1. A eucommia ulmoides suspension cell culture method for improving the content of effective components is characterized by comprising the following steps,

1) inoculating young stems or young leaves of the eucommia aseptic seedlings into a culture medium for culture, and inducing to obtain eucommia callus, wherein a plant growth regulator is added into the culture medium;

2) inoculating the eucommia ulmoides callus into a culture medium to be cultured to obtain eucommia ulmoides suspension cells, wherein a plant growth regulator is added into the culture medium;

3) adding fungus polysaccharide or a mixture of the fungus polysaccharide and methyl jasmonate into the eucommia ulmoides suspension cells, and continuously culturing to obtain the eucommia ulmoides suspension cells with the content of the effective components improved.

2. The culture method according to claim 1, wherein the addition amount of the fungal polysaccharide is 70 to 90mg/L, or the addition amount of the fungal polysaccharide is 30 to 50mg/L and the addition amount of methyl jasmonate is 80 to 120. mu.M/L.

3. The culture method according to claim 1 or 2, wherein the fungal polysaccharide is prepared by subjecting a Staphylococus botrytis dothidea to fermentation culture, collecting mycelia, and drying to obtain a dried mycelia; extracting with water under reflux, filtering, mixing the filtrate with ethanol, standing, filtering, dissolving the precipitate with water, eluting protein, centrifuging, collecting the upper layer solution, and freeze drying to obtain fungal polysaccharide.

4. The method according to claim 1, wherein the medium used in the steps 1) and 2) is B5 medium.

5. The method according to claim 1, wherein the plant growth regulator is a mixture of 6-BA and NAA or 2, 4-D.

6. The method according to claim 1, wherein the plant growth regulator is a mixture of 6-BA and NAA.

7. The culture method according to claim 5 or 6, wherein the amount of 6-BA added is 0.8-1.2mg/L, the amount of NAA added is 0.8-1.2mg/L, and the amount of 2,4-D added is 0.5 mg/L.

Background

Transferring the callus to a liquid medium for culture, called suspension culture, to obtain suspension cells. Various secondary metabolites can be produced by using the suspension cells. In order to achieve high yields of the desired metabolites from suspension cells, improvements are generally made in several ways, such as breeding high-producing cell lines and adding elicitors.

Eucommia ulmoides cell suspension culture and the accumulation of major secondary metabolites, Qiu dao, academic papers of university of Jiangxi, which describe the kinds of elicitors, including glycoproteins, such as glycosylated bovine serum albumin oxide and lysozyme oxide, protein elicitors, such as collagenase and chain protease, polysaccharides, such as fusarium mycelium, ethylene, salicylic acid, chitin and chitosan, and oligosaccharides thereof, microorganism elicitors, such as fungal elicitors, and metal ions, such as copper, calcium, magnesium ions, etc.

Eucommia bark is a rare traditional Chinese medicine, secondary metabolites mainly comprise lignans, iridoids, flavonoids, phenylpropanoids, polysaccharides, phenols, triterpenes, steroids, organic acids, amino acids, trace elements and the like, and the optimal culture conditions obtained by optimizing a eucommia bark cell suspension system through tests are B5+0.5mg/L NAA, 0.6 mg/L6-BA, 3% sucrose as a carbon source, pH 5.52 and the culture period of 12 days. The initial optimal inoculum size was 1.6 g.DCW/L. The maximum values of the metabolites are 16.63mg/g (113.08mg/L) of flavone and 3.93mg/g (26.57mg/L) of chlorogenic acid respectively. Optimization of callus induction and enrichment culture conditions of eucommia leaf, scientific technology and engineering, 13 th volume, 7 th phase, discusses the influence of the combination of four substances, namely 6-BA, NAA, 2,4-D and PVP, on callus and cells of eucommia. The results show that: (1) the four substances have the influence on the callus induction of eucommia ulmoides in the sequence of 6-BA>NAA>PVP, in order of effect on proliferation 6-BA>NAA>2, 4-D. The optimal culture medium for inducing callus by using eucommia leaf is MS +0.5 mg.L^-16-BA+0.5mg·L^-1NAA+2％PVP。

The study on the production of secondary metabolites by eucommia ulmoides cell culture, quality of Zhuyun, Master scholar thesie of south China university, researches the influence of additives on the growth of suspension cells and the content of aucubin, and the conclusion shows that: the sugar is one of the keys influencing the success of plant tissue culture, and the type and the amount of the sugar not only influence the growth speed and the growth amount of the culture, but also influence the metabolic level and the synthesis of secondary metabolites. When sucrose is used as a carbon source, the growth of cells is most suitable, the content of aucubin is the largest, and the effects of other two carbon sources are not as good as that of sucrose. However, sucrose has a certain inhibiting effect on the generation of aucubin. The result of the preliminary optimization of the condition for accumulating aucubin is as follows: MS +20g/L sucrose + pH 5.8+2.0 mg/L2, 4-D +0.5 mg/L6-BA, and the content of aucubin can reach 60mg/L at the maximum. The addition of 100mg/L hydrolyzed casein and 15mg/L yeast extract can promote the synthesis of aucubin. The content of chlorogenic acid reaches the highest in 15d, and the preliminary optimization result of the chlorogenic acid accumulation condition is as follows: MS +35g/L sucrose + pH 5.3+1.0 mg/L6-BA. The addition of 400mg/L hydrolyzed casein and 10mg/L yeast extract can promote the synthesis of chlorogenic acid. The addition of the precursor has certain influence on the synthesis of the secondary metabolite, and different concentration gradients can be analyzed to improve the content of the secondary metabolite.

H₂O₂The research on the influence of flavone content and peroxidase activity in callus of eucommia ulmoides, Deng Yun and the like, molecular plant breeding, 2018, volume 16, stage 3 shows that 5-10mmol/L H is obtained₂O₂The total flavone content is highest when the callus of eucommia is cultured for 10 days, and when the callus of eucommia is cultured for 5-10 days and exogenous H is added₂O₂The POD activity was highest at a concentration of 10 mmol/L. Correlation analysis shows that the correlation coefficient between the total flavone content of eucommia ulmoides callus and POD activity is r ═ 0.42061, and the correlation coefficient has certain correlation, and 5-10mmol/L H is presumed₂O₂Can stimulate the accumulation of flavone by improving POD enzyme activity in callus of eucommia.

The GC-MS/SIM method is used for determining amino acids, aromas and the like in eucommia leaves in different producing areas, and the amino acid content in eucommia leaves in different producing areas is researched at the No. 4 of No. 10 of 4 months of 2012 in Zhongnan pharmacy, and the total amino acid content is 9.95% at most.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a eucommia ulmoides suspension cell culture method for improving the content of effective components, which can effectively improve the activity of peroxidase in eucommia ulmoides suspension cells and improve the content of substances such as amino acid and the like.

The invention relates to a method for culturing eucommia ulmoides suspension cells with improved content of effective components, which comprises the following steps,

1) inoculating young stems or young leaves of the eucommia aseptic seedlings into a culture medium for culture, and inducing to obtain eucommia callus, wherein a plant growth regulator is added into the culture medium;

2) inoculating the eucommia ulmoides callus into a culture medium to be cultured to obtain eucommia ulmoides suspension cells, wherein a plant growth regulator is added into the culture medium;

3) adding fungus polysaccharide or a mixture of the fungus polysaccharide and methyl jasmonate into the eucommia ulmoides suspension cells, and continuously culturing to obtain the eucommia ulmoides suspension cells with the content of the effective components improved.

Preferably, the addition amount of the fungal polysaccharide is 70-90mg/L, or the addition amount of the fungal polysaccharide is 30-50mg/L and the addition amount of the methyl jasmonate is 80-120 mu M/L.

Preferably, the preparation method of the fungal polysaccharide comprises the steps of carrying out fermentation culture on the Botryosphaeria dothidea, collecting mycelium, and drying to obtain a mycelium dried substance; extracting with water under reflux, filtering, mixing the filtrate with ethanol, standing, filtering, dissolving the precipitate with water, eluting protein, centrifuging, collecting the upper layer solution, and freeze drying to obtain fungal polysaccharide.

Preferably, the culture medium of step 1) and step 2) is B5 culture medium.

Preferably, the plant growth regulator is 6-BA, and NAA or a mixture of 2, 4-D. Preferably, the plant growth regulator is a mixture of 6-BA and NAA.

Preferably, the addition amount of 6-BA is 0.8-1.2mg/L, the addition amount of NAA is 0.8-1.2mg/L, and the addition amount of 2,4-D is 0.5 mg/L.

The method has the beneficial effects that the fungus polysaccharide or the composition of the fungus polysaccharide and methyl jasmonate is added into the eucommia ulmoides suspension cells to induce the eucommia ulmoides suspension cells to generate a large amount of active ingredients, and the active ingredients mainly comprise peroxidase, amino acid, arctiin, naringin and hesperidin, so that the method lays a foundation for large-scale industrial production.

Drawings

FIG. 1 is a photograph of callus induced by solid medium.

FIG. 2 is a photograph of callus liquid culture induced suspension cells.

FIG. 3 is a graph showing the change of POD enzyme activity OD values in suspension cells of eucommia ulmoides of different induction treatment groups.

FIG. 4 is a TIC chart of suspension cell samples of eucommia ulmoides from different treatment groups.

FIG. 5 is a graph of the peak area of EIC image of each characteristic ion in different treatment group samples.

In fig. 4, a is the SA (salicylic acid) -treated group; b is MeJA (methyl jasmonate) treatment group; c is a blank group; d is a fungal polysaccharide treatment group.

Detailed Description

Example 1

Preparation of fungal polysaccharide elicitor

Selecting Staphylococus (Botryosphaeria dothidea) for fermentation culture of mycelium, wherein the fermentation amount is 3L, centrifuging, collecting mycelium, and drying. Taking mycelium dry matter, refluxing and extracting for 1-2h (100 ℃ water bath) by 12 times of water, filtering, adding 4 times of 75-80% ethanol with volume concentration into filtrate, standing overnight, filtering, adding water (the weight of the water is 10 times of the weight of the precipitate) into precipitate for dissolving, deproteinizing by adopting a Sevage method (the solvent is chloroform/n-butanol (the volume ratio is 4:1) solution), shaking for 20min, centrifuging, and freeze-drying the upper layer solution to obtain fungal polysaccharide for later use.

Example 2

Culture of eucommia ulmoides suspension cells

Collecting fresh eucommia ulmoides pods, taking out seeds in the eucommia ulmoides pods by using scissors, wiping the surfaces with alcohol with the volume concentration of 75%, and carrying out surface disinfection. And (3) treating the mixture for 30s with ethanol with the volume concentration of 70% in an ultra-clean workbench under the aseptic condition, and then washing the mixture for 3-5 times with the aseptic water. And treating the mixture for 3 to 5min by using a sodium hypochlorite solution with the mass concentration of 6 percent, washing the mixture for 5 to 6 times by using sterile water, finally soaking the mixture for 6 to 10min by using a mercuric chloride solution with the mass concentration of 0.1 percent, washing the mixture for 5 to 6 times by using the sterile water, and then inoculating the mixture into a culture medium for culture. The sterile seedling after seed germination is used for the induction experiment of callus of eucommia, and young stem leaves are taken.

Effect of different media on the Germination of eucommia ulmoides seeds

Adopting MS, B5 and WPM 3 culture mediums, respectively adding 1.0 mg/L6-BA +0.1mg/L NAA into the culture mediums, inoculating 6 granules into each bottle of the treated eucommia seeds, and inoculating 10 bottles of different culture mediums. And after 20d, investigating the germination rate of the eucommia seeds. After the eucommia seeds are inoculated for 7 days, the seeds begin to germinate and sprout. After 15 days, complete plants were grown and the germination rates of the seeds were different on different media (Table 1). Germination rates were investigated at 20d with low salt B5 being highest and high salt MS being lowest.

TABLE 1 Effect of different media on the Germination of eucommia ulmoides seeds

Effect of different hormones on germination of eucommia seed

The B5 culture medium is adopted as a basic culture medium, and five different hormones of 1.0 mg/L6-BA, 1.0mg/L NAA, 1.0 mg/L2, 4-D, 1.0mg/L KT and 0.5 mg/L6-BA +0.5mg/L NAA are respectively added into the culture medium. And (3) sterilizing eucommia seeds, inoculating the sterilized eucommia seeds into different culture mediums, inoculating 5 granules into each bottle, and inoculating 5 bottles into different culture mediums. And after 20d, investigating the germination rate of the eucommia seeds. After 7 days of inoculation, the seeds were observed to begin to germinate. After 15 days, complete plants were grown and the germination rates of the seeds were different on different media (Table 2). The germination rate was highest at 20d investigated with 6-BA + NAA, KT was relatively low.

TABLE 2 Effect of different hormones on the Germination of eucommia ulmoides Oliver seeds

Influence of different hormones on the induction of callus growth of eucommia ulmoides

On the basis of germination of eucommia seed, aseptic seedling is taken, young stem is cut into small segments of about 0.7cm, leaf is cut into 0.5cm × 0.5cm square blocks, and the explant is inoculated to wound. Adopting a B5 culture medium and 2 explants of the young stem and the young leaf of the eucommia sterile seedling, and respectively adding 1.0 mg/L6-BA +1.0mg/L NAA, 1.0mg/L IBA, 0.5 mg/L2, 4-D and 1.0mg/L KT of four different hormones. The effect of different hormones on callus induction rates was studied. Each bottle was inoculated with 5 blocks of sterile seedling explants. Each explant was inoculated with 10 flasks per hormone, and the callus induction rate was investigated for each stem and leaf 20 d.

After the young leaves are inoculated for 7 days, young stems expand, two ends of the leaves begin to wrinkle, milky callus grows out gradually at main veins, and after 15 days, a plurality of longitudinal cracks appear on the surfaces of thicker stem sections, and the initially appearing callus is milky. The rice-grain milky white callus grows out from the leaf margin of the leaf, most obvious in the vein (see figure 1). As the culture medium time is prolonged, the callus tissues appear different colors on the culture medium with different hormones, and the callus growth rate, the callus growth condition and the color of 4 auxin treated explants have obvious differences after 20 days (Table 3). Explants in the hormone KT developed roots, buds and began to wither, and very few calli in the hormone IBA lost luster and died. Through careful observation, the inductivity of culture media with different hormones to callus of explants of eucommia aseptic seedlings is better than that of hormones NAA and 2,4-D, IBA is lower, and the inductivity of KT is 0. Then inoculating the callus to B5+6-BA + NAA or 2,4-D liquid culture medium suitable for callus culture or culturing suspension cells (figure 2), and updating the culture medium every 7D to obtain the suspension cells.

TABLE 3 Effect of different hormones on the induction of callus growth of eucommia ulmoides

Detection of related enzyme activity in suspension cells after induction of signal molecules and non-signal molecules

6 groups of equal amount of suspension cells with good growth state (original culture medium washed by sterile water) are all added with a proper amount of fresh 2,4-D culture solution, salicylic acid (signal molecule) SA (the concentration of salicylic acid is controlled to be 200 mu M/L), methyl jasmonate MeJA (the concentration of methyl jasmonate is controlled to be 200 mu M/L), fungal polysaccharide (non-signal molecule) (the addition amount of fungal polysaccharide is 80mg/L), mixture of fungal polysaccharide (the addition amount of fungal polysaccharide is 40mg/L) and salicylic acid (the concentration of salicylic acid is controlled to be 100 mu M/L), mixture of fungal polysaccharide (the addition amount of fungal polysaccharide is 40mg/L) and methyl jasmonate (the concentration of methyl jasmonate is controlled to be 100 mu M/L), and equal amount of sterile water is added for blank control. And after 48h of induction, taking out, repeatedly washing with sterile water for several times in a mode of centrifugation, adding sterile water for rinsing and then centrifuging, washing out the culture medium on the surface of the plant cell, and then carrying out the next step of intracellular POD enzyme activity determination experiment. Taking 0.1g of suspended cell material, shearing, putting into a mortar, adding a proper amount of phosphate buffer solution, grinding into homogenate, extracting the residue once by using 5mL of phosphate buffer solution, filtering at low temperature to obtain supernatant, namely crude enzyme solution, fixing the volume to 10mL, and storing at low temperature for later use.

Taking 2 test tubes, adding 3mL of reaction mixed solution (the preparation of the reaction mixed solution is that 50mL of 100mmol/L phosphate buffer solution (pH 6.0) is taken to be placed in a beaker, 28 mu L of guaiacol is added, heating and stirring are carried out on a magnetic stirrer until the guaiacol is dissolved, after the solution is cooled, 19 mu L of 30% hydrogen peroxide is added, the mixture is uniformly mixed and is stored in a refrigerator for standby) and 1mL of phosphate buffer solution as a control, and adding 3mL of the reaction mixed solution and 1mL of the solution to be tested into the other 1 test tube. And (3) rapidly mixing the solutions in the two test tubes, pouring the mixture into a cuvette, placing the cuvette in a sample chamber of a spectrophotometer, immediately starting a stopwatch to record time, measuring an absorbance (OD) value at 470nm, reading every 10s, and recording the absorbance value for 4 min.

In OD Change per minute (Δ A)₄₇₀Min/g.min) represents the enzyme activity, and can also be expressed as 1 peroxidase activity unit (U) in terms of a change in OD value of 0.01 per minute.

Peroxidase Activity (U/g.min)^-1)＝ΔA₄₇₀xVT/(W × VS × 0.01 × t) wherein:

ΔA₄₇₀-change in OD over reaction time.

VT-Total volume of enzyme extract (mL).

W-plant fresh weight (g).

VS-volume of enzyme solution taken up (mL) at assay.

t-reaction time (min).

The POD enzyme activity OD value in suspension cells of eucommia ulmoides cells of six different experimental treatment groups is shown in the following figure 3.

From the slope of the curve in the figure, the POD enzyme activity of the fungus polysaccharide and methyl jasmonate mixture group is the highest, compared with a single substance, the POD enzyme activity of the fungus polysaccharide induction group is higher, and then the POD enzyme activity is a blank control group, and then the POD enzyme activity is a salicylic acid treatment group, and the worst POD enzyme activity is a methyl jasmonate treatment group. According to the experimental data, the suspension cell intracellular peroxidase activities of the blank control group, the salicylic acid induction group, the methyl jasmonate induction group and the fungal polysaccharide induction group are calculated to be 812.31, 594.45, 180.57 and 895.69 (U/g.min) respectively^-1) The intracellular peroxidase activities of the suspended cells of the group consisting of the mixture of fungal polysaccharide and salicylic acid and the mixture of fungal polysaccharide and methyl jasmonate were 876.66 and 918.83 (U/g.min), respectively^-1)。

The experimental result shows that the fungal polysaccharide has a promoting effect on the growth of eucommia ulmoides suspension cells. Compared with a blank control, the peroxidase activities of the suspension cells of the salicylic acid induction group and the methyl jasmonate induction group are smaller, the two groups have certain inhibition effects, the methyl jasmonate can induce to generate peroxidase at the early stage, and has certain inhibition effects at the later stage.

The combination of fungal polysaccharide and salicylic acid or methyl jasmonate, both had the effect of inducing peroxidase production relative to sterile water, probably primarily due to fungal polysaccharide induction. The combination of the fungal polysaccharide and methyl jasmonate has the induction capacity equivalent to that of 2 times of the fungal polysaccharide, and the possible reason is that the synergistic effect of the fungal polysaccharide and methyl jasmonate jointly promotes the change of enzyme activity through exogenous stimulation and intrinsic signal transduction response.

Example 3

Metabolite detection of suspension cells before and after induction

And detecting metabolites of eucommia ulmoides suspension cells of the four treatment groups by using a UPLC-Q-TOF/MS method.

Sample treatment: 0.5g of suspension cells of each of 6 treatment groups are taken, precisely weighed, placed in a conical flask with a plug, precisely added with 50mL of methanol with the mass concentration of 50%, tightly plugged, weighed, ultrasonically treated (the power is 250W, the frequency is 50kHz) for 40min, cooled, weighed again, supplemented with the methanol with the mass concentration of 50% to make up the lost weight, filtered, and the subsequent filtrate is taken and injected into UPLC-Q-TOF/MS for detection.

HPLC detection conditions: the chromatographic column is Agilent Eclipse XDB-C₁₈(2.1X 100mm, 3.5 μm), column temperature 30 deg.C, detection wavelength 210nm, sample injection amount 4 μ L, flow rate 0.3 mL/min^-1The mobile phase is acetonitrile (A) -0.01 percent formic acid solution (B); the gradient elution procedure is shown in table 4 below.

TABLE 4 gradient elution schedule

MS detection conditions: ESI source, positive ion mode scan, scan range m/z 100-. Parameters are as follows: gas Temp:350 ℃, Drying Gas:6 mL/min^-1，Nebulizer:60psi，Sheath Gas Temp:350℃，Sheath Gas Flow:12mL·min^-1VCap 4000V, Skimmer 65V, Fragmentor 130V, colloid Energy 5V, 10V. See fig. 4.

By combining mass spectrum maps, 8 common compounds are identified from eucommia ulmoides suspension cells of different treatment groups, and the mass spectrum result analysis of 8 common peaks shows that two ionization forms [ M + H ]]⁺And [ M + Na]⁺And calculating the element composition of each main component, and performing primary identification by analysis and comparison of compounds, wherein 8 compounds are valine (1), leucine (2), phenylalanine (3), tryptophan (4), 2-hydroxyphenylacetic acid (5), arctiin (6), naringin (7) and hesperidin (8).

By extracting the 8 characteristic ion graphs and integrating the EIC graphs, comparing the peak areas of the EIC graphs of the characteristic ions in different treatment groups (see figure 5), the content of the compounds 1, 6, 7 and 8 in the fungal polysaccharide induction treatment group suspension cells is higher, the content of the compounds 3, 4 and 5 in the methyl jasmonate induction treatment group samples is higher, the content of the compound 2 in the blank control group samples is higher, the content of the compounds 1, 5, 6, 7 and 8 in the fungal polysaccharide and salicylic acid group and the content of the compounds in the fungal polysaccharide and methyl jasmonate group suspension cells are higher, but the content of the compounds in the fungal polysaccharide and methyl jasmonate induction treatment group suspension cells is higher, and the secondary metabolism accumulation of the amino acid compounds is supposed to be higher under the salicylic acid induction condition; under the induction condition of fungal polysaccharide, the content of flavone and lignan compounds is high, and the fungal polysaccharide can be matched with salicylic acid group or methyl jasmonate to induce the generation of compounds 1, 5, 6, 7 and 8, so that the effect of improving the content of effective components is achieved.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the spirit of the present disclosure, features from the above embodiments or from different embodiments may also be combined, steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments in this application as described above, which are not provided in detail for the sake of brevity.

It is intended that the one or more embodiments of the present application embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements, and the like that may be made without departing from the spirit and principles of one or more embodiments of the present disclosure are intended to be included within the scope of the present disclosure.