Induction culture method for lavender suspension cells having high antioxidant activity

Abstract

An induction medium and induction culture method for lavender suspension cells having high antioxidant activity. Suspension cells having a high ABTS free radical scavenging rate can be cultured by optimizing the formulation of a medium, and the contents of total phenols, total flavonoids and total triterpenoids are relatively high. Additionally, lavender suspension cells induced by using the medium grow rapidly and have a stable system, and extracts thereof can significantly reduce redundant ROS generated by oxidative stress of HaCaT cells, exhibit good biological antioxidant activity, and provide a reference for the development of lavender secondary metabolite resources and the like.

Description

Method for inducing culture of lavender suspension cells with high antioxidation activity TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing culture of lavender suspension cells with high antioxidation activity. BACKGROUND

[0002] Lavender is a plant of the Labiatae family, and is originally from the Mediterranean and Europe. It is one of the commonly used aromatic and medicinal plants, and has volatile oils, flavonoids, phenolic acids, phenylpropanoids and other bioactive substances. In China, it is mainly planted in Yili, Xinjiang. At the present stage, the development of lavender is mainly focused on the extraction of essential oils in calices, and the active ingredients in stems and leaves have not been effectively utilized. In addition, due to the long growth cycle and the susceptibility to the environment, the propagation and research of lavender are limited, and plant tissue culture technology provides a solution to this problem. Compared with adult plants, the secondary metabolites synthesized by cells obtained through tissue culture have diversity and are more conducive to the regulation of the synthesis of secondary metabolites in cells, such as the production of anticancer drug paclitaxel by Taxus cells and the enrichment of ginsenosides by ginseng cells. However, there are few reports on the regulation of the synthesis of secondary metabolites in lavender suspension cells. SUMMARY

[0003] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0004] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0005] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a method for inducing culture of lavender suspension cells with high antioxidation activity.

[0006] To solve the above technical problems, the present application provides the following technical scheme: comprising,

[0007] The lavender seeds are inoculated in a sterile seedling culture medium after pretreatment to obtain explants;

[0008] The explants are inoculated in a high-temperature sterilized callus induction culture medium to obtain callus;

[0009] The callus is screened and placed in a cell suspension culture medium for culture to obtain suspension cells;

[0010] The suspension cells are weighed and subcultured on a subculture medium to realize the proliferation of the suspension cells;

[0011] The suspension cell induction medium is MS medium + 0.5-8 mg / L 6-BA + 0.125-4 mg / L 2,4-D + 0.125-8 mg / L jasmonic acid methyl ester.

[0012] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the suspension cell induction medium is MS medium + 0.5-8 mg / L 6-BA + 0.125-4 mg / L 2,4-D + 1-8 mg / L jasmonic acid methyl ester.

[0013] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the suspension cell induction medium is MS medium + 0.5-8 mg / L 6-BA + 0.125-4 mg / L 2,4-D + 2-4 mg / L jasmonic acid methyl ester.

[0014] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the explant is the leaf after germination of the aseptic seedling.

[0015] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the callus induction medium is MS medium + 1-2 mg / L 6-BA + 0.25-0.5 mg / L 2,4-D.

[0016] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the culture cycle of the subculture is 7-10 days.

[0017] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the ABTS free radical scavenging rate of the suspension cell after proliferation is obviously improved, and the ABTS free radical half-inhibitory concentration is <0.5 mg / mL.

[0018] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the induction culture method can promote the suspension cell to produce secondary metabolites, including total phenols, total flavonoids and total triterpenes.

[0019] As a preferred scheme of the induction culture method of the lavender suspension cell with high antioxidant activity, the lavender suspension cell obtained by the induction culture method has a good repair effect on H2O2 damaged cells.

[0020] Another object of the present application is to overcome the deficiencies in the prior art and provide an application of the induction culture method in the research and development of secondary metabolites of lavender resources.

[0021] The present application has the following advantages:

[0022] (1) The plant tissue culture technology is used to solve the problem that the lavender seeds are not easy to germinate, and to break through the limitation of the natural environment on the growth of lavender, so that the lavender cells can be obtained in a short time.

[0023] (2) By adding 2mg / L methyl jasmonate as an inducer in the culture medium, the lavender suspension cells with high antioxidant activity are cultured, the ABTS free radical scavenging rate is obviously improved, and the ABTS free radical half-inhibition concentration is less than 0.5mg / mL; 2mg / L methyl jasmonate can promote the suspension cells to produce secondary metabolites such as total phenols, total flavonoids and total triterpenes, and the obtained lavender suspension cells have significant difference in metabolic pathways (metabolic signal transduction pathway and ABC transporter pathway) compared with the blank group.

[0024] (3) The lavender suspension cells obtained by the induction culture method can significantly reduce the excess ROS produced by cell oxidative stress, repair the oxidative damage of HaCaT cells, and show good biological antioxidant activity. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0026] Fig. 1 is a schematic diagram of the culture process of lavender suspension cells in Example 1 of the present application.

[0027] Fig. 2 is a growth curve and cell viability change diagram of lavender suspension cells in Example 1 of the present application.

[0028] Fig. 3 is a mass change diagram of lavender suspension cells before and after culture in Example 1 of the present application.

[0029] Fig. 4 is an ABTS free radical scavenging rate diagram of lavender suspension cell extracts in Example 1 of the present application.

[0030] Fig. 5 is a total phenol, total flavonoid and total triterpene content change diagram of lavender suspension cells before and after culture in Example 1 of the present application.

[0031] Fig. 6 is a diagram of repair of oxidative damage cells and ROS content of lavender suspension cell extracts in Example 1 of the present application.

[0032] Figure 7 is a metabolic pathway annotation chart between the suspended cells without adding the inducer (CK) and the cells cultured by 2 mg / L MeJA in Example 1 of the present application.

[0033] Figure 8 is a chart of ABTS free radical scavenging rate of the suspended cells induced by different inducers at the same concentration in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0034] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the description examples.

[0035] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, therefore the present application is not limited to the specific examples disclosed below.

[0036] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is separate or alternative to other embodiments.

[0037] The materials and reagents used in the embodiments of the present application are from the following sources:

[0038] Digital electronic analytical balance, produced by Shanghai Ping Instrument Factory; centrifuge, produced by Germany Eppendorf Company; electric heating constant temperature blast drying oven, produced by Shanghai Jinghong Instrument and Equipment Co., Ltd.; pH meter, produced by Switzerland METTLER TOLEDO Company; multifunctional microwell plate enzyme label instrument, produced by the United States BioTek Company; super-clean workbench, produced by Haier Biomedical Co., Ltd.; high-temperature sterilization pot, produced by Zimeng Instrument Co., Ltd.; freeze dryer, ultraviolet crosslinking instrument, produced by Ningbo Xinzhi Biological Technology Co., Ltd.; oscillation incubator, produced by Shanghai Zhicheng Analytical Instruments Co., Ltd.; high-speed tissue grinder, Beijing Tianguan Biotech Co., Ltd.; flow cytometer, the United States BD Biosciences Company.

[0039] Lavandula angustifolia seeds were purchased from Hillside Horticulture Co., Ltd. and were produced in Xinjiang. Gibberellin was purchased from Maobao Biological Technology Co., Ltd. Sodium hypochlorite, 2,3,5-triphenyltetrazolium chloride (TTC), 30% hydrogen peroxide, anhydrous ethanol, dimethyl sulfoxide (DMSO), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were all purchased from National Pharmaceutical Reagent Co., Ltd. MS and MS (without agar) medium were purchased from HIBIO Biological Technology Co., Ltd. DMEM high-sugar medium was purchased from Thermo Fisher Scientific, USA. Penicillin-streptomycin double antibody was purchased from Invitrogen Gibco, USA. Fetal bovine serum (FBS) was purchased from New Zealand NEWZERUM Co.

[0040] The MS medium formula in the application is as follows:

[0041] The determination method of the cell growth curve in the application is as follows: the weight of the callus is taken as the fresh weight of the cells, and the weight is taken every 2 days until the 14th day. The growth days are taken as the horizontal coordinate, and the fresh weight of the cells is taken as the vertical coordinate to draw the cell growth curve.

[0042] The cell growth activity detection method in the application is as follows: TTC staining method is used, 485nm is detected, and the cell activity is expressed by absorbance. The detection is taken every 2 days until the 14th day. The growth days are taken as the horizontal coordinate, and the absorbance is taken as the vertical coordinate to draw the cell growth activity curve.

[0043] The ABTS free radical scavenging rate determination method in the application is as follows: 2.45mmol / L potassium persulfate solution is used to prepare 7mmol / L ABTS concentrated solution. After 16h of reaction, the ABTS concentrated solution is diluted with water to an absorbance of about 0.7 at 734nm. 50μL of cell extract and 150μL of ABTS diluent are added to a 96-well plate, and the reaction is carried out at room temperature. Each group has 3 parallel samples, which are recorded as A1. The control group uses ethanol instead of the extract, which is recorded as A0. After 15min of reaction, the absorbance at 734nm is detected by an enzyme marker, and the ABTS free radical inhibition rate calculation formula is as follows:

[0044] The ABTS free radical scavenging rate calculation formula in the application is as follows: ABTS free radical inhibition rate % = [(1-A1 / A0)]x100%

[0045] The H2O2 damaged cell survival rate determination method in the application is as follows:

[0046] 2x10 5HaCaT cells were inoculated in 96-well plates at 100 μL per well, and cultured for 24 h until the cells adhered, and then the culture solution was discarded, and 100 μL of the extract was diluted with DMEM to high concentration (250 mg / mL), medium concentration (50 mg / mL), and low concentration (25 mg / mL) of the cell extract. The control group was the culture solution, and the blank group was the culture solution without inoculation of cells. After incubation for 24 h, the supernatant was discarded, 100 μL of 1.1 mmol / L H2O2 solution was added to each well, and after stimulation for 2 h, the culture solution containing H2O2 was discarded, and PBS was used for washing twice, and then 100 μL of MTT solution (0.5 mg / mL) was added. After incubation for 4 h, the supernatant was removed, 100 μL of DMSO was added, and the absorbance was detected at 490 nm after shaking in the enzyme marker. The cell survival rate was calculated according to the following formula:

[0047] In the formula, As is the absorbance of the sample group, Ac is the absorbance of the control group, and Ab is the absorbance of the blank group.

[0048] In the present application, the ROS content determination and analysis method in HaCaT cells is as follows:

[0049] HaCaT cells were inoculated in 6-well plates at 5×105 per well, and cultured for 24 h until the cells adhered, and then the culture solution was discarded, and 100 μL of the extract was diluted with DMEM to high concentration (250 mg / mL), medium concentration (50 mg / mL), and low concentration (25 mg / mL) of the cell extract. The control group and the model group were the culture solution, the model group cells were cells stimulated only by H2O2 without addition of the drug, and the blank group was the culture solution without inoculation of cells. After incubation for 24 h, the supernatant was discarded, 100 μL of 1.1 mmol / L H2O2 solution was added to each well, and after stimulation for 2 h, the culture solution containing H2O2 was discarded, and PBS was used for washing twice, and then 0.5 μmmol / L DCFH-DA (diluted with complete culture medium) was added for incubation at 37 °C for 30 min, and then the DCFH-DA was washed thoroughly with PBS; the cells were digested, collected in an EP tube, vortexed after addition of PBS, centrifuged to remove the supernatant, and repeated for 3-4 times; finally, an appropriate amount of PBS was added, the cells were blown and mixed uniformly, and the ROS was detected by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. DCFH-DA is dichlorodihydrofluorescein diacetate.

[0050] In the present application, the determination method of metabolomics analysis is as follows:

[0051] Chromatographic conditions: ACQUITY UPLC BEH Amide column (1.7 μm x 2.1 mm x 100 mm); column temperature 25℃, flow rate 0.5 mL / min, injection volume 2 μL; mobile phase A: water + 25 mmol / L ammonium acetate + 25 mmol / L ammonia water; B phase: acetonitrile. Gradient elution was used, and the program was as follows: 0-0.5 min, 95% B; 0.5-7 min, 95%-65% B; 7-8 min, 65%-40% B; 8-9 min, 40% B; 9-9.1 min, 40%-95% B; 9.1-12 min, 95% B.

[0052] Mass spectrometry conditions: Orbitrap Exploris TM 480mass spectrometer for mass spectrometry, respectively, using electrospray ionization (ESI) positive and negative ion mode detection. ESI source and mass spectrometry setting parameters are as follows: auxiliary heating gas 1 (Gas1): 50, auxiliary heating gas 2 (Gas2): 2, ion source temperature: 350℃, spray voltage (ISVF) positive mode 3500V, negative mode 2800V; primary mass-to-charge ratio detection range: 70-1200Da, resolution: 60000, scan accumulation time: 100ms, secondary using segmented acquisition method, scan range is 70-1200Da, resolution: 60000, scan accumulation time: 100ms, dynamic exclusion time: 4s.

[0053] The total phenol, total flavonoids, and total triterpenes determination method in the application is as follows:

[0054] Total phenol content: 20 μL of the extract was added to 100 μL of 0.1 mol / L Folin phenol solution, mixed with 80 μL of 7.5% Na2CO3 after 10 min, and the absorbance at 760 nm was measured after 2h. Gallic acid was used as a standard, and the total phenol content was expressed as the number of milligrams of gallic acid equivalent per gram of cell dry weight.

[0055] Total flavonoid content: 60 μL of the sample solution to be tested / standard solution was added with 20 μL of 5% NaNO2 and mixed, reacted for 5 min, then 20 μL of 10% Al(Cl)3 was added and mixed, reacted for 5 min, then 4% NaOH was added, the reaction turned red, and the absorbance was measured at 510 nm. Rutin was used as a standard, and the total flavonoid content was expressed as the number of milligrams of rutin equivalent per gram of cell dry weight.

[0056] Total triterpenoids content: 40 μL of sample solution to be tested / standard solution was added in a 96-well plate, and after the solvent was evaporated at 60°C, 20 μL of 5% vanillin-glacial acetic acid solution and 80 μL of perchloric acid were added, and the mixture was placed in a 60°C oven for reaction for 15 min, and finally, the color reaction was terminated by ice-bath for 5 min, and the absorbance was detected at 546 nm. Ursolic acid was used as a standard, and the total triterpenoids content was expressed as the equivalent milligrams of ursolic acid per gram of dry cell weight.

[0057] The ethanol extraction method of the lavender suspension cells in the present application is as follows: the freeze-dried suspension cells are added with an appropriate amount of anhydrous ethanol, and then ground for 1 min by using a plant cell grinder, and then ultrasonically treated for 30 min at 37°C, to obtain an ethanol extract mother liquor with a concentration of 10 mg / mL, which is stored at -4°C for standby use.

[0058] Example 1

[0059] The present example provides a method for inducing culture of lavender suspension cells with high antioxidant activity:

[0060] The lavender seeds are pretreated, and MS medium is prepared, and the pH is adjusted to 5.85, and then the medium is sealed with a sealing film, and then autoclaved at 121°C for 20 min by using high-pressure steam, and then divided and cooled for standby use; an appropriate amount of the lavender seeds is soaked in 400 mg / L gibberellin for 10 h, and then washed with sterile water for 3 times, and then transferred into a clean bench; the seeds are soaked in 3% NaClO for sterilization for 5 min, and then washed with sterile water for 3-5 times, and then dried by using filter paper for standby use; the seeds are inoculated on the MS medium in the clean bench, and then cultured at 25°C under the condition of 16 h light illumination / 8 h darkness for 30 d, and then the leaves of the sterile seedlings are used as explants.

[0061] In the clean bench, the leaves of the sterile seedlings are cut into small square pieces with a width of 4 mm, and then inoculated in a callus induction medium of MS+2 mg / L 6-BA+0.5 mg / L 2,4-D, and then cultured at 25°C under the condition of 16 h light illumination / 8 h darkness for 30 d, to obtain leaf callus.

[0062] The leaf callus is subcultured every 8 d, and the culture conditions are the same as those in the induction culture, and a liquid medium of MS (without agar)+2 mg / L 6-BA+0.5 mg / L 2,4-D is used as the basic medium for the suspension cells, and 2 mg / L MeJA is added for induction, to obtain the lavender suspension cells.

[0063] The inoculation amount of the lavender suspension cells is 1 g / 30 mL, the culture temperature is 25°C, the rotation speed is 100 rpm, and the culture is carried out in total darkness for 7 d for proliferation.

[0064] The lavender suspension cell culture process diagram of the present example is shown in Figure 1. The lavender suspension cell growth curve and cell viability change of the present example are shown in Figure 2. As shown in Figure 2, the growth of the suspension cells conforms to the Logistic growth model, and in the exponential phase (6-10d), the cell growth is rapid, and the cell mass can increase from 2g to 14g, and the maximum growth amount is up to 16g. The mass change of the lavender suspension cells before and after culture of the present example is shown in Figure 3. As shown in Figure 3, the addition of 2mg / L MeJA in the culture medium does not affect the cell growth amount.

[0065] After the leaf suspension cells are freeze-dried, anhydrous ethanol is added for grinding, ultrasonic treatment is performed at 37°C for 30min, and the supernatant is stored at -4°C for standby. The ABTS free radical scavenging rate of the suspension cell extract is determined, as shown in Figure 4. As shown in Figure 4, compared with the CK group (suspension cells cultured without adding inducer), the ABTS free radical scavenging rate of the MJ group (suspension cells cultured by adding 2mg / L MeJA) is increased by more than 3 times. The ABTS free radical half-inhibitory concentration (IC 50 ) is less than 0.5mg / mL.

[0066] The total phenol, total flavonoid, and total triterpene content changes of the lavender suspension cells before and after culture of the present example are shown in Figure 5. As shown in Figure 5, 2mg / L MeJA can promote the production of three secondary metabolites of lavender suspension cells, and the promotion effect on total flavonoids is more significant.

[0067] The lavender suspension cell extract obtained in the present example is used to repair H2O2 damaged cells, and the specific operation is as follows:

[0068] Extract 25μg / mL (MJ 25), 50μg / mL (MJ 50), and 250μg / mL (MJ 250) of the lavender suspension cell liquid obtained by 2mg / L MeJA culture;

[0069] HaCaT cells were selected as H2O2 damaged cells, and H2O2 damaged cells were obtained by treating with 1.1 mM H2O2. Lavender suspension cell extract was used to pretreat HaCaT cells for 24 h, and then the cells were stimulated with 1.1 mM H2O2. The survival rate of HaCaT cells and the content of intracellular ROS were detected, as shown in Figure 6. As shown in Figure 6(a), compared with the model group (Model), the survival rate of HaCaT cells pretreated with lavender suspension cell extract was significantly improved, and the survival rate also increased with the increase of extract concentration, indicating that lavender suspension cell extract could repair the oxidative damage of HaCaT cells. As shown in Figure 6(b), the greater the fluorescence intensity, the higher the ROS content. Compared with the model group, the ROS content of HaCaT cells pretreated with lavender suspension cell extract was significantly reduced, and the higher the concentration, the less the ROS content, indicating that cells cultured with 2 mg / L MeJA (MJ) could effectively reduce the production of intracellular ROS in H2O2 stimulated cells.

[0070] Figure 7 is a metabolic pathway annotation diagram between the suspension cells without adding inducer (CK) and MJ in the present embodiment. As shown in Figure 7, the significant difference metabolic pathways between CK and MJ are metabolic signal transduction pathway (Metabolic pathways) and ABC transporter pathway (ABC transporters).

[0071] Comparative Example 1

[0072] The difference between the present comparative example and Example 1 is that methyl jasmonate in the cell suspension culture medium is replaced by 24-epibrassinolid (24-eBL), methyl-β-cyclodextrin and β-cyclodextrin, and the remaining steps are the same as those of Example 1, thereby obtaining lavender suspension cells induced by the present comparative example.

[0073] After extracting the suspension cells induced by different inducers, the ABTS free radical scavenging rates of the extracts with the same concentration were compared, and the results are shown in Figure 8. As shown in Figure 8, 24-eBL and β-cyclodextrin have no significant promoting effect on the synthesis of antioxidant active substances in lavender suspension cells, and methyl-β-cyclodextrin can promote the synthesis of antioxidant active substances in cells, but the promoting effect is not as good as that of methyl jasmonate.

[0074] Comparative Example 2

[0075] The difference between the present comparative example and Example 1 is that the methyl jasmonate (2 mg / L) in the cell suspension cell culture medium is adjusted to 0.125 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 4 mg / L, 8 mg / L, and the remaining steps are the same as those of Example 1, to obtain lavender suspension cells induced by the present comparative example. The suspension cells induced by different concentrations of methyl jasmonate are compared, and the results are shown in Table 1.

[0076] Table 1 Induction effect of different concentrations of methyl jasmonate

[0077] As can be seen from Table 1, different concentrations of methyl jasmonate can successfully induce lavender suspension cells with certain antioxidant activity. With the increase of the concentration of methyl jasmonate, the ABTS free radical scavenging rate of the suspension cell extract presents a trend of first increasing and then decreasing, and the total phenol, total flavonoid and total triterpene contents change accordingly. 2 mg / L MeJA can significantly regulate the synthesis of antioxidant active substances in leaf suspension cells, and the ABTS free radical scavenging rate is significantly improved, and has no obvious inhibitory effect on the growth of suspension cells. When the concentration of MeJA is 4 mg / L, although it can significantly promote the production of total phenol and total flavonoid, the growth amount of suspension cells is 13.70 g, which is significantly lower than that of the blank group (16.82 g), and inhibits the growth of lavender suspension cells.

[0078] Comparative Example 3

[0079] Methyl-β-cyclodextrin in Comparative Example 1 can promote the synthesis of antioxidant active substances in cells, and the control group is set in the present comparative example: methyl-β-cyclodextrin is used, and the concentrations are selected as 0.25 mg / L, 0.5 mg / L and 1 mg / L (the significance of other concentrations is significantly lower than that of the blank group) which are better in Figure 8, and the remaining steps are the same as those of Example 1. The total phenol, total flavonoid and total triterpene contents are determined.

[0080] Table 2 Induction effect of different concentrations of methyl-β-cyclodextrin

[0081] As can be seen from Table 2, when methyl-β-cyclodextrin is used for induction culture, the contents of the three secondary metabolites are almost the same as those of the blank group and significantly lower than those when MeJA is used for induction, but MeJA can significantly promote the production of the three secondary metabolites.

[0082] Therefore, methyl jasmonate is significantly better than methyl-β-cyclodextrin in promoting the synthesis of antioxidant active substances in cells and the production of secondary metabolites.

[0083] Comparative Examples 4-12

[0084] Adjust the content of 6-BA, 2,4-D and MeJA in the lavender callus suspension cell induction medium, and the other operations are the same as those in Example 1. The growth of the lavender callus suspension cells under different medium component contents is shown in Table 3.

[0085] Table 3 Comparison of the growth of lavender callus under different suspension cell induction medium component contents

[0086] As shown in Table 3, the lavender callus cells obtained by the induction medium of Example 1 have the fastest growth rate and the highest antioxidant activity.

[0087] In summary, the present application uses plant tissue culture technology to solve the problem of difficulty in germination of lavender seeds, and breaks through the limitation of natural environment on the growth of lavender, so that lavender cells can be obtained in a short time. By adding 2 mg / L methyl jasmonate as an inducer in the culture medium, the lavender suspension cells with high antioxidant activity are cultured, and the ABTS free radical scavenging rate is significantly improved, and the ABTS free radical half-inhibition concentration is <0.5 mg / mL. 2 mg / L methyl jasmonate can promote the production of secondary metabolites such as total phenols, total flavonoids and total triterpenes in the suspension cells, and the obtained lavender suspension cells have significant differences in metabolic pathways (metabolic signal transduction pathway and ABC transporter pathway) compared with the blank group. The induced lavender suspension cells can significantly reduce the excess ROS produced by cell oxidative stress, repair the oxidative damage of HaCaT cells, and exhibit good biological antioxidant activity.

[0088] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for inducing culture of high antioxidant activity of Lavandula angustifolia suspension cells, characterized by: The method comprises the following steps: The lavender seeds are inoculated in a sterile seedling culture medium after pretreatment to obtain explants; The explants are inoculated in a high-temperature sterilized callus induction culture medium to obtain callus; The callus is screened and inoculated in a suspension cell induction culture medium to obtain suspension cells; The suspension cells are weighed and subcultured in a subculture medium to proliferate the suspension cells; The suspension cell induction culture medium is MS culture medium+0.5-8 mg / L 6-BA+0.125-4 mg / L 2,4-D+0.125-8 mg / L jasmonic acid methyl ester.

2. The induction culture method of high antioxidative activity of Lavandula angustifolia suspension cells according to claim 1, characterized by: The suspension cell induction culture medium is MS culture medium+0.5-8 mg / L 6-BA+0.125-4 mg / L 2,4-D+1-8 mg / L jasmonic acid methyl ester.

3. The induction culture method of high antioxidative activity of Lavandula angustifolia suspension cells according to claim 1, characterized by: The suspension cell induction culture medium is MS culture medium+0.5-8 mg / L 6-BA+0.125-4 mg / L 2,4-D+2-4 mg / L jasmonic acid methyl ester.

4. The induction culture method of high antioxidative activity of Lavandula angustifolia suspension cells according to claim 1, characterized by: The explants are the leaves of the germinated sterile seedlings.

5. The induction culture method of high antioxidative activity Lavandula angustifolia suspension cells according to claim 1, characterized by: The callus induction culture medium is MS culture medium+1-2 mg / L 6-BA+0.25-0.5 mg / L 2,4-D.

6. The induction culture method of high antioxidative activity Lavandula angustifolia suspension cells according to claim 1, characterized by: The culture period of the subculture is 7-10 days.

7. The induction culture method of high antioxidative activity Lavandula angustifolia suspension cells according to claim 1, characterized by: The ABTS free radical scavenging rate of the proliferated suspension cells is obviously improved, and the ABTS free radical half-inhibitory concentration is less than 0.5 mg / mL.

8. The induction culture method of high antioxidative activity Lavandula angustifolia suspension cells according to claim 1, characterized by: The induction culture method promotes the generation of secondary metabolites, including total phenols, total flavonoids and total triterpenes, by the lavender suspension cells.

9. The induction culture method of high antioxidative activity Lavandula angustifolia suspension cells according to claim 1, characterized by: The lavender suspension cells obtained by the induction culture method have a good repair effect on H2O2 damaged cells.

10. The induction culture method according to any one of claims 1-9 is applied to the research and development of secondary metabolites of lavender resources.

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