CN115851572A - Method for increasing glabridin content in suspension culture cell of liquorice - Google Patents
Method for increasing glabridin content in suspension culture cell of liquorice Download PDFInfo
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Abstract
The invention discloses a method for improving glabridin content in liquorice suspension culture cells, which comprises the steps of culturing aseptic seedlings by using seeds, intercepting cotyledon, stem bud, hypocotyl and other tissues of the aseptic seedlings to induce callus, obtaining loose callus through subculture, and establishing a cell suspension culture system for the callus in a liquid culture medium to carry out shake culture. Adding daidzein (daidzein) as exogenous substrate supplement to the culture medium during suspension culture, and/or adding pterocarya fruit reductase PTR (pterocarpan products enzymes) and/or isopentenyl transferase GmG4DT extracted and separated from Glycyrrhiza glabra into the suspension culture solution; the production of glabridin in suspension culture cells is promoted by the way of jointly adding the substrate and the biological enzyme. After the suspension cells are cultured by the method, the content of the glabridin in the suspension cells reaches 0.10-0.25 percent of the dry cell value, and the growth period of 3-5 years is probably needed for the content of the glabrous greenbrier rhizome whether planted or grown in a wild way.
Description
Technical Field
The invention relates to a method for plant cell suspension culture, in particular to a method for improving the content of glabridin in liquorice suspension culture cells.
Background
Glabradine (Glabradin) is Glycyrrhiza glabraGlycyrrhiza glabraL. a specific isopentenyl isoflavone compound, which has wide biological characteristics, such as antioxidant, anti-inflammation, anti-atherosclerosis, energy metabolism regulation, estrogen regulation, neuroprotection, anti-osteoporosis, whitening and the like, and the content of the isopentenyl isoflavone compound in the glycyrrhiza glabra is 0.1-0.2%. Glabridin is currently used in whitening cosmetics, special medical foods for obese patients, health foods for improving memory and increasing bone density to prevent osteoporosis, and the like.
However, glabridin is only applied to Glycyrrhiza glabra（Glycyrrhiza glabraL. in the above formula, glycyrrhiza uralensis (L.), (Glycyrrhiza uralensisFisch.) and licorice root (bloated fruit)Glycyrrhiza inflataBata.) contains no such component, and only a small part of Sinkiang in China has wild glabrous licorice, so that the raw material source of glabridin in China is very limited. In addition, the liquorice is a perennial herb, the glabridin is used as a secondary metabolite and needs a longer growth time to accumulate, and the wild glabrous liquorice generally needs more than 5-6 years to have higher glabridin content.
The in vitro culture of plant cells can effectively shorten the production period of obtaining secondary metabolites, solve the difference of regional and seasonal changes, and become one of the effective means for obtaining scarce natural products. The research on the cell suspension culture of the liquorice is mature. Studies of Zhanglian and the like find that the callus derived from cotyledon is most suitable for being used as an initial culture of suspension culture of the glycyrrhiza inflata cells, the optimal inoculation amount of the suspension culture is 35g/L, the optimal dosage of the conditioned medium is 1/3 of the total culture volume, and the most suitable culture medium for the growth of the glycyrrhiza inflata suspension cells is an MS culture medium added with the hormone combination of NAA 1.0mg/L and KT 0.5mg/L [1] . The method for suspension culture of Glycyrrhrizae radix cells has been specially studied, and it is found that the hypocotyl of Glycyrrhrizae radix is most suitable for inducing embryogenic callusPerforming suspension culture on the subcultured callus, wherein the suspension culture period is 21 days, the maximum biomass is reached in 18 days, and the content of glycyrrhizic acid and total flavonoids is determined, wherein the result shows that the content of glycyrrhizic acid is 1.138 mg/g, and the content of total flavonoids in licorice can be 6.7 mg/g [2] . The content of the total flavonoids in different calli of glycyrrhiza glabra is compared by the madder and the like, the content of the total flavonoids in the internode calli is the highest, and the hormone combination of 6-BA and NAA and the addition of hydrolyzed milk protein are beneficial to the production of the total flavonoids in the calli of glycyrrhiza glabra [3] . A large number of researches show that the content of certain functional components can be increased in the cell culture process of liquorice by a certain intervention means, the influence of different additives on the formation of flavonoid compounds in liquorice callus culture is researched by the Yangshi sea and the like, and 0.1% of yeast extract, 0.05% of casein hydrolysate, 10 mu mol/L of jasmonic acid and a low-concentration rare earth metal element Eu are found 3+ The content of licorice total flavonoids in the callus tissues can be increased, and the content of the licorice total flavonoids in the callus tissues can be increased by illumination [4-5] . Yugame and the like research the influence of hormone on the total flavone content of glycyrrhiza glabra callus, and the total flavone content in internode callus is the highest and is 2.316% +/-0.015% in MS culture medium added with 6-BA 4.0 mg/L and NAA 1.0mg/L, which shows that the research of producing glycyrrhiza glabra total flavone by callus culture is feasible [6] . Liangwei et al optimized the conditions for producing total flavonoids by Licorice suspension cells, found that when the suspension cell culture medium formulation was MS liquid culture medium + CH 398.608 mg/L +2, 4-D1.044 mg/L +6-BA1.325mg/L, the total flavonoids yield was the highest, 1.28% [7] 。
CN101265462A discloses a culture method for glycyrrhiza uralensis cell industrialization, which makes it possible to obtain effective components by using cell culture technology. CN102532337A discloses a method for obtaining glycyrrhiza polysaccharide by suspension cell culture. CN 111718888A discloses a culture method capable of increasing glycyrrhizic acid content in suspension culture cells of licorice. CN 106367378A discloses a method for culturing glycyrrhiza glabra callus cells capable of improving licoflavone content, which adds p-hydroxyphenylpyruvic acid as a precursor for synthesizing flavonoid compounds in vivo, and uses coronatine as an inducing signal molecule without specific guidance for the synthesis of glabridin. At present, no research is specially aimed at improving the content of glabridin in suspension culture cells of glycyrrhiza glabra.
Reference:
[1] zhanliping, lingling, xu chang fei suspension culture of glycyrrhiza inflata cells [ J ]. Proceedings of hebei university: nature science edition, 2008, 28 (6): 5.
[2] The establishment of a suspension culture line of dactylicapnos root cells and the analysis of active ingredients in suspension cells [ D ]. University of inner Mongolia science and technology, 2010.
[3] Comparison of the total flavone content in different calli from Yuqian, ma 28156, zhao hong Yan, glycyrrhiza glabra [ J ] seeds, 2011, 30 (7): 4.
[4] Influence of different additives on flavonoid formation in callus culture of Glycyrrhiza uralensis Fisch.J. J.Pharmacology J.2006, 041 (002): 96-99)
[5] Influence of different physicochemical factors on the growth of licorice callus and flavonoid synthesis [ J ]. Proceedings of Jilin university of agriculture, 2006.
[6] Influence of hormones on the total flavone content of glycyrrhiza glabra callus [ J ] proceedings of the university of river: nature science edition, 2011, 29 (4): 4.
[7] Licorice suspension cell production condition optimization and inoxidizability research [ J ]. North horticulture, 2022 (014): 000.
Disclosure of Invention
In order to solve the increasing consumption demand of glabridin in cosmetics, health-care food and special medical food, the problem that wild glabrous licorice resources in China are scarce and the growing period of artificially planted glabrous licorice is long and the glabridin content is low, the invention aims to seek an induction culture mode capable of stimulating the synthesis of glabridin and firstly provides and implements a culture method for improving the secondary metabolite glabridin in suspension culture cells of glabrous licorice.
The technical scheme adopted by the invention is as follows:
a method for increasing glabridin content in suspension culture cell of Glycyrrhrizae radix comprises culturing aseptic seedling with seed, cutting cotyledon, stem bud, hypocotyl, etc. of aseptic seedling, inducing callus, subculturing to obtain loose callus, and culturing the callus in liquid culture medium to obtain cell suspension culture system for shake culture. Adding daidzein (daidzein) as exogenous substrate supplement to the culture medium during suspension culture, and/or adding glabridin to the suspension culture solution to synthesize key biological enzyme, wherein the key biological enzyme is pterocarpus fruit reductase PTR (pterocarpan reductases enzymes) and/or isopentenyl transferase GmG4DT extracted and separated from Glycyrrhiza glabra; the production of glabridin in suspension culture cells is promoted by the way of jointly adding the substrate and the biological enzyme.
A method for improving the content of glabridin in suspension culture cells of liquorice comprises the following concrete implementation steps:
step 1, obtaining aseptic seedlings: selecting plump seeds of wild Glycyrrhiza glabra L.in Xinjiang, treating with concentrated sulfuric acid for 40min, washing with clear water, treating with 0.1% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times; inoculating to MS basal medium added with 30g/L sucrose and 6g/L agar, adjusting pH value to 5.8-6.0, culturing in dark at 25 + -2 deg.C for 5d, culturing at 25 + -2 deg.C under illumination of 2000-4000Lux at 25 + -2 deg.C for 16h/d for 20-25d, increasing hypocotyl length to 2-3cm, and developing cotyledon to obtain Glycyrrhiza glabra aseptic seedling;
step 2, callus induction culture: taking hypocotyl and cotyledon of aseptic seedling as explant, cutting into small segments or blocks, inoculating into MS basal medium containing 0.5mg/L NAA, 0.6 mg/L6-BA, 30g/L sucrose, 6g/L agar and pH of 5.8-6.0, and culturing in dark at 25 + -2 deg.C for 20 days to induce callus; then subculturing twice to obtain a large amount of glycyrrhiza glabra callus;
step 3, callus suspension shaking culture: selecting the pale yellow callus which grows vigorously and has loose texture in the step 2, and transferring the pale yellow callus to a liquid culture medium containing 0.5mg/L NAA, 1.0 mg/L6-BA, 0.8 mg/L2, 4-D and 30g/L sucrose for suspension shaking culture; the inoculation amount is 40g/L, the rotating speed of a shaking table is 120 rpm, the pH value of a liquid culture medium is 5.8-6.0, and the culture temperature is 25 +/-2 ℃. Subculturing for 1 time every 20 days, and continuously subculturing for three times;
step 4, inducing and improving the glabridin content in the suspension culture system cells: transferring the glycyrrhiza glabra cells in the glycyrrhiza glabra suspension culture system in the step 3 into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 5-20mg/L daidzein and/or 0.1-1.0 mu g/L pterocarya reductase PTR, pterocarpan reductase enzymes and/or 0.05-0.5 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culture, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotation speed of a shaking table is 120 rpm; after 15-20 days of culture, separating out cells with nylon net, extracting and analyzing glabridin.
The daidzein source is leguminous plant soybean which is easy to plant, high in content and low in cost.
The pterocarpus marsupium reductase PTR is derived from leaves of Glycyrrhiza glabra, and the isopentenyl transferase GmG4DT is derived from roots of Glycyrrhiza glabra.
After the culture is finished and the cultured cells are separated and taken out, the culture solution adopts a semi-permeable membrane with the cut-off molecular weight of 10000Da to recover the biological enzyme for the preparation of the culture solution of the next batch.
The invention adds soybean aglycone (daidzein) from soybean into the liquid culture medium of suspension culture as an exogenous substrate supplement, supplements the concentration of key intermediate soybean aglycone synthesized by glabridin in suspension culture cells, accelerates the progress of relevant biochemical reaction in the culture cells and promotes the generation of glabridin. The pterocarpus fruit reductase PTR (pterocarpan reductase enzymes) and the isopentenyl transferase GmG4DT extracted and separated from the glycyrrhiza glabra are added into the suspension culture solution, and the pterocarpus fruit reductase PTR and the isopentenyl transferase are used as biological enzyme assistance for promoting the synthesis of the glabridin, so that the defect of the critical biological enzyme of the isoflavone secondary metabolite induced by the injury stimulation of the wild growth environment of a plant body to the glycyrrhiza glabra plant body, which is deficient in the culture environment of suspension cells, is compensated, and the generation of the glabridin is further promoted.
After the culture is finished, the nylon net is used for separating out cells to extract and analyze glabridin in the cells, the culture solution after the culture is finished adopts a semi-permeable membrane with the cut-off molecular weight of 10000Da to recover the biological enzyme, the biological enzyme is used for preparing the culture solution in the next batch, the repeated utilization rate of the biological enzyme is increased, and the biological enzyme can be supplemented properly in the process.
In conclusion, the invention supplements daidzein in soybean of leguminous plant with wide source and low price and the same genus as Glycyrrhiza glabra as an exogenous substrate for the first time, and the daidzein is used as an essential intermediate for in vivo synthesis of glabridin plants, and the introduction of daidzein greatly promotes the corresponding biochemical reaction; the pterocarpus fruit reductase PTR and the isopentenyl transferase GmG4DT which are two key biological enzymes beneficial to the synthesis of the glabridin and are derived from different parts of the liquorice are added into a culture medium of a cell suspension culture system as exogenous supplements for the first time, so that the biosynthesis of the glabridin is promoted together, the difference between the cell suspension culture and the stimulation of the field growth environment of plants is made up, and the generation of the glabridin in cells cultured in a suspension manner is also promoted to a great extent. After the suspension cells are cultured by the method, the content of the glabridin in the suspension cells reaches 0.10-0.25% of the cell stem value, and the growth period of 3-5 years is probably needed for the content of the glabrous greenbrier rhizome whether planted or grown in a wild way. Compared with the prior art, the method has the advantages of short period, no limitation of regions, time and climate and easy industrialization, and the industrialization of the glabridin production by the suspension cell culture mode becomes possible.
Detailed Description
The present invention is further illustrated by the following examples, but is not limited to the following examples.
Example 1, a method for increasing glabridin content in suspension culture cells of licorice, comprising the following steps:
step 1, obtaining aseptic seedlings: selecting plump seeds of wild Glycyrrhiza glabra L.in Xinjiang, treating with concentrated sulfuric acid for about 40min, washing with clear water, treating with 0.1% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times. Inoculating to MS basal medium added with 30g/L sucrose and 6g/L agar, adjusting pH value to 5.8-6.0, culturing in dark at 25 + -2 deg.C for about 5d, culturing at 25 + -2 deg.C under illumination intensity of 2000-4000Lux for 16h/d for 20-25d, and developing cotyledon to obtain Glycyrrhiza glabra aseptic seedling;
step 2, callus induction culture: the hypocotyl and cotyledon of the aseptic seedling are taken as explants, cut into small sections (blocks) and inoculated into MS basal medium containing 0.5mg/L NAA, 0.6 mg/L6-BA, 30g/L sucrose, 6g/L agar and 5.8-6.0 of pH value, and cultured in the dark at the temperature of 25 +/-2 ℃ for about 20 days to induce the callus. Then subculturing twice to obtain a large amount of glycyrrhiza glabra callus.
Step 3, callus suspension shaking culture: and (3) selecting the pale yellow callus which grows vigorously and has loose texture in the step (2), and transferring the pale yellow callus into a liquid culture medium containing 0.5mg/L NAA, 1.0 mg/L6-BA, 0.8 mg/L2, 4-D and 30g/L sucrose for suspension shaking culture. The inoculation amount is 40g/L, the rotating speed of a shaking table is 120 rpm, the pH value of a liquid culture medium is 5.8-6.0, and the culture temperature is 25 +/-2 ℃. Subculture every 20 days for 1 time, and subculture was continued for three times.
Step 4, inducing and improving the glabridin content in the suspension culture system cells: and (3) transferring the glycyrrhiza glabra cells in the liquid culture medium in which the glycyrrhiza glabra is suspended in the step (3) into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 5mg/L daidzein, 0.5 mu g/L pterocarya fruit reductase PTR (pterocarpan products enzymes) and 30g/L sucrose for culture, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotation speed of a shaking table is 120 rpm. Separating cells on the 20 th day, and measuring the content of the glabridin, wherein the result shows that the glabridin content accounts for 0.07 percent of the dry weight of the cells.
Embodiment 2, a method for increasing glabridin content in suspension culture cells of licorice, comprising the following steps:
steps 1 to 3 are the same as in example 1
Step 4, inducing and improving the glabridin content in the suspension culture system cells: transferring the glycyrrhiza glabra cells in the liquid culture medium in which the glycyrrhiza glabra is suspended in the step 3 into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 10mg/L daidzein, 0.05 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culture, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotating speed of a shaking table is 120 rpm. Separating cells on the 20 th day, and measuring the content of the glabridin, wherein the result shows that the glabridin content accounts for 0.10 percent of the dry weight of the cells.
Embodiment 3, a method for increasing glabridin content in suspension culture cells of licorice, comprising the following steps:
steps 1 to 3 are the same as in example 1
Step 4, inducing and improving the glabridin content in the suspension culture system cells: and (4) transferring the glycyrrhiza glabra cells in the liquid culture medium in the step (3) into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 10mg/L daidzein, 0.2 mu g/L pterocarya fruit reductase PTR (pterocarpan reductases enzymes), 0.1 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culturing, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotation speed of a shaking table is 120 rpm. The content of glabridin is measured by separating cells from the culture solution by the nylon mesh on the 20 th day, and the result shows that the content of glabridin accounts for 0.12% of the dry weight of the cells.
Embodiment 4, a method for increasing glabridin content in suspension culture cells of licorice, comprising the following steps:
steps 1 to 3 are the same as in example 1
Step 4, inducing and improving the glabridin content in the suspension culture system cells: transferring the glycyrrhiza glabra cells in the liquid culture medium in which the glycyrrhiza glabra is suspended in the step 3 into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 15mg/L daidzein, 1.0 mu g/L pterocarya fruit reductase PTR (pterocarpan products enzymes), 0.5 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culture, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotating speed of a shaking table is 120 rpm. Separating cells on the 20 th day, and measuring the content of the glabridin, wherein the result shows that the glabridin content accounts for 0.22% of the dry weight of the cells.
Embodiment 5, a method for increasing glabridin content in suspension culture cells of licorice, comprising the following steps:
steps 1 to 3 are the same as in example 1
Step 4, inducing and improving the glabridin content in the suspension culture system cells: and (3) transferring the glycyrrhiza glabra cells in the liquid culture medium in the step (3) to an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 20mg/L daidzein, 0.1 mu g/L pterocarya fruit reductase PTR (pterocarpan products enzymes), 0.2 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culturing, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotation speed of a shaking table is 120 rpm. The content of glabridin is measured by separating cells from the culture solution by the nylon mesh on the 20 th day, and the result shows that the glabridin content accounts for 0.25% of the dry weight of the cells.
In examples 1-5 above:
the daidzein source is leguminous plant soybean which is easy to plant, high in content and low in cost.
The pterocarpus marsupium reductase PTR is derived from leaves of Glycyrrhiza glabra, and the isopentenyl transferase GmG4DT is derived from roots of Glycyrrhiza glabra.
After the culture is completed and the cultured cells are separated and taken out, the culture solution adopts a semi-permeable membrane with the cut-off molecular weight of 10000Da to recover the biological enzyme, and the biological enzyme is used for preparing the culture solution of the next batch, so that the reutilization of the biological enzyme is achieved. The specific method comprises the following steps:
step 1, the culture solution obtained after the cells are separated from the nylon net in the embodiment 5 is put into a pretreated cellulose CE dialysis bag with the molecular weight cutoff of 10000Da, the temperature is 5-8 ℃, the bag is put into pure water to be stirred and dialyzed, and the pure water is replaced for 4 times every 3 hours.
And 2, preparing an MS culture medium by using the water solution containing the biological enzyme recovered by the dialysis membrane in the first step, adding 0.8mg/L NAA, 1.0 mg/L6-BA and 30g/L sucrose, and adding 20mg/L daidzein to prepare a new liquid suspension culture medium.
And 3, inoculating the newly well-dispersed subculture callus cells into the culture medium obtained in the previous step, wherein the culture temperature is 25 +/-2 ℃, and the rotating speed of a shaking table is 120 rpm. The content of glabridin is measured by separating cells from the culture solution by the nylon mesh on the 20 th day, and the result shows that the content of glabridin accounts for 0.23% of the dry weight of the cells.
The comparative examples are as follows:
step 1, obtaining aseptic seedlings: selecting plump seeds of wild Glycyrrhiza glabra L.in Xinjiang, treating with concentrated sulfuric acid for about 40min, washing with clear water, treating with 0.1% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times. Inoculating to MS basal medium added with 30g/L sucrose and 6g/L agar, adjusting pH value to 5.8-6.0, culturing in dark at 25 + -2 deg.C for about 5d, culturing at 25 + -2 deg.C under illumination intensity of 2000-4000Lux for 16h/d for 20-25d, and developing cotyledon to obtain Glycyrrhiza glabra aseptic seedling;
step 2, callus induction culture: the hypocotyl and cotyledon of the aseptic seedling are taken as explants, cut into small sections (blocks) and inoculated into MS basal medium containing 0.5mg/L NAA, 0.6 mg/L6-BA, 30g/L sucrose, 6g/L agar and 5.8-6.0 of pH value, and cultured in the dark at the temperature of 25 +/-2 ℃ for about 20 days to induce the callus. Then subculturing twice to obtain a large amount of glycyrrhiza glabra callus.
Step 3, callus suspension shaking culture: and (3) selecting the pale yellow callus which grows vigorously and has loose texture in the step (2), and transferring the pale yellow callus into a liquid culture medium containing 0.5mg/L NAA, 1.0 mg/L6-BA, 0.8 mg/L2, 4-D and 30g/L sucrose for suspension shaking culture. The inoculation amount is 40g/L, the rotating speed of a shaking table is 120 rpm, the pH value of a liquid culture medium is 5.8-6.0, and the culture temperature is 25 +/-2 ℃. Subculture every 20 days for 1 time, and subculture was continued for three times.
Step 4, inducing and improving the glabridin content in the suspension culture system cells: transferring the glycyrrhiza glabra cells in the liquid culture medium in which the glycyrrhiza uralensis is suspended in the step 3 to an induction culture medium which is based on an MS culture medium and added with 0.8mg/L NAA, 1.0 mg/L6-BA and 30g/L sucrose for culturing, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotating speed of a shaking table is 120 rpm. Separating cells on the 20 th day, and measuring the content of the glabridin, wherein the result shows that the glabridin content accounts for 0.04% of the dry weight of the cells.
From the results, it can be seen that the content of glabridin in suspension cells can be effectively increased by introducing appropriate concentrations of daidzein, pterocarpan reductase PTR (pterocarpan reductase enzymes) and isopentenyl transferase GmG4DT into the suspension cell culture medium.
Claims (5)
1. A method for improving glabridin content in suspension culture cells of liquorice is characterized in that after aseptic seedlings are cultured by seeds, tissues such as cotyledons, stem buds and hypocotyls of the aseptic seedlings are cut out to carry out callus induction, loose callus is obtained by subculture, and then the callus is subjected to shake culture in a liquid culture medium to establish a cell suspension culture system; adding daidzein (daidzein) as exogenous substrate supplement to the culture medium during suspension culture, and/or adding glabridin to the suspension culture solution to synthesize key biological enzyme, wherein the key biological enzyme is pterocarpus fruit reductase PTR (pterocarpan reductases enzymes) and/or isopentenyl transferase GmG4DT extracted and separated from Glycyrrhiza glabra; the production of glabridin in suspension culture cells is promoted by the way of jointly adding the substrate and the biological enzyme.
2. The method for increasing the content of glabridin in suspension culture cells of licorice according to claim 1, wherein the method is implemented by the following steps:
step 1, obtaining aseptic seedlings: selecting plump seeds of wild Glycyrrhiza glabra L.in Xinjiang, treating with concentrated sulfuric acid for 40min, washing with clear water, treating with 0.1% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times; inoculating to MS basal medium added with 30g/L sucrose and 6g/L agar, adjusting pH value to 5.8-6.0, culturing in dark at 25 + -2 deg.C for 5d, culturing at 25 + -2 deg.C under illumination of 2000-4000Lux at 25 + -2 deg.C for 16h/d for 20-25d, increasing hypocotyl length to 2-3cm, and developing cotyledon to obtain Glycyrrhiza glabra aseptic seedling;
step 2, callus induction culture: taking hypocotyl and cotyledon of aseptic seedling as explant, cutting into small segments or blocks, inoculating into MS basal medium containing 0.5mg/L NAA, 0.6 mg/L6-BA, 30g/L sucrose, 6g/L agar and pH of 5.8-6.0, and culturing in dark at 25 + -2 deg.C for 20 days to induce callus; then subculturing twice to obtain a large amount of glycyrrhiza glabra callus;
step 3, callus suspension shaking culture: selecting the pale yellow callus which grows vigorously and has loose texture in the step 2, and transferring the pale yellow callus into a liquid culture medium containing 0.5mg/L NAA, 1.0 mg/L6-BA, 0.8 mg/L2, 4-D and 30g/L sucrose for suspension shaking culture; the inoculation amount is 40g/L, the rotating speed of a shaking table is 120 rpm, the pH of a liquid culture medium is 5.8-6.0, and the culture temperature is 25 +/-2 ℃; subculturing for 1 time every 20 days, and continuously subculturing for three times;
step 4, inducing and improving the glabridin content in the suspension culture system cells: transferring the glycyrrhiza glabra cells in the glycyrrhiza glabra suspension culture system in the step 3 into an induction culture medium which is based on an MS culture medium and is added with 0.8mg/L NAA, 1.0 mg/L6-BA, 5-20mg/L daidzein and/or 0.1-1.0 mu g/L pterocarya reductase PTR, pterocarpan reductase enzymes and/or 0.05-0.5 mu g/L isopentenyl transferase GmG4DT and 30g/L sucrose for culture, wherein the pH value of the culture medium is 5.8-6.0, the culture temperature is 25 +/-2 ℃, and the rotation speed of a shaking table is 120 rpm; after 15-20 days of culture, separating out cells with nylon net, extracting and analyzing glabridin.
3. The method of claim 2, wherein the daidzein source is soybean, which is a leguminous plant that is easily planted, has a high content and is low cost.
4. The method of claim 2, wherein the pterocarpus validus reductase PTR is derived from the leaf of Glycyrrhiza glabra and the prenyltransferase GmG4DT is derived from the root of Glycyrrhiza glabra.
5. The method for increasing glabridin content in suspension culture cell of licorice according to any one of claims 1-4, wherein after the culture is completed and the cultured cell is separated and taken out, the culture fluid adopts a semi-permeable membrane with molecular weight cut-off of 10000Da to recover the biological enzyme for the preparation of the culture fluid of the next batch.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN116574668B (en) * | 2023-06-13 | 2023-12-05 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
| CN117448254B (en) * | 2023-10-23 | 2024-04-09 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
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