Methods to increase flavonoid production in longan embryonic callus using exogenous quercetin or vitexin

Abstract

This invention provides a method for increasing flavonoid production in longan embryogenic callus using exogenous quercetin or vitexin, belonging to the field of plant tissue culture. Key metabolites quercetin and vitexin were screened from extensive targeted metabolomics data of longan embryogenic callus treated with arginine and D-arginine. Quercetin or vitexin was added to the subculture medium of longan embryogenic callus, and the growth and flavonoid content of longan embryogenic callus at different treatment times were measured. The relative flavonoid production rate was calculated, thereby achieving the goal of high flavonoid production. The key metabolites quercetin and vitexin identified in this invention can significantly increase flavonoid production in longan embryogenic callus and have significant application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for increasing the flavonoid yield of longan embryogenic callus using exogenous quercetin and vitexin. Background Technology

[0002] Embryogenic callus is a type of callus with embryogenic characteristics, characterized by its round shape, prominent nuclei, high nucleus-to-cytoplasmic ratio, small vacuoles, vigorous cell division, and abundant nutrients. It is an important material for the industrialized production of plant secondary metabolites. At the end of the 20th century, Lai Zhongxiong et al. (1997) established a subculture system for loose embryogenic callus from longan. Longan is rich in flavonoids, which possess various pharmacological activities such as anticancer, anti-inflammatory, and antioxidant properties, showing broad application prospects in pharmaceutical development and food health products. Previously, we found that spermine treatment significantly promoted the growth and flavonoid accumulation of longan embryogenic callus, while its biosynthesis inhibitor, D-arginine treatment significantly inhibited the growth and flavonoid accumulation of longan embryogenic callus. Therefore, we conducted extensive targeted metabolomics analysis on longan embryogenic callus under different treatments, identifying a total of 946 metabolites. Among them, quercetin and vitexin were among the metabolites with the most significant differences in content, suggesting that quercetin and vitexin (molecular structures as shown in the attached instructions) are likely to be the most abundant metabolites. Figure 1 (As shown) Quercetin may be involved in regulating the growth of embryogenic callus and the accumulation of flavonoids in longan. Quercetin is a widely distributed flavonol compound in the plant kingdom with various biological activities. Modern medicine considers quercetin to be an effective component for relieving cough, asthma, lowering blood pressure, and fighting cancer. Vitexin is also a natural flavonoid compound and is often used to treat cardiovascular diseases. Currently, there are no reports on the correlation between exogenous quercetin and vitexin treatment and the accumulation of functional metabolites in plants.

[0003] Based on existing technology, this invention adds quercetin and vitexin to the culture medium for subculture of longan embryogenic callus, and measures the growth and flavonoid content of longan embryogenic callus at different treatment times to calculate the relative flavonoid production rate, so as to produce more plant flavonoids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing the flavonoid yield of longan embryonic callus by using exogenous quercetin or vitexin.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for increasing flavonoid production in longan embryogenic callus using exogenous quercetin or vitexin. This includes two aspects: screening for exogenous quercetin and vitexin, and the culture cycle of longan embryogenic callus.

[0007] Key metabolites quercetin and vitexin were screened from extensive targeted metabolomics data of longan embryonic callus treated with spermine and D-arginine. Spermine treatment increased the growth and flavonoid content of longan embryonic callus, as evidenced by increased quercetin and vitexin content in the callus. D-arginine treatment inhibited the growth and flavonoid content of longan embryonic callus, as evidenced by decreased quercetin and vitexin content in the callus.

[0008] The method for obtaining longan embryogenic callus was as follows: Longan embryogenic callus was induced from the embryos of young fruits of the 'Honghezi' longan variety 40-50 days after flowering by Researcher Lai Zhongxiong of Fujian Agriculture and Forestry University and has been preserved long-term at the Institute of Horticultural Plant Bioengineering. The embryogenic callus was alternately cultured in MS medium supplemented with 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar, and pH 5.8, and in MS medium supplemented with 1.0 mg / L 2,4-D, 0.5 mg / L kinetin, 5 mg / L AgNO3, 20 g / L sucrose, 7 g / L agar, and pH 5.8. The culture conditions were dark culture at 25℃ for 20 days, which enabled long-term preservation of longan embryogenic callus. Longan embryogenic callus subcultured for 20 days was used for subsequent processing.

[0009] The preparation method of quercetin and vitexin stock solutions is as follows:

[0010] 50 μmol∙mL −1 Preparation of quercetin stock solution: Weigh 0.1511 g of quercetin using an analytical balance, dissolve it in DMSO, and bring the volume up to 10 mL.

[0011] 50 μmol∙mL −1 Preparation of vitexin stock solution: Weigh 0.21621 g of vitexin using an analytical balance, dissolve it in DMSO, and bring the volume to 10 mL.

[0012] The culture medium for subculturing longan embryogenic callus was prepared by autoclaving the subculture medium and then mixing it with quercetin or vitexin stock solution at approximately 50°C to prepare 10 μmol / L quercetin and vitexin subculture mediums. A subculture medium with an equal volume of DMSO was used as a control. The different treatment media were poured into petri dishes and allowed to cool and solidify before use. Longan embryogenic callus subcultured for 20 days was used for treatment. The initial inoculum size was 0.1 g, divided into 4 clusters per petri dish. Dark culture was performed at 25°C for 20, 25, 30, and 35 days.

[0013] The advantages of this invention are:

[0014] (1) It was determined that exogenous quercetin and vitexin have the effect of promoting the production of flavonoids in longan embryonic callus;

[0015] (2) The optimal culture period for promoting the production of flavonoids from longan embryonic callus by exogenous quercetin and vitexin was determined. Attached Figure Description

[0016] Figure 1 These are the molecular structural formulas of quercetin and vitexin. Detailed Implementation

[0017] To make the above-mentioned features and advantages of the present invention more apparent and understandable, specific embodiments are described below in detail. Unless otherwise specified, the methods of the present invention are conventional methods in the art.

[0018] A method for increasing flavonoid production in longan embryonic callus using exogenous quercetin or vitexin includes the following steps:

[0019] (1) Longan embryogenic callus was alternately cultured in MS medium supplemented with 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar and pH 5.8 and MS medium supplemented with 1.0 mg / L 2,4-D, 0.5 mg / L kinetin, 5 mg / L AgNO3, 20 g / L sucrose, 7 g / L agar and pH 5.8. The culture conditions were dark culture at 25℃ and the culture cycle was 20 days, so that the longan embryogenic callus could be continuously subcultured and proliferated.

[0020] (2) Longan embryogenic callus that has been subcultured for 20 days was inoculated into a culture dish containing 20 mL of MS medium supplemented with 10 μmol / L quercetin or vitexin, 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar, and pH 5.8. Each medium was sterilized by autoclaving and used after cooling and solidification. The initial inoculation amount of longan embryogenic callus was 0.1 g, distributed in 4 positions in the culture dish, and cultured in the dark at 25°C for 30 days.

[0021] Example 1

[0022] A method for increasing flavonoid production in longan embryogenic callus using exogenous quercetin and vitexin includes two aspects: treatment with exogenous quercetin and vitexin and the culture cycle of longan embryogenic callus.

[0023] Longan embryogenic callus obtained for the experiment: Young fruits of the 'Hongkerzi' longan variety, 40-50 days after flowering, were collected. After routine surface disinfection, the embryos were aseptically removed and inoculated into MS medium supplemented with 2.0 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, and pH 5.8. The culture was carried out in the dark at 25°C for 40 days to obtain loose, fine-grained embryogenic callus. The longan embryogenic callus was then alternately cultured in MS medium supplemented with 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar, and pH 5.8, and in MS medium supplemented with 1.0 mg / L 2,4-D, 0.5 mg / L kinetin, 5 mg / L AgNO3, 20 g / L sucrose, 7 g / L agar, and pH 5.8. The culture conditions were dark culture at 25°C for 20 days. d, to continuously proliferate the embryogenic callus of longan; the embryogenic callus of longan cultured for 20 days was used for subsequent treatment experiments.

[0024] The preparation method of quercetin and vitexin stock solutions is as follows:

[0025] 50 μmol∙mL −1 Preparation of quercetin stock solution: Weigh 0.1511 g of quercetin using an analytical balance, dissolve it in DMSO, and bring the volume up to 10 mL.

[0026] 50 μmol∙mL −1 Preparation of vitexin stock solution: Weigh 0.21621 g of vitexin using an analytical balance, dissolve it in DMSO, and bring the volume to 10 mL.

[0027] The culture medium for subculturing longan embryogenic callus was prepared by autoclaving the subculture medium and then mixing it with quercetin or vitexin stock solution at approximately 50°C to prepare 10 μmol / L quercetin and vitexin subculture mediums. A subculture medium with an equal volume of DMSO was used as a control. The different treatment media were poured into petri dishes and allowed to cool and solidify before use. Longan embryogenic callus subcultured for 20 days was used for treatment, with an initial inoculum size of 0.1 g per dish, divided into four clusters. The callus was cultured in the dark at 25°C for 20, 25, 30, and 35 days. The growth of longan embryogenic callus in each dish was accurately measured using an analytical balance. Each treatment was inoculated into 20 petri dishes, and the experiment was repeated three times.

[0028] The quercetin subculture medium or vitexin subculture medium is MS medium supplemented with 10 μmol / L quercetin or vitexin, 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar, and pH 5.8.

[0029] 1. The specific procedures for determining flavonoids in longan embryonic callus are as follows:

[0030] 1) Sampling and drying: Samples were taken at 20, 25, 30 and 35 days after the culture of longan embryogenic callus. The growth of each treatment was measured using an analytical balance and then dried in an oven at 50°C to constant weight. The dried samples were then ground in a mortar and passed through a 40-mesh sieve for later use.

[0031] 2) Flavonoid extraction: Weigh 0.0200 g of sample powder using an analytical balance, add 2 mL of flavonoid extraction solution (60% ethanol solution by volume), shake and extract at 60℃ for 2 h, centrifuge (10000 g, 10 minutes), and the supernatant is the flavonoid extraction solution;

[0032] 3) Flavonoid content determination: The flavonoid content of the samples was determined using the "Plant Flavonoid Reagent Kit" from Suzhou Keming Biotechnology Co., Ltd.

[0033] Effect test:

[0034] When the subculture medium for longan embryonic callus was sterilized at high temperature and cooled to about 50°C, quercetin stock solution and vitexin stock solution were added to prepare 10 μmol / L quercetin subculture medium and vitexin subculture medium (200 μL of quercetin stock solution and vitexin stock solution were added per liter of treatment medium, respectively). Subculture medium with an equal volume of DMSO was used as the control (CK).

[0035] 1) Effects of exogenous addition of quercetin and vitexin on the growth of embryogenic callus in longan.

[0036] Table 1 shows the changes in the growth of longan embryogenic callus under different culture media and different culture cycles. The overall trend of longan embryogenic callus growth is an inverted V-shape, with the maximum growth at a culture cycle of 30 days. Exogenous addition of 10 μmol / L quercetin and vitexin both promoted the growth of longan embryogenic callus, with the maximum growth at a culture cycle of 30 days. The growth under quercetin treatment was 36.58% higher than the control (CK) and 96.02% higher than the CK cultured for 20 days. The growth under vitexin treatment was 16.05% higher than the CK and 66.55% higher than the CK cultured for 20 days. This indicates that in this invention, both exogenous quercetin and vitexin can achieve the goal of increasing the growth of longan embryogenic callus, with exogenous quercetin showing a better effect.

[0037] Table 1. Growth amount (g) of longan embryogenic callus under different culture medium treatments.

[0038]

[0039] 2) Effects of exogenous addition of quercetin and vitexin on the accumulation of flavonoids in longan embryonic callus.

[0040] Table 2 shows the changes in flavonoid content in longan embryonic callus under different treatment media and different culture cycles. The trend of flavonoid content in longan embryonic callus also showed an inverted "V" shape, with the highest flavonoid content at a culture cycle of 30 days. Exogenous addition of 10 μmol / L quercetin and vitexin both promoted the flavonoid content in longan embryonic callus, and the flavonoid content was the highest at a culture cycle of 30 days. Among them, the flavonoid content in the quercetin treatment was 20.83% higher than that in the control (CK) and 61.46% higher than that in the CK cultured for 20 days. The flavonoid content in the vitexin treatment was 6.77% higher than that in the CK and 42.67% higher than that in the CK cultured for 20 days. This indicates that in this invention, both exogenous quercetin and vitexin can achieve the purpose of increasing the flavonoid content in longan embryonic callus, with exogenous quercetin showing a better promoting effect.

[0041] Table 2 Flavonoid content (mg·g) of longan embryogenic callus under different culture media −1 )

[0042]

[0043] 3) Since the relative flavonoid production rate of longan embryogenic callus is related to its growth rate, flavonoid content, and culture period, the following formula is used to further calculate the relative flavonoid production rate of longan embryogenic callus:

[0044] Daily yield of flavonoids = (growth rate × flavonoid content) ÷ number of cultivation days;

[0045] Relative production rate of flavonoids = (Daily flavonoid production of the treatment group) ÷ Daily flavonoid production of the control group (CK) over 20 days.

[0046] The effects of exogenous addition of quercetin and vitexin on the relative flavonoid production rate of longan embryogenic callus. Table 3 shows the changes in the relative flavonoid production rate of longan embryogenic callus under different treatment media and different culture cycles. The trend of the relative flavonoid production rate of longan embryogenic callus also showed an inverted "V" shape, with the highest flavonoid content at a culture cycle of 30 days. Exogenous addition of 10 μmol / L quercetin and vitexin both promoted the relative flavonoid production rate of longan embryogenic callus, and the maximum relative flavonoid production rate was observed at a culture cycle of 30 days. Specifically, the relative flavonoid production rate under quercetin treatment was 65.08% higher than the control (CK) and 111.23% higher than the CK cultured for 20 days. The relative flavonoid production rate under vitexin treatment was 23.95% higher than the CK and 58.61% higher than the CK cultured for 20 days. This invention demonstrates that both exogenous quercetin and vitexin can increase the relative production rate of flavonoids in longan embryonic callus tissue, with exogenous quercetin showing a better promoting effect, thus achieving the goal of high-yield plant flavonoids.

[0047] Table 3. Relative flavonoid productivity of longan embryogenic callus under different culture media.

[0048]

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for increasing flavonoid production in longan embryonic callus using exogenous quercetin or vitexin, characterized in that, Includes the following steps: (1) Longan embryogenic callus was alternately cultured in MS medium supplemented with 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar and pH 5.8 and MS medium supplemented with 1.0 mg / L 2,4-D, 0.5 mg / L kinetin, 5 mg / L AgNO3, 20 g / L sucrose, 7 g / L agar and pH 5.

8. The culture conditions were dark culture at 25℃ and the culture period was 20 days, so that the longan embryogenic callus could be continuously subcultured and proliferated. (2) Longan embryogenic callus that has been subcultured for 20 days was inoculated into a culture dish containing 20 mL of MS medium with 10 μmol / L quercetin or vitexin, 1.0 mg / L 2,4-D, 20 g / L sucrose, 7 g / L agar, and pH 5.

8. Each medium was sterilized by autoclaving and used after cooling and solidification. The initial inoculation amount of longan embryogenic callus was 0.1 g, distributed in 4 positions in the culture dish, and cultured in the dark at 25℃ for 30 days.

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