Artemisia argyi extract as well as preparation method and application thereof in promoting growth of crops

Abstract

According to the folium artemisiae argyi extract, the preparation method thereof and the application of the folium artemisiae argyi extract in growth promotion, the folium artemisiae argyi extract required by a plant biostimulant comes from folium artemisiae argyi, the folium artemisiae argyi extract has the advantages of being rich in source, easy to extract and the like, green and sustainable agricultural development is facilitated, and a new scheme is provided for yield increase of crops. According to the extract, leaves and stems of traditional Chinese medicine folium artemisiae argyi serve as raw materials and are subjected to two times of ultrasonic extraction (absolute ethyl alcohol), filtrate is combined, then a crude extract is prepared, a purified component is a 60% ethanol component obtained by separating the crude extract through macroporous resin, the purified component mainly comprises a flavonoid component, and the content of eupatilin is the highest. The plant height, leaf length and fresh weight of rice, pakchoi, cotton and soybean are all improved after the fertilizer is applied according to the application mode mentioned in the invention.

Description

Technical Field

[0001] This invention belongs to the field of plant biostimulant technology, specifically relating to an Artemisia argyi extract, its preparation method, and its application in promoting crop growth. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Plant biostimulants are a class of natural or biologically derived substances that are neither fertilizers nor pesticides. They act on plants or their rhizosphere environment to enhance plant physiological processes, improve stress resistance, nutrient absorption efficiency, and crop quality. Plant biostimulants include humic acids, protein hydrolysates and other nitrogenous compounds, seaweed and plant extracts, chitosan and other biopolymers, inorganic compounds, and beneficial fungi and bacteria. As a green and environmentally friendly agricultural input, plant biostimulants have broad application prospects. They can not only improve crop yield and quality but also reduce the use of chemical pesticides and fertilizers, which is of great significance for environmental protection and sustainable agricultural development.

[0004] Plants in nature produce a large number of active small-molecule organic compounds that are closely related to growth regulation, nutrient absorption, disease resistance, and stress resistance under different growth cycles and environments. These compounds have been extensively extracted as biostimulants. However, the development of existing plant extracts into plant biostimulants generally faces problems such as limited sources, complex extraction processes, high costs, and unclear active substances.

[0005] Ai ( Artemisia argyi Artemisia argyi (L. et Van) is extremely easy to propagate and grows, with a wide natural distribution, found almost throughout China except for extremely arid and cold regions. The leaves, stems, and stalks of Artemisia argyi contain various active substances such as flavonoids, organic acids, amino acids, polysaccharides, and phenylpropanoids, and have the potential to be developed into plant biostimulants.

[0006] Existing research on Artemisia argyi extract reveals that it contains isocyanin and brown cyanidin, and isocyanin has the effects of heat dissipation, analgesia, hemostasis, anticoagulation and treatment of gastric ulcers; some literature discloses that flavonoids extracted from Artemisia argyi have antitumor and antibacterial and anti-inflammatory effects.

[0007] There is a lack of further research on the application of Artemisia argyi extract in other fields. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the purpose of this invention is to provide an Artemisia argyi extract, its preparation method, and its application in promoting growth. This invention provides a novel application of Artemisia argyi extract as a plant biostimulant that promotes crop growth. The technical solution provided by this invention is as follows: As a first aspect of the present invention, a method for preparing Artemisia argyi extract is provided, comprising the following steps: S1, Preparation of Artemisia argyi crude extract (AEE): The stems and leaves of Artemisia argyi were powdered and soaked in anhydrous ethanol for 20-30 hours, followed by ultrasonic extraction. The content of iso-euphorbia flavonoids was higher than 40%, approximately 40%~50%, and the content of cyanidin was higher than 10%, approximately 10%~15%. S2, Preparation of purified Artemisia argyi component (Fr-60%): obtained by separating the crude Artemisia argyi extract (AEE) prepared in step S1 through macroporous adsorption resin.

[0009] In step S1, the wormwood is Qi Ai.

[0010] In step S1, the ultrasonic extraction conditions are: 25-30℃, ultrasonic extraction power of 200W-300W, extraction for 20-40 minutes; preferably, ultrasonic extraction is performed twice.

[0011] In step S2, the resin is eluted sequentially with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol aqueous solutions until it is nearly colorless; the macroporous resin used is D101.

[0012] In step S2, the component separated by 50%-70% ethanol aqueous solution is selected as the refined extract of Artemisia argyi, preferably the component separated by 60% ethanol aqueous solution.

[0013] In step S2, eluents of different concentrations are collected, excess elution solvent is removed by concentration, and the extract is obtained by freeze-drying.

[0014] As a second aspect of the invention, it provides an Artemisia argyi extract prepared by the method for preparing the Artemisia argyi extract, comprising crude Artemisia argyi extract (AEE) and purified Artemisia argyi fraction (Fr-60%).

[0015] The above-mentioned purified component (Fr-60%) contains a variety of flavonoids, among which iso-zellin accounts for more than 62% and brown cyanidin accounts for more than 4%, which are active substances that may play a major role in promoting growth.

[0016] As a third aspect of the invention, it is provided that the Artemisia extract described in the second aspect is used to promote crop growth.

[0017] As a fourth aspect of the present invention, a method for promoting crop growth is provided, wherein the crop is treated by soaking, spraying or root dipping with the Artemisia extract described in the second aspect.

[0018] Furthermore, in the third and fourth aspects, the crops include rice, bok choy, cotton, and soybeans.

[0019] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This application provides methods for preparing crude and purified Artemisia argyi extracts and their effects on plant growth promotion. The obtained extracts can effectively improve the plant height, leaf length, and fresh weight of rice, Chinese cabbage, cotton, and soybeans through seed soaking and spraying, and have broad-spectrum effects. Root dipping treatment can also improve the plant height, leaf length, and fresh weight of Chinese cabbage, cotton, and soybeans. 2. The extract provided by this invention is a plant biostimulant derived from the traditional Chinese medicine Artemisia argyi. It is economical, readily available, green, environmentally friendly, safe, non-toxic, and easy to use, providing a new solution for increasing crop yields. After using the method of this invention, the crop leaves are dark green, the plant is tall, and the leaf length and fresh weight are significantly increased. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 These are the high-performance liquid chromatography (HPLC) results of Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%); where A is the chromatogram of the standard, B is the chromatogram of Artemisia argyi crude extract (AEE), and C is the chromatogram of Artemisia argyi purified extract (Fr-60%).

[0022] Figure 2 This is a schematic diagram showing the growth of rice and pak choy after 30 days of seed soaking treatment with crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%). In the diagram, A is a photo of rice growth, B is the plant height measurement result of rice, C is the leaf length measurement result of rice, and D is the whole plant fresh weight measurement result of rice. E is a photo of pak choy growth, F is the plant height measurement result of pak choy, G is the leaf length measurement result of pak choy, and H is the whole plant fresh weight measurement result of pak choy.

[0023] Figure 3 This is a schematic diagram showing the growth of cotton and soybeans after 30 days of treatment with crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%). In the diagram, A is a photo of soybean growth, B is the measurement result of soybean plant height, C is the measurement result of soybean leaf length, and D is the measurement result of soybean whole plant fresh weight; E is a photo of cotton growth, F is the measurement result of cotton plant height, G is the measurement result of cotton leaf length, and H is the measurement result of cotton whole plant fresh weight.

[0024] Figure 4This is a schematic diagram showing the growth of rice and pak choy 30 days after spraying with crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%). In the diagram, A is a photo of rice growth, B is the plant height measurement result of rice, C is the leaf length measurement result of rice, and D is the whole plant fresh weight measurement result of rice. E is a photo of pak choy growth, F is the plant height measurement result of pak choy, G is the leaf length measurement result of pak choy, and H is the whole plant fresh weight measurement result of pak choy.

[0025] Figure 5 This is a schematic diagram showing the growth of cotton and soybeans 30 days after spraying with crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%). In the diagram, A is a photo of soybean growth, B is the measurement result of soybean plant height, C is the measurement result of soybean leaf length, and D is the measurement result of soybean whole plant fresh weight; E is a photo of cotton growth, F is the measurement result of cotton plant height, G is the measurement result of cotton leaf length, and H is the measurement result of cotton whole plant fresh weight.

[0026] Figure 6 This is a schematic diagram showing the growth of pakchoi and cotton 30 days after root dipping with Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%). In the diagram, A is a growth photo of pakchoi, B is the plant height measurement result of pakchoi, C is the leaf length measurement result of pakchoi, and D is the whole plant fresh weight measurement result of pakchoi; E is a growth photo of cotton, F is the plant height measurement result of cotton, G is the leaf length measurement result of cotton, and H is the whole plant fresh weight measurement result of cotton.

[0027] Figure 7 This is a schematic diagram showing the growth of soybeans after 30 days of root dipping with Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%). In the diagram, A is a photo of soybean growth, B is the measurement result of soybean plant height, C is the measurement result of soybean leaf length, and D is the measurement result of soybean whole plant fresh weight.

[0028] Figure 8 The germination rates of rice, bok choy, cotton, and soybean seeds after soaking in Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%) for 3 days are shown in Figure A, which is a germination photograph, and Figure B is a bar chart. Detailed Implementation

[0029] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] The present invention will be further described below with reference to the embodiments.

[0031] This invention provides an Artemisia argyi extract and uses it as a plant biostimulant.

[0032] In some embodiments of the present invention, a crude Artemisia argyi extract (AEE) is provided, which is an ultrasonic extract of Artemisia argyi root or stem after pulverization, and the extraction solvent is anhydrous ethanol.

[0033] In some embodiments of the present invention, a method for preparing the above-mentioned crude Artemisia argyi extract (AEE) is provided, wherein the stems and leaves of Artemisia argyi are powdered and soaked in anhydrous ethanol for 20-30 h, followed by ultrasonic extraction. The ultrasonic extraction conditions are: extraction power of 200W-300W, temperature of 25-30℃, extraction time of 20-40 min, and extraction times of 2 times.

[0034] In some embodiments of the present invention, the product obtained by this step contains more than 40% isoflavone, approximately 40% to 50%, and more than 10% brown cyanidin, approximately 10% to 15%. In some embodiments, the contents of isoflavone and brown cyanidin are 42% and 12%, respectively.

[0035] In another embodiment of the present invention, a purified Artemisia argyi component (Fr-60%) based on the above-mentioned crude Artemisia argyi extract (AEE) is provided. This component is obtained by separating the above-mentioned crude Artemisia argyi extract (AEE) through a macroporous adsorption resin, and a 50%-70% ethanol aqueous solution component is selected as the refined Artemisia argyi extract, preferably a 60% ethanol aqueous solution separation component.

[0036] Another embodiment of the present invention provides a method for preparing the above-mentioned purified arsenic, comprising the following steps: (1) The above-mentioned crude Artemisia argyi extract (AEE) was packed into a macroporous adsorption resin column and eluted with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol aqueous solutions in sequence until it was nearly colorless; the macroporous resin used was D101.

[0037] (2) Collect the eluents of each concentration separately, concentrate to remove excess eluent solvent, and freeze-dry to obtain the refined extract of Artemisia argyi; preferably, the excess eluent solvent is removed by rotary evaporation under reduced pressure.

[0038] The above-mentioned purified component (Fr-60%) contains a variety of flavonoids, among which isoflavone has the highest content and is the active substance that may play the main role in promoting growth.

[0039] In another embodiment of the present invention, the application of the above-mentioned crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%) in promoting the growth of rice, Chinese cabbage, cotton and soybean is provided.

[0040] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0041] The materials and reagents used in this embodiment of the invention are as follows: 1. The mugwort powder was purchased from Qichun-Huichun Qiai Health Industry Development Co., Ltd. This company's mugwort powder is mainly produced by crushing and sieving the leaves and stems of mugwort.

[0042] 2. Tested seeds: Nipponbare rice ( Oryza sativa L. ssp. japonica. cv. Nipponbare), Chinese cabbage (Yifenghe-Siji Huangyang Chinese cabbage), cotton (Minfeng-Chuangyoumian 168), soybean (Shouhe-Zhonghuang 37).

[0043] 3. Preparation of drug-containing solution: Weigh out a certain amount of dried crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%), dissolve them in ethanol (the amount of ethanol should not exceed 5% of the total volume), add sterile water to prepare a drug-containing solution with a concentration of 1 mg / mL, sonicate at 50℃ for 30 min to aid dissolution, and then add 0.025% Tween 80 (v:v). The control group consisted of no extract, only ethanol and Tween 80.

[0044] Example 1 Preparation of Artemisia argyi extract (AEE): Weigh 375 g of crushed Artemisia argyi stems and leaves, add 1.5 L of anhydrous ethanol and soak for 24 h, then perform ultrasonic extraction. The ultrasonic extraction conditions are: extraction power of 250 W, extraction temperature of 28℃, and extraction time of 0.5 h. Filter and repeat the extraction on the residue once in the same way. Combine the filtrates, filter and concentrate to a thick consistency, and freeze dry to obtain crude Artemisia argyi extract (AEE).

[0045] Example 2 Preparation of purified aril (Fr-60%): Artemisia argyi crude extract (AEE) was mixed with macroporous adsorption resin at a ratio of 1:1.5 and packed into a column at a ratio of 1:10. Separation was performed using D101 type macroporous resin. Elution was carried out sequentially with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol-water solutions. After each gradient elution reached colorless, the next gradient was replaced. The eluted fractions were concentrated by rotary evaporation under reduced pressure at 55°C, and then freeze-dried to obtain the purified and separated Artemisia argyi crude extract (AEE) fraction sample. In this embodiment, the component eluted from the 60% fraction is referred to as the "60% fraction," also known as the "60% fraction distillate," or simply "Fr-60%."

[0046] Example 3 Identification of Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%) To clarify the probiotic basis, high-performance liquid chromatography (HPLC) was used to determine the compounds in crude Artemisia argyi extract (AEE) and purified Artemisia argyi extract (Fr-60%). Chromatographic analysis was performed using an Agilent Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm) at 30 ℃. Gradient elution was performed using a binary elution system of 0.2% formic acid (A) and acetonitrile (B), with an injection volume of 10 μL and a detection wavelength of 330 nm. The specific elution program is shown in Table 1. The compounds were confirmed as iso-euphorbia flavonoids and brown cyanidin by comparison with standards. The content was calculated based on peak area, with the peak areas of brown cyanidin and iso-euphorbia flavonoids divided by the total detected peak area to represent the proportion of each substance in the extract.

[0047] The preparation method of the standard is as follows: Accurately weigh appropriate amounts of each reference standard and prepare a mixed reference solution with methanol containing 0.74 mg / L neochlorogenic acid, 0.13 mg / L chlorogenic acid, 0.34 mg / L cryptochlorogenic acid, 4.02 mg / L caffeic acid, 1.02 mg / L schafod, 1.76 mg / L isochlorogenic acid B, 1.34 mg / L isochlorogenic acid A, 1.28 mg / L isochlorogenic acid C, 0.95 mg / L purpuricin, 0.75 mg / L cyanidin, 0.72 mg / L iso-eupatorin, and 0.56 mg / L vitexin. Analyze the mixed reference solution under chromatographic conditions. The chromatogram is shown below. Figure 1 As shown in Figure A.

[0048] Table 1 Liquid phase elution gradient

[0049] Example 4 Administration of Artemisia argyi crude extract (AEE) and Artemisia argyi purified extract (Fr-60%) Prepare solutions containing 1 mg / mL crude extract of Artemisia argyi (AEE) and purified extract of Artemisia argyi (Fr-60%). First, add a small amount of anhydrous ethanol to dissolve the extract (the amount of ethanol should not exceed 5% of the total volume), and sonicate at 50°C for 30 min to aid dissolution. Then, add sterile water and 0.025% Tween 80 (v:v).

[0050] Application Method 1: Seed soaking: Select plump rice, bok choy, cotton, and soybean seeds, and completely immerse them in the solution containing the medicine. After 4 hours, remove them and plant them in nutrient soil. After 30 days, measure the plant height, leaf length, and fresh weight of the rice, bok choy, cotton, and soybeans respectively.

[0051] Application Method Two: Spraying. Select plump rice, bok choy, cotton, and soybean seeds, keep them moist with gauze to promote germination, and plant them in nutrient soil after germination. One week later, spray the seedlings with a solution containing the pesticide until the leaves are moist. Spray again after a one-week interval, for a total of two sprays. After 30 days, measure the plant height, leaf length, and whole plant fresh weight of rice, bok choy, cotton, and soybeans.

[0052] Application Method 3, Root Dipping: Select plump rice, bok choy, cotton, and soybean seeds, keep them moist with gauze for germination, and plant them in vermiculite after germination. One week later, carefully pull out the seedlings and completely immerse their roots in the medicated solution for 15 minutes before transplanting them into nutrient soil. After 30 days, measure the plant height, leaf length, and whole plant fresh weight of the rice, bok choy, cotton, and soybeans.

[0053] Application Method 4: Select plump rice, bok choy, cotton, and soybean seeds, soak them in the drug-containing solution for 4 hours, then transfer them to petri dishes lined with 20 g of sterilized sand. Add 15 mL of pure water to each petri dish, cover with a layer of gauze to keep moist, and count the germination rate on the 3rd day after treatment.

[0054] like Figure 1 As shown in Figures B and C, liquid chromatography analysis revealed that the crude extract of Artemisia argyi (AEE) contained a small amount of isochlorogenic acid, but was mainly composed of flavonoids. Among these, isochlorophyll and brown cyanidin had the highest peak areas, accounting for 42% and 12% respectively. After purification with macroporous resin, the purified Artemisia argyi extract (Fr-60%) effectively enriched flavonoids, with isochlorophyll content reaching over 62%. Figure 1 It is speculated that the growth-promoting components in Artemisia argyi are mainly flavonoids, such as isozygoflavin. Crude extract of Artemisia argyi (AEE), purified extract of Artemisia argyi (Fr-60%), and seed soaking ( Figure 2 , Figure 3 ) and spraying ( Figure 4 , Figure 5 All treatments can effectively increase plant height, leaf length, and fresh weight in rice, bok choy, cotton, and soybeans, demonstrating broad-spectrum efficacy. Root dipping treatment can also increase plant height, leaf length, and fresh weight in bok choy, cotton, and soybeans, but it is not suitable for rice. Figure 6 , Figure 7Rice plants died after root-dipping transplanting. Among the three treatments—seed soaking, spraying, and root dipping—seed soaking showed the best growth-promoting effect on soybeans and cotton, while spraying was more suitable for rice and bok choy. Soybeans and cotton treated with Artemisia argyi extract (Fr-60%) had plant heights of 56 cm and 24 cm, respectively, and fresh weights of 5.8 g and 9.1 g, respectively. Compared to the control group, the plant height and fresh weight of soybeans treated with Fr-60% were 1.7 times and 2 times higher, respectively, while the plant height and fresh weight of cotton were 1.4 times and 1.7 times higher, respectively. The growth-promoting effect was superior to that of Artemisia argyi crude extract (AEE) and other application methods. The fresh weights of rice and bok choy treated with spraying of Artemisia argyi extract (Fr-60%) were 1.5 g and 4.3 g, respectively, higher than other treatment groups and application methods. Both Artemisia argyi crude extract (AEE) and Artemisia argyi extract (Fr-60%) promoted growth through soaking, spraying, and root dipping, but did not improve seed germination rate. Compared with the control group, the germination rate of pakchoi and soybean increased by 10% and 15% respectively after treatment with crude Artemisia argyi extract (AEE), and the germination rate of pakchoi increased by 10% after treatment with purified Artemisia argyi extract (Fr-60%). The germination rate of the other treatment groups decreased, with the germination rate of cotton decreasing by 5%-25%. Therefore, Artemisia argyi extract is not suitable for improving crop germination rate. Figure 8 The plant biostimulant of this invention is derived from the traditional Chinese medicine Artemisia argyi, which is economical, readily available, green, environmentally friendly, safe, non-toxic, and easy to use, providing a new solution for increasing crop yields. After using the method of this invention, the crop leaves are dark green, the plant is tall, and the leaf length and fresh weight are significantly increased.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an Artemisia argyi extract, characterized in that, Includes the following steps: S1, Preparation of Artemisia argyi crude extract: The stems and leaves of Artemisia argyi were powdered, soaked in anhydrous ethanol, and then extracted by ultrasonication. S2, Preparation of purified Artemisia argyi components: These are obtained by separating the crude Artemisia argyi extract prepared in step S1 using macroporous adsorption resin.

2. The method for preparing Artemisia extract according to claim 1, characterized in that, In step S1, the wormwood is Qi Ai.

3. The method for preparing Artemisia extract according to claim 1, characterized in that, In step S1, the ultrasonic extraction conditions are: 25-30℃, ultrasonic extraction power of 200W-300W, and extraction time of 20-40 minutes.

4. The method for preparing Artemisia extract according to claim 1, characterized in that, In step S2, the resin is eluted sequentially with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol aqueous solutions until it is nearly colorless; the macroporous resin used is D101.

5. The method for preparing Artemisia extract according to claim 1, characterized in that, In step S2, the fraction separated by 50%-70% ethanol aqueous solution is selected as the refined extract of Artemisia argyi.

6. The method for preparing Artemisia extract according to claim 1, characterized in that, In step S2, eluents of different concentrations are collected, excess elution solvent is removed by concentration, and the extract is obtained by freeze-drying. Preferably, the excess elution solvent is removed by rotary evaporation under reduced pressure.

7. The Artemisia argyi extract prepared by the method of any one of claims 1 to 6 comprises crude Artemisia argyi extract and purified Artemisia argyi component; wherein the purified Artemisia argyi component contains more than 60% iso-euphorbia flavonoids.

8. The use of the Artemisia extract according to claim 7 in promoting crop growth.

9. The application according to claim 8, characterized in that, The crops include rice, bok choy, cotton, and soybeans.

10. A method for promoting crop growth, characterized in that, The Artemisia extract described in claim 7 is used to treat crops by soaking seeds, spraying, or dipping roots.

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