％以上，紫丁香苷含量1.1％以上，1，5-二咖啡酰奎尼酸含量2.3％以上，芦丁0.042％以上。
实施例21 培养基后期添加旁路代谢抑制剂提高雪莲培养物中的苯丙素类化合物
培养基配制：
培养液成分为专用培养基(见表1)中添加0.4mg/L 6-BA(6-苄基嘌呤)、2mg/L NAA(萘乙酸)及30g/L蔗糖，pH值为6.0；高温灭菌：121℃，20min。
抑制剂配置：
称取精氨酸10.0g，D-丙氨酸10.0g，5-MT5.0g，柠檬酸钠15.0g溶解到1000mL纯净水中，装入到黑色培养瓶中，121℃灭菌20min备用
培养物的生产：
选取实例4中的细胞系为种子，将其转接到培养瓶进行培养扩繁，培养使用摇床，种子扩繁培养3代以上，将扩繁并稳定继代的种子，转入到生产培养装置中，培养装置为专利公开号为CN208857314U中的培养设备，装有10-50L培养基，通空气0-1m 3/h，培养基为合成培养基(见表1)+0.2mg/L-0.5mg/L 6-BA(6-苄基嘌呤)、2mg/L-4mg/L NAA(萘乙酸)，30g/L蔗糖，接种量为鲜重30g/L，培养温度25±2℃，光照强度1000-2000lux，光照时间12h/天，培养到第13天时，将代谢抑剂按照0.2-5ml/L培养基的比例加入到培养桶内，继续培养2天后对培养物进行收集。
培养物的收集：
培养完成后，对培养液进行过滤，收集上层细胞，50℃烘干24h，测定烘干后的培养物重量15.0g/L。
含量测定：
用分光光度计测定细胞中的总黄酮含量，用HPLC进行检测合成雪莲次生代谢产物，芦丁、绿原酸、紫丁香苷、1，5-二咖啡酰奎尼酸的含量，结果表明雪莲黄酮含量占细胞干重的20％以上，绿原酸含量2.0％以上，紫丁香苷含量1.2％以上，1，5-二咖啡酰奎尼酸含量2.2％以上，芦丁0.052％以上。
表1 合成培养基配方
Figure PCTCN2020122753-appb-000001
表2 MS培养基配方
Figure PCTCN2020122753-appb-000002
表3 1/2MS培养基配方
Figure PCTCN2020122753-appb-000003
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。

Claims (10)

  1. 一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的培养基，其特征在于，所述培养基中，磷元素浓度为0.5-3mmol/L，NO 3 -离子浓度为20-35mmol/L，NH 4 +-离子浓度0-30mmol/L，Ca 2+离子浓度0.5-3mmol/L，Mg 2+离子浓度为0.2-1.5mmol/L，硼离子浓度为0.02-0.1mmol/。
  2. 如权利要求1所述的一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的培养基，其特征在于，所述培养基中，磷元素浓度为2.7mmol/L，NO 3 -离子浓度为30mmol/L，NH 4 +-离子浓度18mmol/L，Ca 2+离子浓度0.6mmol/L，Mg 2+离子浓度为0.3mmol/L，硼离子浓度为0.03mmol/L。
  3. 如权利要求1所述的一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的培养基，其特征在于，所述培养基为(mg/L)：硝酸钾3000，硫酸铵793，磷酸氢二铵340，七水硫酸镁74，二水氯化钙88，碘化钾0.83，硼酸2，四水硫酸锰22.3，七水硫酸亚铁27.8，七水硫酸锌8.6，肌醇100，二水钼酸钠0.25，盐酸硫胺素0.1，五水硫酸铜0.025，盐酸吡哆醇0.5，六水氯化钴0.025，烟酸0.5，乙二胺四乙酸二钠37.3，甘氨酸2。
  4. 一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的诱导剂，其特征在于，所述诱导剂为SNP诱导剂、MeJ诱导剂、SA诱导剂、银离子以及其络合物诱导剂中的一种或几种的组合；其中，SNP诱导剂添加浓度为1-50mmol/L，MeJ诱导剂添加浓度为1-50mg/L，SA诱导剂添加浓度为1-50mg/L，银离子以及其络合物诱导剂添加浓度为1-50mg/L；优选为所述SNP诱导剂添加浓度为10mmol/L，所述MeJ诱导剂添加浓度为20mg/L，SA诱导剂添加浓度为30mg/L，银离子以及其络合物诱导剂添加浓度为20mg/L。
  5. 一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的前体，其特征在于，所述前体为肉桂酸、苯丙氨酸、乙酸钠、酪氨酸、草酸中的一种或几种的组合；其中，肉桂酸添加1-20mg/L，苯丙氨酸添加1-20mg/L，乙酸钠添加1-20mg/L，酪氨酸添加1-50mg/L，草酸添加1-20mg/L；优选为所述肉桂酸添加10mg/L，苯丙氨酸添加10mg/L，乙酸钠添加10mg/L，酪氨酸添加20mg/L，草酸添加10mg/L。
  6. 一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的旁路代谢抑制剂，其特征在于，所述旁路代谢抑制剂为精氨酸、柠檬酸三钠、氟化钠、环己丙氨酸、5-MT的一种或几种的组合；其中，精氨酸0-100mg/L、柠檬酸三钠0-50nmg/L、氟化钠0-100mg/L、环己丙氨酸0-100mg/L、5-MT0-100mg/L。
  7. 如权利要求6所述的一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的旁路代谢抑制剂，其特征在于，所述精氨酸添加10mg/L、柠檬酸三钠添加15mg/L、氟化钠添加15mg/L、环己丙氨酸添加10mg/L、5-MT添加10mg/L。
  8. 一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的方法，其特征在于，所述方法包括 以下步骤：
    (1)原始细胞系的获得：
    采用雪莲的种胚或由种子萌发的植株为外植体，在诱导培养基下诱导愈伤组织并进行反复继代培养，获得雪莲细胞系；
    (2)步骤(1)得到的雪莲细胞系接种到培养基中培养，接种量为1～50g细胞鲜重每升培养基，光照1000-3000lxu，光照时间12-24h/天，100-130转/分的摇床，每7d～20d继代1次，培养3～4代后即可建立起稳定、高产的天山雪莲悬浮细胞培养系；
    (3)大规模培养：选取步骤(2)中的培养物，在培养基中培养，接种量为10～80g细胞鲜重每升培养基，悬浮培养14～20天后，过滤收获细胞；
    其中，所述步骤(2)和步骤(3)的培养基为权利要求1～3任一项所述培养基；或所述培养基为添加权利要求5前体的培养基；或所述培养基为添加权利要求4诱导剂的培养基；或所述培养基为添加权利要求6或7旁路代谢抑制剂的培养基。
  9. 根据权利要求8所述的一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的方法，其特征在于，所述前体为肉桂酸、苯丙氨酸、乙酸钠时，在接种时添加；所述前体为酪氨酸、草酸时，在培养中期时添加；所述诱导剂在培养中期时添加；所述旁路代谢抑制剂在培养后期时添加。
  10. 根据权利要求8所述的一种提高雪莲细胞培养物中黄酮苯丙素类化合物含量的方法，其特征在于，步骤(3)大规模培养前，将细胞接入到培养瓶中含量1-10mm直径的玻璃珠5-20枚的培养瓶中进行培养，培养条件同原细胞，继代过程中淘汰生长不好的培养瓶，10代后建立起耐剪切的稳定、高产的天山雪莲悬浮细胞培养系。
PCT/CN2020/122753 2019-11-28 2020-10-22 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法 WO2021103863A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20894710.1A EP4067479A4 (en) 2019-11-28 2020-10-22 METHOD OF INCREASING THE CONTENT OF FLAVONOID-PHENYLPROPANOID COMPOUNDS IN A SAUSSUREA INVOLUCRATA CELL CULTURE
US17/756,713 US20230287336A1 (en) 2019-11-28 2020-10-22 Method for improving flavonoid phenylpropanoid compounds in saussurea involucrata cell culture

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911194580.1A CN110923190A (zh) 2019-11-28 2019-11-28 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法
CN201911194580.1 2019-11-28

Publications (1)

Publication Number Publication Date
WO2021103863A1 true WO2021103863A1 (zh) 2021-06-03

Family

ID=69846857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/122753 WO2021103863A1 (zh) 2019-11-28 2020-10-22 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法

Country Status (4)

Country Link
US (1) US20230287336A1 (zh)
EP (1) EP4067479A4 (zh)
CN (1) CN110923190A (zh)
WO (1) WO2021103863A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103219A (zh) * 2022-10-25 2023-05-12 山东益衡元健康科技有限公司 一种雪莲干细胞粉的制备方法及其制得的抑菌剂

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110923190A (zh) * 2019-11-28 2020-03-27 大连普瑞康生物技术有限公司 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1557141A (zh) * 2004-02-10 2004-12-29 北京智创信达科技发展有限公司 一种稳定高产雪莲黄酮的紫色水母雪莲细胞系及其用途
CN101688170A (zh) * 2007-04-24 2010-03-31 生物马特里卡公司 用于生命科学的样品存储
US20130177661A1 (en) * 2012-01-05 2013-07-11 National Ilan University Saussurea Involucrate Extract, Pharmaceutical Composition and Use for Anti-Fatigue and Anti-Aging
CN104263690A (zh) * 2014-09-04 2015-01-07 南京标科生物科技有限公司 一种黄葛愈伤组织和悬浮细胞的快速繁殖方法
CN106754633A (zh) * 2017-02-14 2017-05-31 天津艾赛博生物技术有限公司 一种规模化培养天山雪莲细胞高产黄酮的调控方法
CN108504623A (zh) * 2018-04-28 2018-09-07 大连普瑞康生物技术有限公司 一种水母雪莲高产黄酮细胞系及其制备方法
CN208577726U (zh) 2018-04-28 2019-03-05 大连普瑞康生物技术有限公司 一种培养植物细胞系的生物反应器
CN208857314U (zh) 2018-04-28 2019-05-14 大连普瑞康生物技术有限公司 一种植物细胞、器官液体培养装置
CN208857315U (zh) 2018-04-28 2019-05-14 大连普瑞康生物技术有限公司 一种植物细胞和器官液体培养袋
CN110923190A (zh) * 2019-11-28 2020-03-27 大连普瑞康生物技术有限公司 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101139571B (zh) * 2007-08-29 2011-09-14 清华大学 一种生产雪莲次生代谢产物的方法
CN103305454A (zh) * 2012-03-14 2013-09-18 中国医学科学院药物研究所 天山雪莲细胞培养生产紫丁香苷、绿原酸和1,5-二咖啡酰奎尼酸的方法
CN104429943A (zh) * 2014-11-04 2015-03-25 盐城呈祥园艺育苗有限公司 一种非洲菊培养基
CN106947730B (zh) * 2017-04-28 2020-03-27 天津艾赛博生物技术有限公司 一种生产高含量矿物质元素的植物细胞培养方法及其应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1557141A (zh) * 2004-02-10 2004-12-29 北京智创信达科技发展有限公司 一种稳定高产雪莲黄酮的紫色水母雪莲细胞系及其用途
CN101688170A (zh) * 2007-04-24 2010-03-31 生物马特里卡公司 用于生命科学的样品存储
US20130177661A1 (en) * 2012-01-05 2013-07-11 National Ilan University Saussurea Involucrate Extract, Pharmaceutical Composition and Use for Anti-Fatigue and Anti-Aging
CN104263690A (zh) * 2014-09-04 2015-01-07 南京标科生物科技有限公司 一种黄葛愈伤组织和悬浮细胞的快速繁殖方法
CN106754633A (zh) * 2017-02-14 2017-05-31 天津艾赛博生物技术有限公司 一种规模化培养天山雪莲细胞高产黄酮的调控方法
CN108504623A (zh) * 2018-04-28 2018-09-07 大连普瑞康生物技术有限公司 一种水母雪莲高产黄酮细胞系及其制备方法
CN208577726U (zh) 2018-04-28 2019-03-05 大连普瑞康生物技术有限公司 一种培养植物细胞系的生物反应器
CN208857314U (zh) 2018-04-28 2019-05-14 大连普瑞康生物技术有限公司 一种植物细胞、器官液体培养装置
CN208857315U (zh) 2018-04-28 2019-05-14 大连普瑞康生物技术有限公司 一种植物细胞和器官液体培养袋
CN110923190A (zh) * 2019-11-28 2020-03-27 大连普瑞康生物技术有限公司 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHAO-LIN KUO, CHAO-LIN KUO , DINESH-CHANDRA AGRAWAL , HUNG-CHI CHANG, YA-TING CHIU , CHU-PENG HUANG , YI-LIN CHEN, SHIH-HUNG HUANG: "In Vitro Culture and Production of Syringin and Rutin in Saussurea Involucrata (Kar. Et Kir.) – an Endangered Medicinal Plant", BOTANICAL STUDIES, vol. 56, no. 12, 1 December 2015 (2015-12-01), pages 1 - 8, XP055814768, DOI: 10.1186/s40529-015-0092-8 *
KANG EN-XIANG, ZHEN-JIA ZHU, CHUN-YUAN FANG, NIAN-LAI CHEN, XIAO-MEI SUN, SHI-MEI YANG: "Inducing Effects of BTH Treatment on Melon Resistance to Powdery Mildew at Different Growing Stages", CHINA VEGETABLES, vol. 22, 1 January 2010 (2010-01-01), pages 64 - 67, XP055814767 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116103219A (zh) * 2022-10-25 2023-05-12 山东益衡元健康科技有限公司 一种雪莲干细胞粉的制备方法及其制得的抑菌剂
CN116103219B (zh) * 2022-10-25 2023-07-04 山东益衡元健康科技有限公司 一种雪莲干细胞粉的制备方法及其制得的抑菌剂

Also Published As

Publication number Publication date
EP4067479A4 (en) 2023-01-11
US20230287336A1 (en) 2023-09-14
CN110923190A (zh) 2020-03-27
EP4067479A1 (en) 2022-10-05

Similar Documents

Publication Publication Date Title
CN106367378B (zh) 可提高甘草黄酮含量的光果甘草愈伤组织细胞培养方法
CN103609451B (zh) 一种枸杞无菌苗及其愈伤组织的获得方法
CN103396979B (zh) 一种提高硅藻中岩藻黄素产率的培养方法
Verma et al. Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.
CN104920214A (zh) 一种提高三七组培苗皂苷产量的方法
CN102884981B (zh) 两面针组织培养培养基
WO2021103863A1 (zh) 一种提高雪莲细胞培养物黄酮苯丙素类化合物的方法
CN101338298B (zh) 一种雪莲细胞系
CN104351054A (zh) 一种利用led光源促进蝴蝶兰丛生芽快速繁殖的方法
Yin et al. Influence of medium salt strength and nitrogen source on biomass and metabolite accumulation in adventitious root cultures of Pseudostellaria heterophylla
CN105850729A (zh) 一种柳叶马鞭草培育方法
CN102994444B (zh) 一种金钱松细胞的悬浮培养方法
CN102532337A (zh) 一种利用组织培养方法生产甘草多糖的方法
CN101475929B (zh) 一种利用白桦悬浮培养生产齐墩果酸的方法
CN101974479B (zh) 一种培育转基因四合木愈伤组织的方法及其专用培养基
CN108034681A (zh) 利用悬浮培养的地黄茎形成层干细胞生产梓醇的方法
CN107034252B (zh) 一种诱导白木香愈伤细胞产生2-(2-苯乙基)色酮类成分的方法
CN1328951C (zh) 石斛原球茎无激素培养方法
CN102754599B (zh) 利用根诱导原球茎快速繁殖大花蕙兰的方法
CN101768567A (zh) 一种红豆杉细胞系
CN103421737B (zh) 促进扁桃花粉萌发的培养基、培养方法及应用
CN101182542A (zh) 用发根农杆菌遗传转化粘毛黄芩获得产黄芩苷的毛状根的方法
CN107155887A (zh) 一种利用暗紫贝母愈伤组织生产暗紫贝母总生物碱的方法
CN102499095A (zh) 一种大蒜花序轴离体培养直接诱导鳞茎培养基
CN105309316A (zh) 丹参组培苗培养基及丹参组培快繁方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20894710

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020894710

Country of ref document: EP

Effective date: 20220628