Application of choline and activity tracking separation method of choline in xanthium sibiricum
By isolating and identifying choline from Xanthium sibiricum, the problem of unclear influence of invasive plants on soil nitrification was solved, achieving the effect of improving soil nitrification and nitrogen cycling, and providing a novel application of soil stimulants.
Patent Information
- Application Number
- CN202510999430.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-11-07
AI Technical Summary
Existing research has failed to fully understand the impact of invasive alien plants, such as Xanthium sibiricum, on soil nitrification, and the main active substances of their allelopathic effects are unclear, which has affected the in-depth understanding of soil nitrogen transformation mechanisms and the development and utilization of invasive alien plants.
Choline was extracted from Xanthium sibiricum using an active tracking separation method. Choline was isolated and identified through a series of steps, and it was found that it can significantly increase the net nitrification rate of soils in different habitats and promote soil nitrification.
This study deepens our understanding of the mechanisms by which invasive alien plants affect soil nitrogen transformation, provides new ideas for the development and utilization of invasive alien plants, and has application value in the fields of agriculture and ecology. Choline can be used as a novel soil stimulant to promote soil nitrogen cycling, reduce nitrogen fertilizer use, and improve degraded soils.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of agricultural technology, and particularly relates to application of choline and a method for tracking and separating activity of choline in Xanthium strumarium. BACKGROUND
[0002] Alien plant invasion has a significant impact on biodiversity and ecosystem structure and function, and has become a major problem affecting global ecological environment and economy. Nitrogen cycle is the core of the earth's biology and geochemistry, and alien plant invasion can significantly change the nitrogen cycle of the ecosystem. However, we know little about how plant invasion changes this process. Nitrogen cycle mainly includes processes such as nitrogen fixation, nitrification, denitrification, assimilation, dissimilation and ammonification. Among them, nitrification is a series of reactions from NH3 to NH2OH and NO2-oxidation to NO3-, which is the central link of soil nitrogen transformation process. Plants can affect soil nitrification through allelopathy. For example, Brachiaria humidicola inhibits nitrification by secreting a cyclic diterpene substance from the root system to block the reaction process of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). Rice inhibits nitrification by inhibiting the activity of AMO in the ammonia oxidation process through the root exudate 1,9-decadienol, thereby increasing the content of ammonium nitrogen in the rhizosphere soil. On the contrary, some compounds also have the characteristics of promoting soil nitrification. For example, cysteine and methionine can significantly increase the net nitrification rate of alkaline soil, although their effects on soil nitrification vary with different soil types.
[0003] As one of the essential macronutrients, nitrogen plays an irreplaceable role in plant growth and development. Studies have shown that alien plants can accelerate the rate of nitrogen transformation (nitrification) in the invaded area, increasing soil nitrogen availability, which may be an important reason for the successful invasion of alien plants. For example, compared with non-invaded areas, the alien plant Microstegium vimineum can significantly increase the nitrification rate of soil in the invaded area, and promote the invasion through positive feedback with soil nutrient resources. The alien invasive plant Mikania micrantha has a high abundance of nitrogen cycle-related microorganisms (diazotrophs, ammonia-oxidizing and denitrifying bacteria, and ammonia-oxidizing archaea) in the rhizosphere, which enables it to quickly obtain available nitrogen through nitrification, promoting its growth and competitiveness. In addition, some alien invasive plants can also inhibit soil nitrification. Compared with the native plant community in California, the invasive plant Aegilops triuncialis can reduce the abundance of active microorganisms, decrease the availability of soil NO3−, and reduce the nitrification and denitrification potential. Therefore, the invasion of alien plants has a complex impact on soil nitrification, which may vary depending on location, species, and environmental characteristics. It is worth mentioning that different plants (including alien invasive plants) often have different nitrogen preferences. Plants can increase their preferred nitrogen resources (forms) through allelopathy to improve nitrogen use efficiency and promote growth. At present, there are many studies on the effects of alien invasive plants on soil nitrogen transformation, but few studies on how alien invasive plants affect soil nitrogen transformation through allelopathy, and the main active substances of their allelopathy are unclear.
[0004] Xanthium strumarium, also known as Italian cocklebur, is an annual plant belonging to the Asteraceae family. It is native to North America and has rapidly spread in Northeast, North and Northwest China due to its strong adaptability to the environment, causing serious harm to agricultural production and the ecological environment. Xanthium strumarium contains a variety of chemical components, including sesquiterpenes, flavonoids, coumarins, lignans, and thiazines, which have a wide range of pharmacological effects, such as anti-tumor, anti-fungal, anti-viral, anti-inflammatory, and insecticidal effects. Xanthium strumarium also has strong allelopathic effects, with its different tissue (root, stem, leaf, and fruit) leachate and volatile oil having significant inhibitory effects on the seed germination and seedling growth of different crops or weeds. Its water extract can increase the number of soil bacteria and fungi, improve the activity of soil urease and invertase, and increase the content of soil available nitrogen and potassium. Studies have shown that Xanthium strumarium prefers nitrate nitrogen, has stronger nitrate uptake capacity than the native plant Xanthium sibiricum, and is more susceptible to high concentrations of ammonium. However, previous studies on the allelopathic effects of Xanthium strumarium have mainly focused on its effects on the seed germination and seedling growth of native plants, without considering its effects on soil nitrification, and even less on the relationship between its effects on soil nitrification and its nitrogen preference. SUMMARY
[0005] In order to solve the above technical problems, the present application provides a choline application and its active tracking separation method in Xanthium spinosum. The choline is obtained from Xanthium spinosum by the active tracking separation method. It is found for the first time that the choline can significantly improve the net nitrification rate of different habitat soils, thereby promoting the nitrification of the soil. The present application can not only deepen the understanding of the mechanism of the influence of the alien invasive plant on the soil nitrogen transformation, but also provide a new idea for the development and utilization of the alien invasive plant, and reduce the biological safety risk.
[0006] The present application is achieved in this way. The choline application is used to improve the net nitrification rate of different habitat soils, promote the nitrification of the soil, and improve the soil nitrate nitrogen content.
[0007] Preferably, the choline is from Xanthium spinosum.
[0008] An active tracking separation method of choline in Xanthium spinosum is provided, comprising the following steps:
[0009] (1) The whole plant of Xanthium spinosum is taken for ethanol cold immersion extraction. The total extract is obtained after the extraction solution is concentrated. The total extract can significantly improve the net nitrification rate of different habitat soils.
[0010] (2) The total extract obtained in step (1) is suspended with water, and then extracted with petroleum ether and ethyl acetate in sequence. The extraction solutions are concentrated to obtain petroleum ether phase extract, ethyl acetate phase extract and water phase extract, respectively. The water phase extract can significantly improve the net nitrification rate of different habitat soils.
[0011] (3) The water phase extract obtained in step (2) is separated by D101 macroporous adsorption resin, and gradient eluted by ethanol-water system. A total of 100-130 fractions are collected. After TLC and HPLC analysis, they are combined into three parts of Fr. A to Fr. C. The Fr. A part can significantly improve the net nitrification rate of different habitat soils.
[0012] (4) The Fr. A part obtained in step (3) is separated by G-10 dextran gel, and isoelectric eluted by methanol system. A total of 40-60 fractions are collected. After TLC and HPLC analysis, they are combined into seven parts of Fr. A-1 to Fr. A-7. The Fr. A-4 and Fr. A-5 parts can significantly improve the net nitrification rate of different habitat soils.
[0013] (5) Combine Fr. A-4 and Fr. A-5 obtained in step (4) into Fr. AX part, separate by G-10 type dextran gel, and perform isocratic elution in a methanol system, and collect 70-90 fractions, and combine into seven parts of Fr. AX-1 to Fr. AX-7 through TLC and HPLC analysis, wherein the Fr. AX-5 part can significantly improve the net nitrification rate of different habitat soils;
[0014] (6) Separate the Fr. AX-5 part obtained in step (5) by semi-preparative HPLC, and perform isocratic elution in a methanol-water system to obtain three parts of Fr. AX-5-1 to Fr. AX-5-3, wherein the Fr. AX-5-1 part can significantly improve the net nitrification rate of different habitat soils;
[0015] (7) Analyze and identify the Fr. AX-5-1 part obtained in step (6) by UPLC-MS / MS-GNPS and NMR techniques, and determine that the chemical structure is choline.
[0016] Preferably, in step (1), the ethanol cold extraction is performed by 75% ethanol cold extraction for 3 times, and each time is 24 h.
[0017] Preferably, in step (2), petroleum ether and ethyl acetate are sequentially extracted for 5 times.
[0018] Preferably, in step (3), the volume ratio of ethanol to water is 0:100-90:10.
[0019] Preferably, in step (6), the volume ratio of methanol to water is 20:80.
[0020] Compared with the prior art, the present application has important scientific significance and application value, and the beneficial effects mainly embody in the following two aspects:
[0021] I. Scientific significance, the present application firstly extracts and separates choline from Xanthium strumarium by active tracking separation method, which provides a new paradigm for the targeted separation of plant active ingredients. In addition, the present application firstly finds that choline can significantly improve the net nitrification rate of different habitat soils, thereby promoting the nitrification of soil. These deepen our understanding of the mechanism of the influence of alien invasive plants on soil nitrogen transformation, and also provide a new idea for the development and utilization of alien invasive plants.
[0022] II. Application value, the application first discovers that choline can promote the nitrification of soil, so in the field of agriculture, choline can be used as a new type of soil stimulant, reduce the application of nitrogen fertilizer, and is expected to solve the problem of low nitrogen conversion efficiency of continuous cropping barrier soil. In addition, choline can accelerate the reconstruction of nitrogen cycle of degraded soil in the field of ecology, and is expected to have special application value in soil improvement in mining areas and reclamation and other extreme environments. BRIEF DESCRIPTION OF DRAWINGS
[0023] The application will be further described in detail below in combination with the drawings and embodiments:
[0024] Figure 1 is the influence diagram of Xanthium strumarium and Xanthium total extract on soil nitrification. Among them, the white column is Xanthium strumarium, the black column is Xanthium, a) is grassland soil; b) is riverbank soil; c) is broad-leaved forest soil; d) is wasteland soil.
[0025] Figure 2 is the influence diagram of each extract of Xanthium strumarium on soil nitrification. Among them, the white column is water extract, the gray column is petroleum ether extract, and the black column is ethyl acetate extract.
[0026] Figure 3 is the influence diagram of Fr. A to Fr. C three parts of Xanthium strumarium on soil nitrification. Among them, the white column is Fr. A, the gray column is Fr. B, and the black column is Fr. C.
[0027] Figure 4 is the influence diagram of Fr. A-1 to Fr. A-7 seven parts of Xanthium strumarium on soil nitrification. Among them, the white column is 0 g L-1, the horizontal line column is 2 g L-1, the mesh line column is 4 g L-1, and the black column is 8 g L-1.
[0028] Figure 5 is the influence diagram of Fr. AX-1 to Fr. AX-7 seven parts of Xanthium strumarium on soil nitrification.
[0029] Figure 6 is the influence diagram of Fr. AX-5-1 to Fr. AX-5-3 three parts of Xanthium strumarium on soil nitrification. Among them, the white column is 0 g L-1, the horizontal line column is 0.78 g L-1, the mesh line column is 1.56 g L-1, and the black column is 3.12 g L-1.
[0030] Figure 7 is the UPLC-MS / MS-GNPS data analysis diagram of Fr. AX-5-1.
[0031] Figure 8 is the UPLC-MS / MS-GNPS data analysis diagram of choline.
[0032] Figure 9 is the 1H-NMR spectrum of choline.
[0033] Figure 10 is the 13C-NMR spectrum of choline.
[0034] Figure 11 is the effect of choline on soil nitrification. DETAILED DESCRIPTION
[0035] The application will be further described in conjunction with specific examples. These examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples, if not specified, are usually carried out under conventional conditions.
[0036] Example 1: Active tracking separation of choline in Xanthium spinosum
[0037] (1) 100 g of dried whole plant of Xanthium spinosum was subjected to cold extraction with 75% ethanol for 24 h, repeated three times. The total extract 15.8 g was obtained after concentration of the extract. The total extract was diluted with distilled water to obtain solutions with concentrations of 12.0, 8.0, 4.0, 2.0, 1.0, 0.8, 0.4, and 0 g L-1, respectively. The total extract at different concentrations can significantly increase the net nitrification rate of different habitat soils (especially at high concentrations) compared with Xanthium strumarium. Figure 1
[0038] (2) 10 g of the total extract was suspended in 500 mL of water, and then extracted with petroleum ether and ethyl acetate, respectively. The extract was concentrated to obtain petroleum ether phase extract 0.5 g, ethyl acetate phase extract 1.8 g, and water phase extract 7.3 g. Each phase extract was diluted with distilled water to obtain solutions with concentrations of 12.0, 8.0, 4.0, 2.0, 1.0, and 0 g L-1, respectively. The water phase extract can significantly increase the net nitrification rate of different habitat soils. Figure 2
[0039] (3) The water phase extract was separated by D101 macroporous adsorption resin, and gradient elution was performed with distilled water, 30% ethanol water, and 60% ethanol water, respectively. Each 100 mL was collected, and a total of 118 fractions were collected. TLC and HPLC analysis showed that the fractions were combined into three parts, Fr. A to Fr. C. Each part was diluted with distilled water to obtain solutions with concentrations of 16.0, 8.0, 4.0, 2.0, 1.0, and 0 g L-1, respectively. The Fr. A part can significantly increase the net nitrification rate of different habitat soils. Figure 3
[0040] (4) Fr. A part 12.8 g was separated by G-10 type dextran gel, isocratic elution was carried out with methanol system, and every 50 mL was collected, a total of 50 fractions, TLC and HPLC analysis were carried out, and seven parts of Fr. A-1 to Fr. A-7 were combined, and each part was diluted with distilled water to a concentration of 8.0, 4.0, 2.0, 0 g L-1 solution, respectively, wherein Fr. A-4 and Fr. A-5 parts can significantly increase the net nitrification rate of different habitat soils Figure 4 );
[0041] (5) Fr. A-4 and Fr. A-5 were combined as Fr. AX part, and Fr. AX part 3.2 g was separated by G-10 type dextran gel, isocratic elution was carried out with methanol system, and every 10 mL was collected, a total of 85 fractions, TLC and HPLC analysis were carried out, and seven parts of Fr. AX-1 to Fr. AX-7 were combined, and each part was diluted with distilled water to a concentration of 2.0 g L−1 solution, wherein Fr. AX-5 part can significantly increase the net nitrification rate of different habitat soils Figure 5 );
[0042] (6) Fr. AX-5 part was separated by semi-preparative HPLC, isocratic elution was carried out with methanol-water (volume ratio 20:80) system, flow rate 1 mL min-1, 210 nm, to obtain Fr. AX-5-1 to Fr. AX-5-3 three parts, each part was diluted with distilled water to a concentration of 3.12, 1.56, 0.78, 0 g L−1 solution, respectively, wherein Fr. AX-5-1 part can significantly increase the net nitrification rate of different habitat soils Figure 6 ), Fr. AX-5-1 part contains the target compound;
[0043] Example 2: Structure identification of choline in Xanthium strumarium;
[0044] Fr. AX-5-1 part prepared in Example 1 is a white powder, which is tested and analyzed by UPLC-MS / MS-GNPS, and the secondary mass spectrum data is converted into mgf file format, and then uploaded to GNPS database platform (https: / / gnps.ucsd.edu) through FTP client Figure 7 、 Figure 8 ). The results show that the secondary mass spectrum data of Fr. AX-5-1 part is basically consistent with the information of choline in GNPS database. Fr. AX-5-1 part is tested and analyzed by NMR Figure 9 、 Figure 10), 1H-NMR: δ 4. 00 (2H, m, H-1), 3. 51 (2H, m, H-2), 3. 23 (9H, s, 3xN-CH3); 13C-NMR: δ 57. 0 (C-1), 69.0 (C-2), 54.7 (3xN-CH3). The results indicated that the NMR data of Fr. AX-5-1 fraction were basically consistent with the information of choline reported in the literature. In conclusion, the chemical structure of the compound was determined to be choline, and its chemical structure was as follows:
[0045]
[0046] Example 3: Effect of choline in Xanthium strumarium on soil nitrification
[0047] 1. Test materials
[0048] Test sample: choline. Test soil: surface soil (0-10 cm) was collected in Shenyang City (123°33'52"E, 41°48'49"N) in Liaoning Province and brought back to the laboratory for air drying. After passing through a 2 mm sieve, the soil was placed in a polyethylene bag and stored in a 4°C refrigerator for later use.
[0049] 2. Test method
[0050] In a clean beaker (100 mL), 10 g of air-dried soil was added, followed by the addition of 1 mL of choline solution at different concentrations (3.0, 1.5, 1.0, 0.5, 0.1, 0 mg mL-1), respectively. The water content of the soil was adjusted to 60% of the field water capacity, and the beaker was sealed with a hole-preserved film and placed in an artificial climate incubator for 15 days (25°C, 24 h darkness, 80% relative humidity). During this period, the lost water was supplemented every 3 days by weighing to maintain the soil water content at 60%. Each treatment had 3 replicates. After the incubation, 50 mL of 2 mol L-1 KCl solution was added to each beaker, and the mixture was shaken on a constant temperature water bath shaker at 180 r min-1 for 60 min. After standing for 30 min, the filtrate was obtained by filtration, and then the nitrate nitrogen content in the soil was determined using a continuous flow analyzer. The net nitrification rate of the soil (mg N kg-1 d-1) was calculated as follows: [(N1 - N0) × 50 / 1000] / (15 d × 10 / 1000). Wherein, N0 is the initial NO3- concentration in the soil extract, and N1 is the NO3- concentration in the soil extract after 15 days of incubation, both in mg L-1. The effects of different concentrations of choline on the net nitrification rate of the soil were tested by one-way ANOVA. The above analysis was completed in R 4.3.0 and IBM SPSS Statistics 26.0, and the drawing was made using Sigma Plot 10.0.
[0051] 3、Test results
[0052] As can be seen from Figure 11 , the choline involved in the present application can significantly improve the net nitrification rate of the soil in different habitats (grassland, wasteland, riverbank), especially at a high concentration of 3 mg mL-1, and the net nitrification rate increases with the increase of the concentration of choline. The present application first found that choline can promote soil nitrification, so choline can be used as a new type of soil stimulant in the field of agriculture, reducing the application of nitrogen fertilizer, and is expected to solve the problem of low nitrogen conversion efficiency in continuous cropping obstacle soil. In addition, choline can accelerate the reconstruction of nitrogen cycle in degraded soil in the field of ecology, and is expected to have special application value in soil improvement in extreme environments such as mining areas and reclamation.
Claims
1. Use of choline, characterized in that, The application relates to a method for improving the net nitrification rate of different habitat soils, promoting the nitrification of the soils and increasing the soil nitrate nitrogen content.
2. Use of choline according to claim 1, characterized in that, The choline is from Xanthium strumarium.
3. A method for the choline activity trace separation in Xanthium strumarium, characterized by, The method comprises the following steps: (1) taking Xanthium strumarium for ethanol cold extraction, concentrating the extraction liquid to obtain a total extract, and the total extract can significantly improve the net nitrification rate of different habitat soils; (2) suspending the total extract obtained in the step (1) with water, and then extracting with petroleum ether and ethyl acetate in sequence, and then concentrating the extraction liquids to obtain a petroleum ether phase extract, an ethyl acetate phase extract and a water phase extract, wherein the water phase extract can significantly improve the net nitrification rate of different habitat soils; (3) separating the water phase extract obtained in the step (2) by using a D101 macroporous adsorption resin, and gradient eluting with an ethanol-water system, collecting 100-130 fractions, and combining the fractions into three parts of Fr. A to Fr. C through TLC and HPLC analysis, wherein the Fr. A part can significantly improve the net nitrification rate of different habitat soils; (4) separating the Fr. A part obtained in the step (3) by using a G-10 dextran gel, and isocratic eluting with a methanol system, collecting 40-60 fractions, and combining the fractions into seven parts of Fr. A-1 to Fr. A-7 through TLC and HPLC analysis, wherein the Fr. A-4 and Fr. A-5 parts can significantly improve the net nitrification rate of different habitat soils; (5) combining the Fr. A-4 and Fr. A-5 obtained in the step (4) into a Fr. AX part, separating the Fr. AX part by using a G-10 dextran gel, isocratic eluting with a methanol system, collecting 70-90 fractions, and combining the fractions into seven parts of Fr. AX-1 to Fr. AX-7 through TLC and HPLC analysis, wherein the Fr. AX-5 part can significantly improve the net nitrification rate of different habitat soils; (6) separating the Fr. AX-5 part obtained in the step (5) by using a semi-preparative HPLC, isocratic eluting with a methanol-water system, and obtaining three parts of Fr. AX-5-1 to Fr. AX-5-3, wherein the Fr. AX-5-1 part can significantly improve the net nitrification rate of different habitat soils; (7) analyzing and identifying the Fr. AX-5-1 part obtained in the step (6) by using UPLC-MS / MS-GNPS and NMR technologies, and determining that the chemical structure of the Fr. AX-5-1 part is choline.
4. The method of claim 3, wherein the choline is active in the tumorous Xanthium. In the step (1), the ethanol cold extraction is performed for 3 times by using 75% ethanol, and each time is 24 h.
5. The method for tracking and separating choline activity in Xanthium sibiricum according to claim 3, characterized in that, In the step (2), the petroleum ether and the ethyl acetate are extracted for 5 times in sequence.
6. The method of claim 3, wherein the choline is active in the tumor of Xanthium strumarium. In the step (3), the volume ratio of ethanol to water is 0:100-90:
10.
7. The method for tracking and separating choline activity in Xanthium sibiricum according to claim 1, characterized in that, In the step (6), the volume ratio of methanol to water is 20:80.