Epimedium sagittatum cell suspension system as well as construction method and application thereof

Abstract

The invention discloses an epimedium sagittatum cell suspension system as well as a construction method and application thereof, and belongs to the technical field of medicinal plant tissue culture. The construction method of the epimedium sagittatum cell suspension system comprises the following steps: carrying out subculture and suspension culture on epimedium sagittatum calluses to obtain a suspension cell cluster, inoculating the suspension cell cluster into a liquid culture medium, and carrying out shake cultivation to obtain the epimedium sagittatum cell suspension system. On the 6th-8th day of shake cultivation, an elicitor solution is added into the liquid culture medium, and the elicitor comprises jasmonic acid methyl ester, salicylic acid or chitosan. According to the method, the epimedium sagittatum cell suspension system is successfully constructed, the browning problem in the epimedium sagittatum callus culture process is effectively controlled, and a theoretical basis is provided for large-scale culture of epimedium sagittatum so as to solve the current situation problem of short supply of resources at present. Meanwhile, a new direction is provided for improving secondary metabolites of epimedium sagittatum.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal plant tissue culture, and in particular to an Epimedium sagittatum cell suspension system and a construction method and application thereof. Background Art

[0002] Epimedium sagittatum (Sieb. et Zucc.) Maxim. is a perennial herbaceous plant of the genus Epimedium in the Berberidaceae family. The latest research has found that the Chinese herbal medicine Epimedium has anti-cancer activity and is effective against various types of cancer. It is one of the key Chinese herbal medicine varieties developed in my country. However, the raw materials of Epimedium are limited, and it has long relied mainly on wild resources. The large market demand and the growing demand for Epimedium at home and abroad have led to the over-exploitation of wild resources, causing its wild population to be on the verge of extinction, which is not conducive to the sustainable utilization of Epimedium resources. Artificial cultivation of Epimedium has serious seed dormancy, seedling propagation technology is difficult to break through, and economic benefits are low. Under natural conditions, epimedium mainly reproduces asexually, there are few seedlings in the wild, the germination period is long and the germination rate is low. At present, the main way for wild epimedium to maintain population spread and reproduction is still to rely on its underground rhizome cloning. In artificial production, rhizome transplanting is often used for asexual propagation. However, when epimedium reproduces asexually with underground rhizomes, it is easily restricted by fungi, bacteria, growth environment, plant source and other factors. Natural reproduction and artificial planting are far from enough to meet the current market demand, and it is difficult to achieve large-scale production.

[0003] In order to quickly obtain a large number of active ingredients of Epimedium, tissue culture of Epimedium sagittae was carried out. At present, research on tissue culture of Epimedium sagittae has made progress, but the callus of Epimedium sagittae has a long growth cycle, slow proliferation, and low content of effective ingredients, which cannot be cultured on a large scale, and is not enough to alleviate the current situation of insufficient supply of Epimedium sagittae resources. Compared with tissue culture, plant suspension culture has the advantages of fast growth rate, high stability, short test cycle, and strong tolerance. Establishing and optimizing the cell suspension culture system and increasing the content of secondary metabolites of Epimedium sagittae are the decisive factors for large-scale cultivation of Epimedium sagittae and solving the current shortage of resources. To this end, it is necessary to establish a cell suspension system of Epimedium sagittae and optimize the cell line to explore new ways to increase the secondary metabolites of Epimedium sagittae.

[0004] Plant cell suspension culture is an emerging culture technology based on callus tissue, which places the isolated plant tissue in liquid culture medium and cultures and grows it on a shaker. However, it is difficult to induce epimedium explants, and the browning problem is very serious, which affects the subsequent subculture and seedling breeding. Therefore, how to effectively control the browning problem in epimedium tissue culture and successfully establish an epimedium sagittatum cell suspension system is still a problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a cell suspension system of Epimedium sagittatum and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention successfully solves the browning problem of the callus culture process of Epimedium sagittatum, and constructs a cell suspension system of Epimedium sagittatum, which provides a theoretical basis for large-scale cultivation of Epimedium sagittatum to solve the current problem of insufficient supply of resources. At the same time, it provides a new direction for improving the secondary metabolites of Epimedium sagittatum.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing a cell suspension system of Epimedium sagittatum, comprising the following steps:

[0008] Selecting well-growing Epimedium sagittatum callus, obtaining suspension cell clusters after subculture and suspension culture, inoculating the suspension cell clusters into a liquid culture medium for shaking culture, and obtaining the Epimedium sagittatum cell suspension system;

[0009] In the subculture, the culture medium is WPM + 2,4-D 1.5 mg·L -1 +6-BA 1.0mg·L -1 +PVP 0.5~2g·L -1 ;

[0010] In the suspension culture, the culture medium is WPM+6-BA 1.5mg·L -1 +2,4-D 2.0mg·L -1 + sucrose 20g·L -1 ;

[0011] In the shaking culture, the liquid culture medium is WPM+2,4-D 2.0mg·L -1 +6-BA 1.5mg·L -1 +NAA 0.5mg·L -1 + sucrose 30g·L -1 .

[0012] Optionally, in the subculture, the culture medium is WPM + 2,4-D 1.5 mg·L -1 +6-BA 1.0mg·L -1 +PVP1.5g·L -1 .

[0013] Optionally, the inoculation amount of the suspended cell mass in the liquid culture medium is 2 g of the suspended cell mass per 50 mL of the liquid culture medium.

[0014] Optionally, the rotation speed of the shaking incubator is 110 rpm.

[0015] Optionally, the pH of the liquid culture medium is 6.0.

[0016] Optionally, on the 6th to 8th day of the shaking culture, an inducer solution is added to the liquid culture medium, and the inducer includes methyl jasmonate, salicylic acid or chitosan.

[0017] Optionally, the inducer is chitosan.

[0018] The invention also provides an Epimedium sagittatum cell suspension system obtained by the method.

[0019] The present invention also provides application of the Epimedium sagittatum cell suspension system in improving the content of effective ingredients of the Epimedium sagittatum.

[0020] Optionally, the medicinal ingredients include total flavonol glycosides, total flavonoids, total phenols, total alkaloids and total saponins.

[0021] Optionally, the medicinal ingredient is total flavonol glycosides.

[0022] The present invention discloses the following technical effects:

[0023] The present invention successfully constructs a cell suspension system of Epimedium sagittatum and effectively controls the browning problem in the process of Epimedium sagittatum callus culture, providing a theoretical basis for large-scale culture of Epimedium sagittatum to solve the current problem of insufficient supply of resources. At the same time, it provides a new direction for improving the secondary metabolites of Epimedium sagittatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The growth curve of suspension cells of Epimedium sagittatum (A) and the changes of total flavonoids content (B);

[0026] Figure 2 Epimedium sagittatum suspension cells;

[0027] Figure 3Effects of different elicitors on the content of total flavonol glycosides in Epimedium sagittatum suspension cells. Among them, A - C are the contents of epimedin A, epimedin C, and icariin in the experimental group of MEJA elicitor in sequence; D - F are the contents of epimedin A, epimedin C, and icariin in the experimental group of SA elicitor in sequence; G - I are the contents of epimedin A, epimedin C, and icariin in the experimental group of CTS elicitor in sequence; J - L are the contents of total flavonol glycosides in the experimental groups of elicitors MEJA, SA, and CTS in sequence.

[0028] Figure 4 It is a waterfall diagram of each component in Epimedium sagittatum suspension cells induced by different elicitors. Among them, A is the content of metabolites and cell growth in the aqueous extract of Epimedium sagittatum suspension cells treated with different concentrations of MEJA; B is the content of metabolites and cell growth in the aqueous extract of Epimedium sagittatum suspension cells treated with different concentrations of SA; C is the content of metabolites and cell growth in the aqueous extract of Epimedium sagittatum suspension cells treated with different concentrations of CTS.

[0029] Figure 5 Effects of different elicitors on the growth of Epimedium sagittatum suspension cells. Among them, A is the effect of different concentrations of elicitor MEJA on the growth of Epimedium sagittatum suspension cells; B is the effect of different concentrations of elicitor SA on the growth of Epimedium sagittatum suspension cells; C is the effect of different concentrations of elicitor CTS on the growth of Epimedium sagittatum suspension cells.

[0030] Figure 6 Effects of different elicitors on the total phenol content in Epimedium sagittatum suspension cells. Among them, A is the effect of different concentrations of elicitor MEJA on the total phenol content in Epimedium sagittatum suspension cells; B is the effect of different concentrations of elicitor SA on the total phenol content in Epimedium sagittatum suspension cells; C is the effect of different concentrations of elicitor CTS on the total phenol content in Epimedium sagittatum suspension cells.

[0031] Figure 7 Effects of different elicitors on the total flavonoid content in Epimedium sagittatum suspension cells. Among them, A is the effect of different concentrations of elicitor MEJA on the total flavonoid content in Epimedium sagittatum suspension cells; B is the effect of different concentrations of elicitor SA on the total flavonoid content in Epimedium sagittatum suspension cells; C is the effect of different concentrations of elicitor CTS on the total flavonoid content in Epimedium sagittatum suspension cells.

[0032] Figure 8 Effects of different elicitors on the total alkaloid content in Epimedium sagittatum suspension cells. Among them, A is the effect of different concentrations of elicitor MEJA on the total alkaloid content in Epimedium sagittatum suspension cells; B is the effect of different concentrations of elicitor SA on the total alkaloid content in Epimedium sagittatum suspension cells; C is the effect of different concentrations of elicitor CTS on the total alkaloid content in Epimedium sagittatum suspension cells.

[0033] Figure 9 Effects of different elicitors on the total saponin content in Epimedium sagittatum suspension cells. Among them, A shows the effects of elicitor MEJA at different concentrations on the total saponin content in Epimedium sagittatum suspension cells; B shows the effects of elicitor SA at different concentrations on the total saponin content in Epimedium sagittatum suspension cells; C shows the effects of elicitor CTS at different concentrations on the total saponin content in Epimedium sagittatum suspension cells. Detailed implementation manners

[0034] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0035] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the description of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are only exemplary.

[0038] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0039] Example 1 Screening and culture of callus

[0040] The callus of Epimedium sagittatum was previously induced and subcultured in the laboratory of the Engineering and Technology Research Center for Ecological Planting of Genuine Medicinal Materials in Henan Province, Henan University of Chinese Medicine. Callus with good growth and similar size was selected as the experimental material.

[0041] I. Experimental Methods

[0042] 1. Orthogonal Experiment Design

[0043] Calli with good growth and similar sizes were inoculated into media with different hormone ratios. Woody Plant Medium (WPM) was used as the basic medium, with 20 g of sucrose, 5 g of agar, and 0.56 g of calcium nitrate added per liter of the medium. The pH was adjusted to 5.8 - 6.0. For each treatment, 10 bottles were inoculated, with 4 pieces of calli placed in each bottle, and the size of each piece of callus was not less than 5 mm. They were cultured at 25°C for 20 days, and the browning rate and growth status of the calli were counted. Two hormones, 2,4-D and 6-BA, were selected at three levels (concentrations were all mg·L -1 ) for the experiment to investigate the effect of the above two hormones with the function of delaying polyphenol synthesis on the browning of calli induced from Epimedium leaves. According to the L9(3 2 ) orthogonal table, 9 experiments were designed and arranged, and the factor levels were arranged as shown in Table 1.

[0044] Table 1 Factor Levels of Orthogonal Experiment

[0045]

[0046]

[0047] 2. Screening of Anti-browning Agents

[0048] Four anti-browning agents, polyvinylpyrrolidone (PVP), activated carbon (AC), ascorbic acid (VC), and citric acid (CA), were selected. The best subculture medium screened by the orthogonal experiment was used as the blank control. The above four anti-browning agents were added respectively and a blank control was set to induce the calli of Epimedium sagittatum. For each treatment, 10 bottles were inoculated, with 4 pieces of calli placed in each bottle, and the size of each piece of callus was not less than 5 mm. They were cultured at 25°C for 20 days, and the browning rate and growth of the calli were observed and recorded to screen out the best anti-browning agent and its applicable concentration. The types and concentrations of anti-browning agents were designed as shown in Table 2.

[0049] Table 2 Design of Anti-browning Agent Types and Concentrations

[0050]

[0051] 3. Measurement Indexes

[0052] Judgment criteria for visually observing the growth rate of calli: - indicates extremely poor growth state of calli, + indicates slow growth of calli, ++ indicates general growth of calli, and +++ indicates rapid growth of calli.

[0053] Browning rate = Number of browned calli / Number of inoculated calli × 100%

[0054] II. Experimental Results and Analysis

[0055] 1. Screening of the Optimal Hormone Concentration Ratio for the Callus of Epimedium sagittatum

[0056] The browning of callus usually occurs at the initial stage of subculture. Therefore, different hormones and their concentration ratios play an important role in the morphogenesis and regulation of callus. In this invention, two hormones 2,4-D and 6-BA that are widely used in the tissue culture of woody plants and can delay polyphenol synthesis are used in combination to find the optimal hormone ratio. The callus of Epimedium sagittatum was inoculated into the culture media with different hormone ratios. 10 bottles were inoculated for each treatment, 4 pieces of callus were placed in each bottle, and the size of each piece of callus was not less than 5 mm. After culturing at 25 °C for 20 days, it was found that the callus could continue to grow and develop, but there were differences in the browning rate, growth rate, and growth status among the callus inoculated in the culture media with different hormone ratios. Treatment group 8 was the original subculture medium in the laboratory. As can be seen from Table 3, when the concentration of hormone 6-BA was relatively low at 0.5 mg·L -1 and relatively high at 1.5 mg·L -1 , when three gradient concentrations of 2,4-D were added to the culture medium, the effects of the two hormone ratios on callus browning were not obvious; when the added concentration of 6-BA was 1.0 mg·L -1 , the addition of a low concentration of 1.5 mg·L -1 2,4-D had an obvious effect on callus browning, reducing the browning rate by 20%. When the added concentration of 6-BA was 1.0 mg·L -1 , and at the same time, a high concentration of 2.5 mg·L -1 2,4-D was added, the browning rate of callus increased; therefore, the callus inoculated in the culture medium with the hormone ratio of No. 4 had the best growth state, the lowest browning rate, and the fastest growth rate; Considering comprehensively, the optimal hormone ratio culture medium for the proliferation of the callus of Epimedium sagittatum is the No. 4 culture medium, that is, WPM + 2,4-D 1.5 mg·L -1 + 6-BA 1.0 mg·L -1 . Therefore, the culture medium with the hormone ratio of No. 4 was used as the subculture medium in the subsequent experiments.

[0057] Table 3 Effects of Different Hormones and Their Concentration Ratios on the Growth of Callus

[0058]

[0059] 2. Analysis of the Effects of Different Types and Concentrations of Browning Inhibitors on the Browning of the Callus of Epimedium sagittatum

[0060] Through the screening of the optimal hormone concentration ratio for the callus of Epimedium sagittatum, the No. 4 hormone ratio culture medium was obtained, that is, WPM + 2,4-D 1.5 mg·L-1 + 6 - BA 1.0 mg·L -1 is the optimal hormone ratio medium. To ensure good growth of the callus, this medium is selected as the basal medium for the subculture medium to study the browning effects of different types and concentrations of anti - browning agents. The effects of different types of anti - browning agents and their different concentrations on the growth of callus are shown in Table 4.

[0061] Table 4 Browning effects of different types of anti - browning agents and their different concentrations on callus

[0062]

[0063]

[0064] As can be seen from Table 4, when the concentrations of hormones 2,4 - D and 6 - BA in the medium are fixed, adding certain concentrations of three anti - browning agents, VC, PVP, and CA, to the medium all have certain effects on preventing the browning of Epimedium sagittatum callus. The anti - browning effect is PVP > VC > CA, and AC has no effect on inhibiting the browning of Epimedium sagittatum callus at the concentrations used in the experiment. Specifically, when the concentration of PVP is 0.5 - 2 g·L -1 , it can effectively inhibit the browning of callus, and the browning rate of Epimedium sagittatum - induced callus is 0; among them, when the concentration of PVP is 1.5 g·L -1 , the growth condition of Epimedium sagittatum callus is the best, the growth rate is the fastest, and the browning rate is 0; when the concentration of the anti - browning agent CA reaches 0.5 g·L -1 , the anti - browning effect on Epimedium sagittatum - induced callus is the best, the browning rate reaches 0, and the callus is a milky white mass with almost no growth; when the concentration of the anti - browning agent VC is in the range of 0.01 - 0.05 g·L -1 , the browning rate of Epimedium sagittatum - induced callus is lower than that of the blank control group.

[0065] Comprehensively comparing, under a certain hormone concentration ratio in Epimedium sagittatum callus, adding a certain concentration of the anti - browning agent PVP can effectively inhibit the browning of Epimedium sagittatum callus, and the growth condition of the callus becomes better and the growth rate increases. Therefore, WPM + 2,4 - D 1.5 mg·L -1 + 6 - BA 1.0 mg·L -1 is the optimal medium for the proliferation of Epimedium sagittatum callus, PVP is the best anti - browning agent for Epimedium sagittatum callus, and 1.5 g·L -1 of the anti - browning agent PVP is its optimal application concentration.

[0066] Example 2 Construction and optimization of cell suspension system

[0067] I. Experimental method

[0068] 1. Establish an Epimedium sagittatum cell suspension system

[0069] Using the culture medium (WPM + 2,4-D 1.5 mg·L -1 + 6-BA 1.0 mg·L -1 + PVP 1.5 g·L -1 ) screened in Example 1, after uniformly inoculating the Epimedium sagittatum callus for 14 days, select the vigorously growing Epimedium sagittatum callus and inoculate it onto the liquid culture medium in an Erlenmeyer flask on the ultra-clean workbench for suspension culture (120 rpm) in a dark environment. The nutrient ratio of the culture medium is the same as that of the subcultured callus, which is WPM medium + 6-BA 1.5 mg·L -1 + 2,4-D 2.0 mg·L -1 + sucrose 20 g·L -1 , adjust the pH to 5.8 - 6.2, and do not add agar. After 6 - 7 days in the initial stage of suspension culture on the ultra-clean workbench, use forceps to remove large cell clusters, and retain small cell clusters and single cells with good dispersion. Change the culture medium once every 7 days in the initial stage of culture. After 2 - 3 times of initial culture, filter the larger cell clusters through a 30-mesh sieve. Through 4 - 5 times of subculture, a culture containing only single cells or small cell clusters can be obtained. Mix the cells to obtain a suspension cell cluster. After continuing to culture for 5 - 6 days, inoculate it into the liquid culture medium and culture it on a shaker at 110 rpm (add 2 g of suspended cells to every 50 mL of liquid culture medium). The liquid culture medium is WPM medium + 6-BA 1.5 mg·L -1 + 2,4-D 2.0 mg·L -1 + sucrose 30 g·L -1 , pH 6.0.

[0070] 2. Screening of hormone ratios

[0071] Under the above optimal culture conditions, investigate the addition of 2,4-D (1.0, 1.5, 2.0 mg·L -1 ), 6-BA (0.5, 1.0, 1.5 mg·L -1 ), NAA (0, 0.1, 0.5 mg·L -1 ) for L9(3 3 ) orthogonal design optimization. The specific hormone combination scheme is shown in Table 5. After 8 days of culture, collect the cells, measure the cell growth rate, and determine the optimal hormone ratio.

[0072] Table 5 L9(3 3 ) Orthogonal test factor design

[0073]

[0074] 3. Plotting the growth curve of suspended cells and the change curve of total flavonoid content

[0075] Under the above optimal culture conditions, three bottles were taken out every day for a total of 12 days of culture. The fresh weight of the cells and the total flavonoid content were measured, and the growth curve of the fresh weight of the cells and the change curve of the total flavonoids were plotted.

[0076] 4. Determination Indexes and Methods

[0077] Taking the fresh weight of the suspension-cultured cells as the growth index, three replicates were set up.

[0078] Determination of cell fresh weight: At the end of suspension culture, the culture solution was filtered through a 500-mesh filter screen to collect the cells. The cells were rinsed 2-3 times with distilled water to wash away the surface culture solution, and the excess water was blotted with filter paper. The fresh weight of the cells was obtained by weighing.

[0079] Cell growth rate = (fresh weight of harvested cells - fresh weight of inoculated cells) / fresh weight of inoculated cells × 100%.

[0080] Determination of total flavonoid content: The suspension cells of Epimedium sagittatum were dried by vacuum freeze-drying method.

[0081] Take 0.05 g of the dry sample of the suspension cells of Epimedium sagittatum, accurately add 2 mL of dilute ethanol, weigh, ultrasonically treat (power 400 w, frequency 50 kHz) for 1 h, cool to room temperature, centrifuge at 8000 rpm for 10 min at 4 °C, and take the supernatant as the test solution for use.

[0082] Referring to the content determination item of the Chinese Pharmacopoeia (2020 Edition). Establish a standard curve for the total flavonoids of Epimedium sagittatum: y = 0.0357x + 0.0233 (r 2 = 0.9993) (standard curve range: 0.8 - 103 μg·mL -1 ).

[0083] II. Results

[0084] 1. Effect of Hormone Ratio on the Growth of Suspension Cells

[0085] Through orthogonal experiments, the present invention found that the combined application of multiple hormones significantly improved the growth of the suspension cells of Epimedium sagittatum, as shown in Table 6.

[0086] Table 6 Effect of Hormone Ratio on the Growth of Suspension Cultured Cells of Epimedium sagittatum

[0087]

[0088] Note: The data represent the mean ± standard deviation of three replicates.

[0089] In cell suspension culture, the synergistic effect of cytokinin and auxin is usually required. Different combinations and concentrations of hormones have significant effects on cell growth, proliferation, and differentiation. As shown in Table 6, the combined use of multiple auxins is beneficial to the growth of suspension cells. The influencing factors of exogenous hormone types from major to minor are 2,4-D > NAA > 6-BA, that is, the concentration of 2,4-D has the greatest impact on the growth state of Epimedium sagittatum suspension cells. In the medium for cell suspension proliferation, the hormone composition is 2,4-D 2.0 mg·L -1 + 6-BA 1.5 mg·L -1 + NAA 0.5 mg·L -1 When this is the case, the average fresh weight of harvested cells can reach 9.913 g, and the growth is the best.

[0090] 2. Dynamic growth curve of total flavonoid content in Epimedium sagittatum suspension cells

[0091] During the culture process of Epimedium sagittatum suspension cells, there are obvious differences in the changes of cell fresh weight and total flavonoid content. The growth curve of Epimedium sagittatum suspension cells basically presents an "S" shape. As Figure 1 shown, from 0 to 3 days, the cell growth rate is slow, which is the growth lag phase of cells, and the cells are adapting to the new environment; from 4 to 7 days, the cells grow rapidly, which is the logarithmic growth phase; from 8 to 12 days, the cell growth is gentle, and there is a downward trend starting from 10 days, which is the stationary phase. The change curve of total flavonoid content in Epimedium sagittatum suspension cells is not completely synchronized with the growth curve, showing a trend of first decreasing, then increasing, and then decreasing throughout the culture period. During the growth lag phase, Epimedium sagittatum suspension cells are adapting to the new environment, and the accumulated content of flavonoids gradually decreases; when entering the logarithmic phase, due to cell division and proliferation of Epimedium sagittatum suspension cells, the total flavonoid content drops sharply. As the cells adapt to the environment and proliferate, the total flavonoid accumulation gradually increases, and the total flavonoid content reaches the maximum value on the 8th day; with the increase of the culture days, the total flavonoid content gradually decreases. This may be because in the early stage, the suspension cells adapt to the new environment and some cells die. In the later stage, the cells adapt to the new medium. As the growth and metabolic activities accelerate, the total flavonoids are continuously accumulated and reach the peak on the 8th day, which is 31.49 mg·g -1 At this time, the Epimedium sagittatum suspension cells are seen Figure 2 , with bright cell color and good growth state. Continuing the culture, the color of the culture solution gradually fades and browning occurs. The growth curve of Epimedium sagittatum suspension cells shows that the optimal culture period for Epimedium sagittatum is 8 - 9 days, at which time the cell growth state is the best, and the fresh weight and total flavonoid content are relatively large.

[0092] In summary, under the conditions of an inoculation amount of 2 g, a sucrose concentration of 30 g·L -1 , 110 rpm, and pH = 6.0, the optimal culture conditions for cell suspension proliferation screened are WPM + 2,4-D 2.0 mg·L-1 +6-BA 1.5mg·L -1 +NAA0.5mg·L -1 This result shows that the combined use of multiple auxins is beneficial to the growth of suspension cells, and the suspension cells of Epimedium sagittatum cultured under this condition are bright yellow, highly active, and divide vigorously. According to the optimal culture conditions, the cell growth curve was drawn. The total flavonoid accumulation of Epimedium sagittatum suspension cells reached a peak on the 8th day of inoculation, and the cells were in good growth state at this time. Selecting a suspension cell system with good growth state and high flavonoid content for the next step of induction and regulation is the basis for achieving efficient production of effective ingredients of Epimedium sagittatum.

[0093] Example 3 Effects of different elicitors on the content of metabolites in suspension cells of Epimedium sagittatum

[0094] The secondary metabolites of medicinal plants have important economic value and are the main source components for exerting drug effects. Among them, the type and action concentration of inducers have a great influence on the accumulation of secondary metabolites of suspended cells. The present invention adds three inducers, namely methyl jasmonate (MEJA), salicylic acid (SA) and chitosan (CTS), to explore the influence of these three inducers on the growth of suspended cells and the accumulation of metabolites of Epimedium sagittatum from the perspective of inducer type and action concentration, and measures its main effective components (total flavonol glycosides), and measures the content of secondary metabolites (total flavonoids, total phenols, total alkaloids, and total saponins), and screens out the best inducer type and action concentration, so as to provide theoretical data reference for realizing large-scale production of effective components of Epimedium sagittatum.

[0095] 1. Test methods

[0096] 1. Preparation of inducers

[0097] Methyl jasmonate (MEJA) inducer: Accurately pipette 235.0 μL of MEJA and dilute to 10 mL with 95% ethanol to prepare 100 mmol / L -1 The solution was used as the mother solution. The mother solution was sterilized by filtering through a 0.22 μm microporous filter membrane and set aside. The culture solution concentration was adjusted to 25, 50, 100, and 200 μmol·L -1 , and the control group was taken without MEJA.

[0098] Salicylic acid (SA) inducer: Accurately weigh 138.0 mg SA powder, dissolve in an appropriate amount of 95% ethanol, and dilute to 10 mL with distilled water to prepare 100 mmol / L -1 The solution was used as the mother solution. The mother solution was sterilized by filtering through a 0.22 μm microporous filter membrane and set aside. The culture solution concentration was adjusted to 25, 50, 100, and 150 μmol·L -1 , and the control group was taken without adding SA.

[0099] Chitosan (CTS) elicitor: Accurately weigh 0.2 g of CTS powder and place it in a 100 mL Erlenmeyer flask. Place the Erlenmeyer flask in a 60 °C water bath. Add 0.4 mL of glacial acetic acid dropwise to 0.2 g of chitosan in the Erlenmeyer flask within 15 min, then add distilled water and wait for the chitosan powder to dissolve. Make the volume up to 40 mL, cool it, add NaOH solution to adjust the pH to 5.8, and then sterilize it at 121 °C and 0.1 MPa for 20 min to obtain the elicitor solution, which is reserved for use. When in use, make the concentration of the culture solution 10, 25, 50, 75, 100 mg·L -1 , and use the one without adding CTS as the control group.

[0100] Add the elicitor solution in the middle and late stages of the shaking culture growth of Epimedium sagittatum suspension cells (the 6th - 8th day). After 2 days, collect the suspension cells with a 500 - mesh filter cloth, measure the fresh weight of the cells, store some in a -80 °C refrigerator for standby, and store some in a -20 °C refrigerator for standby.

[0101] 2. Sample treatment

[0102] Sample treatment: After freezing the Epimedium sagittatum suspension cells in a -20 °C refrigerator for 24 h, use the vacuum freeze - drying method to dry the Epimedium sagittatum suspension cells in a vacuum freeze - dryer, grind them, and pass through a No. 3 pharmacopoeia sieve.

[0103] Preparation of aqueous extract: Weigh 0.02 g of the dry sample of Epimedium sagittatum suspension cells, accurately add 2 mL of distilled water, perform ultrasonic treatment (power 400 w, frequency 50 kHz) for 30 min, cool to room temperature, centrifuge at 8000 rpm for 10 min at 4 °C, and take the supernatant as the test solution for use. (The test solution is used for the determination of the content of related primary metabolites, and its concentration is adjusted according to specific indicators, with 3 replicates).

[0104] Preparation of ethanol extract: Take the dry sample of Epimedium sagittatum suspension cells, accurately weigh 0.05 g, accurately add 2 mL of dilute ethanol, weigh it, perform ultrasonic treatment (power 400 w, frequency 50 kHz) for 1 h, cool to room temperature, centrifuge at 8000 rpm for 10 min at 4 °C, and take the supernatant as the test solution for use. (The test solution is used for the determination of the content of related secondary metabolites, and its concentration is adjusted according to specific indicators, with 3 replicates).

[0105] 3. Determination indexes

[0106] Determine the total flavonol glycosides, reducing sugars, soluble proteins, total phenols, total flavonoids, total alkaloids, and total saponins in the Epimedium sagittatum suspension cells.

[0107] II. Results

[0108] 1. Effects of different elicitors on the content of total flavonol glycosides in Epimedium sagittatum suspension cells

[0109] After treatment with different elicitors, the contents of total flavonol glycosides in Epimedium sagittatum suspension cells varied. In the content determination item of Epimedium sagittatum in the Pharmacopoeia, the total flavonol glycosides include epimedin A, epimedin B, epimedin C and icariin. In this invention, epimedin A, epimedin C and icariin were detected, while epimedin B was not detected. The content detection results are shown in Figure 3 .

[0110] It can be seen from Figure 3 that in Epimedium sagittatum suspension cells, the contents of total flavonol glycosides showed different changing trends with the increase of the concentrations of the three elicitors. The most important pharmacodynamic component of Epimedium sagittatum is total flavonol glycosides. The optimal concentrations of MEJA, SA and CTS for inducing total flavonol glycosides in Epimedium sagittatum are 50 μmol·L -1 , 50 μmol·L -1 , 10 mg·L -1 respectively. The inducing effect of 50 μmol·L -1 MEJA is the best.

[0111] 2. Growth and metabolite contents of Epimedium sagittatum suspension cells under different elicitor treatments

[0112] There were significant differences in the contents of various components of Epimedium sagittatum suspension cells under different elicitors. The waterfall chart of the measurement results is as shown in Figure 4 .

[0113] Figure 4 A in Figure 4 is the metabolite content and cell growth in the water extract of Epimedium sagittatum suspension cells treated with different concentrations of MEJA, Figure 4 B in

[0114] 3. Effects of different elicitors on the growth of Epimedium sagittatum suspension cells

[0115] It can be seen from Figure 5Analysis shows that under different elicitor treatment groups, the trend of the fresh weight of Epimedium sagittatum suspension cells changing with the elicitor concentration is not consistent. In the suspension cells of Epimedium sagittatum, the fresh weight of cells in the experimental group with the addition of MEJA elicitor showed a downward trend as its concentration increased, but overall tended to be stable. There was no significant difference in the fresh weight of cells in the elicitor treatment group compared with the control group (P>0.05); within the SA elicitor experimental group, as the added SA concentration increased, the fresh weight of its cells showed an overall downward trend. When the SA concentration was 100 μmol·L -1 and 150 μmol·L -1 the harvested cell fresh weight was significantly lower than that of the control group (P<0.01); within the CTS elicitor experimental group, as the added CTS concentration increased, the fresh weight of its cells showed a trend of first increasing and then decreasing. When the CTS concentration was 50 mg·L -1 and 75 mg·L -1 the harvested cell fresh weight was significantly higher than that of the control group (P<0.001). When the CTS concentration was 100 mg·L -1 the harvested cell fresh weight was significantly higher than that of the control group (P<0.05). The inhibitory effects of MEJA and SA on the growth of Epimedium sagittatum suspension cells may be due to the toxicity of the elicitor to the cells, which can cause apoptosis of some cells. The promoting effect of CTS may be because chitosan provides energy for the cells.

[0116] 4. Effects of different elicitors on the contents of total phenols, total flavonoids, total alkaloids and total saponins in Epimedium sagittatum suspension cells

[0117] Figure 6 shows the effects of elicitors on the total phenol content in Epimedium sagittatum suspension cells, Figure 7 shows the effects of elicitors on the total flavonoid content in Epimedium sagittatum suspension cells, Figure 8 shows the effects of elicitors on the total alkaloid content in Epimedium sagittatum suspension cells. Figure 9 shows the effects of elicitors on the total saponin content in Epimedium sagittatum suspension cells.

[0118] As Figures 6 to 9 can be seen, MEJA can significantly promote the synthesis of total phenols, total flavonoids, total alkaloids and total saponins in Epimedium sagittatum suspension cells, while the effects of SA and CTS on the synthesis of total phenols vary according to the added elicitor concentration, and they show inhibitory effects on the synthesis of total flavonoids. Appropriate concentration of SA promotes the synthesis of total alkaloids. Low concentration of CTS inhibits the synthesis of total alkaloids, and appropriate concentration of CTS significantly promotes the synthesis of total alkaloids and total saponins. Low concentration of SA promotes the synthesis of total saponins, and high concentration of SA inhibits the synthesis of total saponins. The contents of total phenols, total alkaloids and total saponins in Epimedium sagittatum suspension cells treated with 200 μmol·L -1 MEJA reached the maximum values, which were 57.17, 2.58, 19.15 mg·g-1 , which were 2.19, 3.27, and 1.40 times that of the control group. After treatment with 100 μmol·L -1 , the total flavonoid content of Epimedium sagittatum suspension cells treated with MEJA reached the maximum value, which was 36.06 mg·g -1 , which was 1.50 times that of the control group. After treatment with 50 μmolL -1 , the total alkaloid content of Epimedium sagittatum suspension cells treated with SA reached the maximum value, which was 2.80 mg·g -1 , which was 1.28 times that of the control group. After treatment with 75 mgL -1 , the total alkaloid content of Epimedium sagittatum suspension cells treated with CTS reached the maximum value, which was 1.95 mg·g -1 , which was 1.24 times that of the control group. After treatment with 25 μmolL -1 , the total saponin content of Epimedium sagittatum suspension cells treated with SA reached the maximum value, which was 21.89 mg·g -1 , which was 1.11 times that of the control group.

[0119] In summary, the three elicitors in this example had different effects on the growth and metabolism of Epimedium sagittatum suspension cells. MEJA and SA inhibited cell growth, while CTS significantly promoted cell growth. At appropriate concentrations, the three elicitors could significantly promote the synthesis of total flavonol glycosides. When the elicitor concentration was within the appropriate range, it could significantly increase the contents of secondary metabolites such as total flavonoids, total phenols, total alkaloids, and total saponins in Epimedium sagittatum suspension cells.

[0120] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a cell suspension system of Epimedium sagittatum, characterized in that: The following steps are involved: Selecting well-growing Epimedium sagittatum callus, obtaining suspension cell clusters after subculture and suspension culture, inoculating the suspension cell clusters into a liquid culture medium for shaking culture, and obtaining the Epimedium sagittatum cell suspension system; In the subculture, the culture medium is WPM + 2,4-D 1.5 mg·L -1 +6-BA 1.0mg·L -1 +PVP 0.5~2g·L -1 ; In the suspension culture, the culture medium is WPM+6-BA 1.5mg·L -1 +2,4-D 2.0mg·L -1 + sucrose 20g·L -1 ; In the shaking culture, the liquid culture medium is WPM+2,4-D 2.0mg·L -1 +6-BA 1.5mg·L -1 +NAA0.5mg·L -1 + sucrose 30g·L -1 .

2. The method according to claim 1, characterized in that In the subculture, the culture medium is WPM + 2,4-D 1.5 mg·L -1 +6-BA 1.0mg·L -1 +PVP 1.5g·L -1 .

3. The method according to claim 1, characterized in that The inoculation amount of the suspended cell mass in the liquid culture medium is 2 g of the suspended cell mass per 50 mL of the liquid culture medium.

4. The method according to claim 1, characterized in that: The rotation speed of the shaking culture is 110 rpm, and the pH of the liquid culture medium is 6.

0.

5. The method according to claim 1, characterized in that On the 6th to 8th day of the shaking culture, an inducer solution is added to the liquid culture medium, wherein the inducer comprises methyl jasmonate, salicylic acid or chitosan.

6. The method according to claim 5, characterized in that The inducer is chitosan.

7. The Epimedium sagittatum cell suspension system obtained by the method according to any one of claims 1 to 6.

8. Use of the Epimedium sagittatum cell suspension system according to claim 7 in increasing the content of effective ingredients in Epimedium sagittatum.

9. The use according to claim 8, characterized in that: The medicinal ingredients include total flavonol glycosides, total flavonoids, total phenols, total alkaloids and total saponins.

10. The use according to claim 8, characterized in that: The medicinal ingredients are total flavonol glycosides.

Images