Method for increasing flavonoid substances in suaeda salsa root exudates under saline-alkali stress and application

Abstract

The invention relates to the field of plant physiological ecology and phytochemistry research, in particular to a method for increasing flavonoid substances in suaeda salsa root exudates under saline-alkali stress and application. And carrying out stress treatment on the suaeda salsa plant for 6-8 weeks by using a NaCl solution with the concentration of 50-300 mmol / L or an alkali solution with the pH value of 8.0-9.0, and collecting a culture solution to obtain the root exudates rich in target flavonoid substances. According to the method, the relative content of the target flavonoid substance 3, 5-Di-O-methyl-8-prenylafelectin-4beta-ol in the suaeda salsa root exudates can be remarkably increased, and compared with a control group, the relative content of the target flavonoid substance 3, 5-Di-O-methyl-8-prenylafelectin-4beta-ol in the suaeda salsa root exudates can be increased by 44.64% through low-salt (100 mmol / L NaCl) treatment and can be increased by 28.9% through high-alkali (pH 9.0) treatment.

Description

Technical Field

[0001] This invention relates to the fields of plant physiological ecology and phytochemistry research, and more specifically, to a method and application for increasing flavonoids in the root exudates of Suaeda salsa in saline-alkali areas under salt-alkali stress. Background Technology

[0002] Suaeda salsa is a typical salt-tolerant halophyte, widely distributed in coastal and inland saline-alkali lands. Studies have shown that Suaeda salsa not only has important ecological restoration value, but its internal components and root exudates also contain various bioactive substances, such as flavonoids. Flavonoids, due to their antioxidant, anti-inflammatory, and antibacterial properties, have significant applications in the pharmaceutical, cosmetic, and functional food industries.

[0003] Currently, methods to increase the flavonoid content in plants mainly include genetic improvement, exogenous hormone treatment, and abiotic stress induction. Among these, abiotic stress induction has received widespread attention due to its simplicity and low cost. Salt-alkali stress, as a common abiotic stress, can affect the synthesis and accumulation of plant secondary metabolites. However, there are currently no systematic reports on targeted regulation methods for flavonoids in the root exudates of *Suaeda salsa* in saline-alkali soil, especially lacking precise induction techniques targeting specific flavonoid components. Summary of the Invention

[0004] This invention aims to address the aforementioned problems in the prior art by providing a method for regulating the flavonoid content in root exudates of *Suaeda salsa* under salt-alkali stress. This method can significantly increase the content of the target flavonoid—3,5-Di-O-methyl-8-prenylafzelechin-4β-ol—in root exudates, and is simple to operate, low in cost, and suitable for large-scale application.

[0005] Through experimental research, the inventors unexpectedly discovered that applying specific intensities of salt or alkali stress to Suaeda salsa plants can significantly activate their root secondary metabolic pathways, promoting the secretion of specific flavonoids into the rhizosphere. In particular, increasing the NaCl concentration to approximately 100 mmol / L or adjusting the pH to 8.5–9.0 significantly increased the secretion of the target flavonoids.

[0006] In a first aspect, the present invention provides a method for increasing the content of flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress, comprising: Salt-alkali stress was applied to hydroponically grown Suaeda salsa plants, wherein the salt-alkali stress was selected from salt stress, alkali stress, or a combination thereof; and the flavonoids were collected from the root exudates of the plants.

[0007] In a preferred embodiment, the flavonoid is collected from the root exudate of the plant as 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol.

[0008] In a preferred embodiment, the salt stress is achieved by adding NaCl to the nutrient solution to create a salt stress environment with a NaCl concentration of 50–300 mmol / L; more preferably, the NaCl concentration is 100 mmol / L.

[0009] In a preferred embodiment, the alkaline stress is to adjust the pH of the nutrient solution to 8.0–9.0; more preferably, the pH is selected from 8.5 or 9.0.

[0010] In a preferred embodiment, the salt-alkali stress is applied in a gradual manner: for salt stress, the NaCl concentration is gradually increased to the target concentration at a rate of 10 to 100 mmol / L per day; for alkali stress, the pH value is gradually adjusted to the target pH value at a rate of 0.3 to 0.7 per day.

[0011] More preferably, the rate of increase of NaCl is 50 mmol / L per day, and the rate of increase of pH value is 0.5 per day. This is to avoid stress damage caused by direct stress and to facilitate the efficient induction and accumulation of the target flavonoid.

[0012] In a preferred embodiment, the Suaeda salsa plant is a seedling with a height of 2-4 cm.

[0013] In a preferred embodiment, the treatment period for salt and alkali stress is 6 to 8 weeks; more preferably 7 weeks. During this stage, Suaeda salsa is in the late growth stage and about to enter the flowering stage, possessing complete metabolic regulation capabilities.

[0014] In a preferred embodiment, the collection of root exudates includes: washing the roots of the stressed plants 2-3 times with deionized water, transferring them to a container containing the collection solution and culturing them in the dark for 10-14 hours, and collecting the collection solution as a root exudate enrichment solution. More preferably, the root exudate is transferred to a container containing the collection solution and cultured in the dark for 12 hours, and then the collection solution is collected as a root exudate enrichment solution. This avoids saturation degradation due to prolonged culture time, and the dark treatment also effectively controls the influence of circadian rhythms.

[0015] The collected solution is a sterile culture medium with the same salt concentration or pH value as the nutrient solution used in the stress treatment.

[0016] In a second aspect, the invention relates to the application of a NaCl solution containing 50–300 mmol / L or a nutrient solution with a pH of 8.0–9.0 in the preparation of a product that increases the content of 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol in the root exudates of Suaeda salsa.

[0017] The significant advantages of this invention compared to the prior art are as follows: This invention provides a method for regulating the flavonoid content in root exudates of *Suaeda salsa* through salt-alkali stress. The method employs salt-alkali stress treatment, with low-salt (100 mmol / L NaCl) or high-alkali (pH 9.0) treatments showing the best results. Experiments of this invention have shown that moderate salt or alkali stress can effectively activate the secondary metabolic pathways of *Suaeda salsa*, promoting the secretion of the target flavonoid compound 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol from the roots, thereby significantly increasing its relative content in the root exudates.

[0018] Furthermore, this invention employs a gradual stress method in a hydroponic system, allowing plants to gradually adapt to adverse environments and avoiding growth inhibition caused by direct stress, thereby achieving efficient and stable secretion of the target flavonoids. Experimental results show that, compared with the control group, the relative content of the target flavonoids increased to 29.26% under low-salt (100 mmol / L NaCl) treatment, an increase of 44.64%; and to 26.09% under high-alkali treatment, an increase of 28.9%. The method provided by this invention helps to increase the yield of the target flavonoids, and is simple to operate, low in cost, and highly targeted, enabling the green and large-scale production of rare flavonoid compounds. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in multiple embodiments of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0021] Unless otherwise specified, specific techniques or conditions in the embodiments described herein shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all conventional commercially available products.

[0022] This invention provides a method for increasing the content of 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol in root exudates of Suaeda salsa in saline-alkali soil through salt-alkali stress, specifically including the following steps: (1) Seed preparation To disinfect the surface of Suaeda salsa seeds, immerse them in a 70-80% ethanol solution for 20-60 seconds, then in a 1-3% sodium hypochlorite solution for 5-15 minutes. Finally, rinse them 3-5 times with sterile distilled water. After disinfection, soak the seeds in distilled water for 1-4 hours to allow them to fully absorb water. The type, concentration, and treatment time of the disinfectant can be adjusted according to the specific batch of seeds, as long as surface disinfection is achieved.

[0023] (2) Establishment of a hydroponic system for Suaeda salsa in saline-alkali soil For germination, a substrate culture method is used: sterilized quartz sand or other inert substrate is evenly spread at the bottom of the seedling container, an appropriate amount of water is added to moisten the substrate, the seeds are evenly sown, and then the container is kept moist. When the seedlings grow to about 2-4 cm, they are transplanted, and seedlings with uniform growth are selected and transferred to hydroponic containers.

[0024] Hydroponics is used during the seedling stage: Nutrient solutions commonly used in this field can be selected, such as 1 / 2 concentration Hoagland nutrient solution or Japanese horticultural formulations. Cultivation conditions can be controlled as follows: 12–18 hours of light per day, temperature 20–28°C, light intensity 8000–12000 lux, and relative humidity 40–60%. Those skilled in the art can routinely optimize these conditions according to the plant's growth status.

[0025] (3) Gradient salt-alkali stress treatment After the seedlings resume growth, they are subjected to salt-alkali stress treatment. The salt-alkali stress is selected from salt stress, alkali stress, or a combination thereof. Salt stress: Add NaCl to the nutrient solution to make the NaCl concentration in the nutrient solution reach 50-300 mmol / L; Alkali stress: Add Na2CO3 to the nutrient solution and adjust the pH of the nutrient solution to 8.0-9.0.

[0026] The stress treatment was applied gradually: for salt stress, the NaCl concentration was gradually increased to the target concentration at a rate of 10–100 mmol / L per day; for alkali stress, the pH value was gradually adjusted to the target pH value at a rate of 0.3–0.7 per day.

[0027] The culture medium was replenished daily based on the plant's transpiration rate to maintain the predetermined stress level, and the culture medium was changed every 2–4 days. The treatment cycle was 6–8 weeks.

[0028] (4) Collection and treatment of root exudates After the stress treatment, select healthy plants and wash the roots repeatedly with deionized water 2-4 times. Immerse the roots in a container containing the collection solution and culture in the dark for 10-14 hours, then collect the root exudate enrichment solution.

[0029] The features and performance of the present invention will be further described in detail below with reference to some embodiments.

[0030] Examples 1-7: Effects of different salt and alkali stress treatments on the flavonoid content in the root exudates of Suaeda salsa: (1) Seed preparation: The Suaeda salsa seeds used in this embodiment of the invention were collected in November 2024 from Haixing County, Cangzhou City, Hebei Province (38°09'56"N", 117°33'57"E). After being air-dried, they were stored in a refrigerator at 4°C. The plant seeds were disinfected in 75% ethanol solution for 30 s, disinfected with 2% sodium hypochlorite solution for 10 min, and then rinsed 5 times with sterile distilled water. The treated seeds were placed in centrifuge tubes and soaked in distilled water for 2 h to allow the seeds to fully absorb water.

[0031] (2) Establishment of the hydroponic system for Suaeda salsa: During the germination stage, a substrate culture method was adopted. Sterilized quartz sand was evenly spread at the bottom of the seedling box, distilled water was added to moisten the substrate, and the seeds were evenly sown in the seedling box. The opening was sealed with plastic wrap to reduce water evaporation. When the seedlings grew to about 3 cm, they were transplanted. Seedlings with uniform growth were selected and transferred to 250 mL brown plastic bottles, with 3 seedlings per bottle. During the seedling stage, a hydroponic method was adopted, using 1 / 2 concentration Hoagland nutrient solution (Qingdao Haibo HB8870-1) for culture, pH=7.0, light 16 h, dark 8 h, temperature 25℃, light intensity 10000 lux, and relative humidity 45%.

[0032] (3) Gradual salt-alkali stress treatment: Examples 1-7 were set up as control (CK), low salt (SL), medium salt (SM), high salt (SH), low alkali (AL), medium alkali (AM), and high alkali (AH), respectively, for a total of 7 treatment groups. Each group was subjected to gradual stress treatment with different concentrations of NaCl and Na2CO3 solutions. The NaCl treatment solution concentration was increased by 50 mmol / L per day, and the pH of the Na2CO3 treatment solution was increased by 0.5 per day. Each treatment reached the predetermined stress level on the same day (specific parameters are shown in Table 1). The culture medium was replenished daily according to the plant transpiration to maintain the predetermined treatment concentration. The culture medium was changed every 3 days. Each treatment was set up with 5 biological replicates, and the treatment period was 7 weeks.

[0033] Table 1 Salt / alkali stress levels in each treatment group

[0034] (4) Collection and treatment of root exudates: After 7 weeks of stress treatment, healthy plants were selected from each group and their roots were washed three times with deionized water. The roots were then immersed in brown plastic bottles containing 50 mL of culture medium (with the same salt and alkali stress level as the original culture medium) and cultured in the dark for 12 h. The root exudate enrichment was collected, stored in 50 mL cryovials, treated with liquid nitrogen, and stored at -80℃ for subsequent metabolomics analysis.

[0035] (5) Determination of flavonoids: The relative content of 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol in root exudates was determined by liquid chromatography-mass spectrometry (LC-MS). The results of the relative content of target flavonoids under different treatments are shown in Table 2.

[0036] Table 2. Effects of different salt-alkali stress treatments on the relative content of target flavonoids

[0037] (6) Results Analysis 1) Both salt stress and alkali stress can effectively increase the content of the target flavonoid 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol in the root exudates of Suaeda salsa in saline-alkali soil. Salt stress is effective in the range of 100–300 mmol / L, with the best effect at 100 mmol / L, where the relative content of the target flavonoid reached 29.26%, an increase of 44.64% compared with the control group. Alkali stress is significantly effective in the pH range of 8.5–9.0, with the best effect at pH 9.0, where the relative content of the target flavonoid reached 26.09%, an increase of 28.97% compared with the control group.

[0038] 2) The effects of stress were closely related to concentration and pH. Treatment with 200 mmol / L NaCl showed a weak effect, while treatment at pH 8.0 showed no significant effect. This indicates that both salt and alkali stress require specific intensities to effectively activate secondary metabolic pathways, and excessively high or low concentrations / pH values ​​are detrimental to the accumulation of target flavonoids. Treatment with 100 mM NaCl increased the content of target flavonoids by 44.64%, significantly higher than the 28.97% increase under alkali stress. However, alkali stress can still serve as an effective regulatory tool, especially in scenarios where high-salt environments need to be avoided, where it has unique advantages.

[0039] In summary, this invention achieves targeted regulation of the content of a specific rare flavonoid, 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol, in the root exudates of *Suaeda salsa* in saline-alkali soil through a combination of salt stress or alkali stress and a gradual approach. Both salt stress (100–300 mM) and alkali stress (pH 8.5–9.0) can serve as effective induction methods, with 100 mM NaCl treatment showing the best results. The method of this invention is simple to operate, low in cost, and suitable for the green production of rare flavonoids.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for increasing flavonoid content in root exudates of *Suaeda salsa* under salt-alkali stress, characterized in that, After treating Suaeda salsa plants with 50–300 mmol / L NaCl solution or alkaline solution with pH 8.0–9.0 for 6–8 weeks, root exudates rich in the aforementioned flavonoids were obtained.

2. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 1, characterized in that, The flavonoid is 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol.

3. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 2, characterized in that, The *Suaeda salsa* plants were cultivated using hydroponics, with the NaCl concentration in the nutrient solution controlled at 50–300 mmol / L, or the pH value in the nutrient solution controlled at 8.0–9.

0.

4. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, The concentration of NaCl in the nutrient solution is controlled at 100 mmol / L.

5. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, The pH value of the nutrient solution is controlled at 8.5 or 9.

0.

6. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, In the stress treatment, the NaCl concentration is increased by 50 mmol / L per day to the target concentration, or the pH value is increased by 0.5 per day to the target pH value.

7. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, The stress treatment period is 7 weeks.

8. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, The *Suaeda salsa* plants mentioned are seedlings, with a height of 2–4 cm.

9. The method for increasing flavonoids in the root exudates of *Suaeda salsa* under salt-alkali stress according to claim 3, characterized in that, The method for obtaining root exudates includes washing the roots of plants subjected to stress treatment, transferring them to clean water for 12 hours in the dark, and collecting the culture solution.

10. Application of NaCl solution containing 50–300 mmol / L or nutrient solution with pH 8.0–9.0 in the preparation of products that increase the content of 3,5-Di-O-methyl-8-prenylafzelechin-4β-ol in root exudates of Suaeda salsa.

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