Echium phenol extract, preparation method and application in botanical herbicide

Abstract

The present invention provides a phenolic extract from Echium sibiricum and a preparation method thereof. A novel phenolic extract is disclosed. The present invention also provides the use of the phenolic extract in a botanical herbicide. The phenolic extract's inhibitory effect on other plants can be used to create a highly effective botanical herbicide based on the phenolic extract, maintaining environmental friendliness and safety against non-target organisms. At 0.06 g / ml, the herbicide inhibited weed seedling length by 27.9% to 42.6% and root length by 37.9% to 45.3%, resulting in a 15.5% to 46.9% decrease in chlorophyll content and a 25.5% to 36.2% decrease in photosynthetic rate. Twenty-one days after treatment, the herbicide at 0.06 g / ml reduced the chlorophyll content of the weed seedlings by 39.5% to 67% and the photosynthetic rate by 41.1% to 74.8%.

Description

Technical Field

[0001] The present invention provides an echium phenol extract and a preparation method thereof, and discloses a new echium phenol extract. The present invention also provides application of the echium phenol extract in a botanical herbicide, belonging to the technical field of botanical herbicides. Background Art

[0002] In agricultural production, weeds compete with crops for resources, severely impacting crop growth and yield. Traditional chemical herbicides pose challenges such as environmental pollution, increased weed resistance, and harm to non-target organisms. With increasing awareness of environmental protection and ecological security, the development of green, effective, and safe botanical herbicides has become a key research area in agriculture.

[0003] Previous studies have found that Echium vulgare contains secondary metabolites with allelopathic effects, and its extracts have an impact on the growth of various plants. However, its potential as a botanical herbicide has not been fully explored, and there is a lack of optimization of core technologies. Summary of the Invention

[0004] The invention discloses an echium phenol extract and a preparation method thereof, and obtains the echium phenol extract. The echium phenol extract has an inhibitory effect on other plants, and further provides a high-efficiency botanical herbicide based on the echium phenol extract.

[0005] The phenolic extract of Echium ochraceum described in this invention contains multiple phenolic compounds, such as phenolic acids (benzoic acid, protocatechuic acid, trans-ferulic acid, etc.), flavonoids (naringin, kaempferol, etc.), and flavonols (catechin, epicatechin). This extract synergizes with azadirachtin to inhibit seed germination and seedling growth of common weeds.

[0006] HPLC-MS analysis showed multiple characteristic peaks corresponding to different phenolic compounds, and the types and contents of the phenolic compounds were determined. There were 21 phenolic compounds, including nine phenolic acids, including benzoic acid (Rt = 11.178 ± 0.42 min, m / z =191.00, concentration was 2130 μg / g), protocatechuic acid (Rt = 7.385 min, m / z = 153.00, concentration was 15.88 ± 0.54 μg / g), vanillic acid (Rt = 12.993 min, m / z = 179.00, concentration was 71.01 ± 1.32 μg / g), chlorogenic acid (Rt = 14.960 min, m / z = 515.00, concentration was 8.191 ± 0.36 μg / g), p-coumaric acid (Rt = 17.087 min, m / z = The concentrations of the following compounds were detected: 1) trans-ferulic acid (Rt = 18.744 min, m / z = 193.00, 323.4 ± 1.96 μg / cm³), rosmarinic acid (Rt = 22.209 min, m / z = 359.00, 16.25 ± 0.98 μg / g), p-hydroxybenzoic acid (Rt = 23.754 min, m / z = 717.00, 29.29 ± 0.6 μg / g), and catechol (Rt = 23.754 min, m / z = 515.00, 228.1 ± 1.75 μg / g).There were 10 flavonoids, including naringin (Rt = 21.634 min, m / z = 579.00, concentration was 2.507 ± 0.09 μg / g), kaempferol (quercetin-3-O-galactoside, Rt = 22.910 min, m / z =463.00, concentration was 535 ± 0.71 μg / g), rutin (Rt = 22.888 min, m / z = 609.00, concentration was 4.781 ± 0.12 μg / g), apigenin-7-O-glucoside (Rt = 24.302 min, m / z = 431.00, concentration was 4.02 ± 0.61 μg / g), quercetin (Rt = 25.112 min, m / z = 447.00, with a concentration of 1523 ± 0.77 μg / g), naringenin (Rt = 26.977 min, m / z = 271.00, with a concentration of 1.56 ± 0.12 μg / g), hesperidin (Rt = 29.763 min, m / z = 285.00, with a concentration of 6.302 ± 1.60 μg / g), quercetin (Rt = 23.451 min, m / z = 329.00, with a concentration of 583.5 ± 5.18 μg / g), hesperetin (Rt = 31.852 min, m / z =269.00, with a concentration of 7.427 ± 2.14 μg / g), and acacetin (Rt = 37.061 min, m / cm³ = 283.00, with a concentration of 69.5 ± Two flavonols, (+)-catechin (Rt = 9.189 min, m / z = 289.00, concentration: 377 ± 1.45 μg / g) and epicatechin (Rt = 13.795 min, m / z = 289.00, concentration: 18.56 ± 2.14 μg / g), were also detected.

[0007] These different types of phenolics, each with specific chemical structures and properties, work synergistically in plant allelopathic interactions, conferring the extract's herbicidal activity and distinguishing it from other plant extracts. This extract, in synergistic action with azadirachtin, inhibits seed germination and seedling growth of common weeds.

[0008] Select blue thistle plants in full bloom, wash them, and air-dry them at a temperature of 20-25°C and a relative humidity of 50-60% until the moisture content is 10-15%. Then cut them into uniform pieces less than 2 cm in size.

[0009] Ultrasonic extraction of Echium plants was performed using a water / ethanol composite solvent system: the volume ratio of water to ethanol was 4:1-5:1; the solid-liquid ratio was 1:10-1:12; the ultrasonic power was 300-400W, the temperature was 35-40°C, the extraction time was 40-50 minutes, and the crude extract was filtered.

[0010] The crude extract is subjected to adsorption separation using a macroporous adsorption resin (such as AB-8 type); the crude extract is passed through a chromatography column filled with the macroporous adsorption resin at a flow rate of 1-1.5 BV / h, and is eluted with an ethanol aqueous solution with a volume fraction of 35-45%, and the eluate containing phenolic substances is collected and concentrated under reduced pressure to obtain a refined echium phenolic extract.

[0011] The present invention discloses a high-efficiency botanical herbicide based on echium phenol extract, which is prepared from the following substances in parts by mass:

[0012] 100 parts of echium phenolic extract, 6-8 parts of azadirachtin, 3-4 parts of xanthan gum and sodium carboxymethyl cellulose (1:1 mixture);

[0013] For spray formulations, add sodium dodecylbenzenesulfonate: 1.5 - 2.5 parts;

[0014] For wettable powder formulations, add 35-45 parts of diatomaceous earth.

[0015] Azadirachtin is used as a synergist, exhibiting a synergistic effect with the echium phenolic extract. The addition level of azadirachtin is 6-8% of the extract's mass. Azadirachtin possesses multiple biological activities and can interfere with weed physiological processes. On one hand, azadirachtin can affect weed hormone balance, for example, inhibiting the synthesis or action of growth-promoting hormones such as auxin and gibberellins. Auxin and gibberellins play a key role in plant growth and development, promoting cell elongation and division and regulating plant growth direction and morphological development. When azadirachtin inhibits the action of these hormones, weed growth is hindered. On the other hand, azadirachtin can also disrupt the weed cell membrane structure, interfering with intracellular transport and metabolic processes, leading to cellular dysfunction. The cell membrane is a crucial barrier for the exchange of substances and transmission of information between cells and the external environment. Once the cell membrane structure is disrupted, the flow of substances into and out of the cell is impaired, and normal metabolic activity is disrupted.

[0016] Xanthan gum and sodium carboxymethyl cellulose can form a network structure in the solution, which can prevent the effective ingredients in the extract from precipitating or stratifying. At the same time, during the spraying or watering process, it can also make the herbicide better adhere to the plant surface and improve its effect.

[0017] Echium phenolic extracts inherently have low toxicity to non-target organisms. After optimizing their formulation, their impact on non-target organisms, such as beneficial insects, birds, and soil microorganisms, is further minimized. Phenolic compounds primarily act on weed physiological processes, with minimal impact on the physiological systems of non-target organisms. Azadirachtin has pest repellent and inhibitory effects, minimal harm to beneficial organisms, and does not disrupt the structure and function of soil microbial communities. It is relatively selective for non-target organisms.

[0018] The present invention provides a phenolic extract of echium oleiferum. The phenolic extract's inhibitory effects on other plants can be used to create a highly effective botanical herbicide based on the phenolic extract, maintaining environmental friendliness and safety against non-target organisms. At 0.06 g / ml, the herbicide inhibited weed seedling length by 27.9% to 42.6% and root length by 37.9% to 45.3%, resulting in a 15.5% to 46.9% decrease in chlorophyll content and a 25.5% to 36.2% decrease in photosynthetic rate. Twenty-one days after treatment, the herbicide at 0.06 g / ml reduced the chlorophyll content of the weed seedlings by 39.5% to 67% and the photosynthetic rate by 41.1% to 74.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the plant structure of Echium in full bloom;

[0020] Figure 2 .Flow chart of optimized preparation process of echium extract;

[0021] Figure 3 : A bar graph showing the effect of blue thistle botanical herbicide on the germination rate of different weed seeds, showing the comparison of weed seed germination rate under different concentrations of blue thistle botanical herbicide and the control;

[0022] Figure 4: A bar chart showing the effects of echium botanical herbicides on weed seedling length and root length during the T2 period, showing the inhibitory or promoting effects of different concentrations of echium botanical herbicides on seedling length and root length.

[0023] Figure 5: A graph showing the effects of echium botanical herbicides on chlorophyll content in potted weeds, showing the inhibitory trend of chlorophyll content at different concentrations of echium botanical herbicides at different times;

[0024] Figure 6: A chart showing the effects of blue thistle botanical herbicides on the photosynthetic rate of potted weeds, showing the inhibitory trend of different concentrations of blue thistle botanical herbicides on the photosynthetic rate at different times. DETAILED DESCRIPTION

[0025] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0026] Example 1, preparation of phenolic extracts (specific process as shown in Figure 1 )

[0027] Test results: HPLC-MS analysis showed multiple characteristic peaks corresponding to different phenolic compounds, and the types and contents could be determined. There were 21 phenolic compounds, including nine phenolic acids, including benzoic acid (Rt = 11.178 ± 0.42 min, m / z = 191.00, concentration was 2130 μg / g), protocatechuic acid (Rt = 7.385 min, m / z = 153.00, concentration was 15.88 ± 0.54 μg / g), vanillic acid (Rt = 12.993 min, m / z = 179.00, concentration was 71.01 ± 1.32 μg / g), chlorogenic acid (Rt = 14.960 min, m / z = 515.00, concentration was 8.191 ± 0.36 μg / g), p-coumaric acid (Rt = 17.087 min, m / z = The peak values ​​of the HPLC-MS / MS were 14.877 min, m / z = 144.00, and the concentrations were 23.71 ± 1.77 μg / g, 16.37 ± 1.91 μg / g, 16.70 ± 1.99 μg / g, 24.77 ± 2.81 μg / g, 26.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 29.71 ± 1.71 μg / g, 24.77 ± 2.97 μg / g, 26.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 26.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 29.77 ± 2.97 μg / g, 26.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 29.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 26.77 ± 2.97 μg / g, 28.97 ± 3.81 μg / g, 29.Ten flavonoids were detected, including naringin (Rt = 21.634 min, m / z = 579.00, concentration was 2.507 ± 0.09 μg / g), kaempferol (quercetin-3-O-galactoside, Rt = 22.910 min, m / z = 463.00, concentration was 535 ± 0.71 μg / g), rutin (Rt = 22.888 min, m / z = 609.00, concentration was 4.781 ± 0.12 μg / g), apigenin-7-O-glucoside (Rt = 24.302 min, m / z =431.00, concentration was 4.02 ± 0.61 μg / g), quercetin (Rt = 25.112 min, m / z = 447.00, 1523 ± 0.77 μg / g), naringenin (Rt = 26.977 min, m / z = 271.00, 1.56 ± 0.12 μg / g), hesperidin (Rt = 29.763 min, m / z = 285.00, 6.302 ± 1.60 μg / g), quercetin (Rt = 23.451 min, m / z = 329.00, 583.5 ± 5.18 μg / g), hesperetin (Rt = 31.852 min, m / z = 269.00, 7.427 ± 2.14 μg / g), and acacetin (Rt = 37.061 min, m / cm³ = 283.00, 69.5 ± 1.89 μg / g). Two flavonols, (+)-catechin (Rt = 9.189 min, m / z = 289.00, concentration: 377 ± 1.45 μg / g) and epicatechin (Rt = 13.795 min, m / z = 289.00, concentration: 18.56 ± 2.14 μg / g).

[0028] These different types of phenolic compounds, each with specific chemical structures and properties, work synergistically in plant allelopathic effects, conferring herbicidal activity that distinguishes the extract from other plant extracts. This extract, in synergistic action with azadirachtin, inhibits seed germination and seedling growth of common weeds. Example 2

[0029] By optimizing the extraction method, the extraction rate and purity of phenolic compounds were increased, significantly enhancing the herbicide's effectiveness. For common weeds such as barnyard grass (Echinochloa crusgalli), foxtail grass (Setaria viridis), and purslane (Portulaca oleracea), concentrations of 0.01-0.06g / mL significantly inhibited seed germination and seedling growth. Compared to traditional echium extracts, under the same weed species and treatment time, the herbicide of this invention increased the inhibition of weed germination by 15-25% ( Figure 3 Compared with the blank control, the inhibition degree of seedling growth reached 20~45% at the experimental concentration ( Figure 4 ).

[0030] Azadirachtin and phenolic extracts act synergistically to more effectively disrupt weeds' physiological metabolic processes. This impacts both the light and dark phases of photosynthesis. During the light phase, azadirachtin and phenolic extracts may interfere with chlorophyll's function, reducing its efficiency in absorbing, transferring, and converting light energy. Chlorophyll is a key substance in photosynthesis that absorbs light energy and converts it into chemical energy, powering photosynthesis. When chlorophyll function is disrupted, the absorption, transfer, and conversion of light energy are hampered, reducing the amount of ATP and NADPH produced during photosynthesis. During the dark phase, azadirachtin may inhibit the activity of key enzymes involved in carbon fixation, such as RuBisCO, hindering carbon dioxide fixation and carbohydrate synthesis. Carbon fixation is the core process of the dark phase of photosynthesis. Through the action of RuBisCO, carbon dioxide is fixed and converted into organic matter. When RuBisCO activity is inhibited, carbon dioxide fixation is reduced, which in turn affects plant growth and development.

[0031] Regarding respiration, it interferes with the function of mitochondria within weed cells, inhibiting electron transfer in the respiratory chain and reducing ATP production. This deprives weed cells of energy and impacts their normal physiological activities. Mitochondria are the primary site of cellular respiration, a crucial energy source for cells. When mitochondrial function is disrupted and electron transfer is blocked, ATP production decreases, leading to energy shortages and the inability of cells to carry out normal physiological activities, ultimately hindering weed growth or even death.

[0032] The positive effects of the present invention are shown by the following experiments:

[0033] Seed germination experiment: Echinochloa crus-galli, Setaria viridis, and Purslane were selected as experimental subjects. These weeds are common and representative in agricultural production. First, the seeds were disinfected with a 0.1% potassium permanganate solution for 10-15 minutes. Then, they were rinsed with distilled water to remove any residual potassium permanganate solution on the seed surface, which could affect seed germination.

[0034] Sterilized seeds were placed in Petri dishes lined with two layers of filter paper, with 30 seeds placed in each dish, ensuring an even distribution of seeds. Treatments were set up with different concentrations of the botanical herbicide of this invention (spray formulation) (0.01g / mL, 0.02g / mL, 0.03g / mL, 0.04g / mL, and 0.06g / mL) and a control group consisting of distilled water (0.00g / mL). For the treatment groups, the corresponding concentrations of herbicide solution were added each morning and evening to ensure that the seeds were constantly immersed in the solution. For the control group, an equal amount of distilled water was added.

[0035] During the experiment, the germination of seeds was observed and recorded every day, including the germination time, the number of germinations, etc. Figure 3 The inhibitory effect of the herbicide on seed germination was evaluated by comparing the germination rates of treatment groups at different concentrations with the control group. The results showed that at a concentration of 0.06 g / mL, the herbicide of the present invention had the greatest inhibitory effect on the germination rates of barnyard grass, foxtail grass, and purslane, reducing them by 79%, 83.5%, and 80.5%, respectively, compared to the control (ck). At the same concentration, the inhibitory rates of traditional echium extract were 55.7%, 58.1%, and 50.6%, respectively.

[0036] Pot experiments: The above-mentioned weed seeds were grown to the seedling stage under suitable temperature, humidity, and light conditions. Suitable conditions include a temperature of 20-25°C, a relative humidity of 60-70%, a light intensity of 1000-2000 lux, and a photoperiod of 12-16 hours per day. When the seedlings reached a certain height and showed generally consistent growth, seedlings of similar growth were selected and transplanted into pots with different concentrations. Two seedlings were transplanted into each pot, and the experiment was repeated three times to ensure the accuracy and reliability of the experimental results. For spray-type herbicides, a dosage of 10 mL / pot was used for spray treatment. A control group was treated with an equal volume of distilled water. For a period of time after treatment, the growth of the weed seedlings was regularly observed and recorded, and indicators such as seedling length, root length, chlorophyll content, photosynthetic rate, and stomatal conductance were measured. Measurements could be made at T1 (7th day), T2 (14th day), and T3 (21st day) after treatment.

[0037] The inhibitory effect of blue thistle botanical herbicide on the growth of weed seedlings was evaluated by comparing the changes in various indicators between different treatment groups and the control group.

[0038] Conclusion: Reference Figure 4-6 Fourteen days after treatment, the herbicide of the present invention at a concentration of 0.06 g / ml inhibited weed seedling growth by 27.9% to 42.6% and root length by 37.9% to 45.3%, resulting in a 15.5% to 46.9% decrease in chlorophyll content and a 25.5% to 36.2% decrease in photosynthetic rate. Twenty-one days after treatment, the herbicide of the present invention at a concentration of 0.06 g / ml reduced the chlorophyll content of the weed seedlings by 39.5% to 67% and the photosynthetic rate by 41.1% to 74.8%.

Claims

1. Use of echium phenol extract as an effective active ingredient in the preparation of herbicides, characterized in that The method for extracting the echium phenolic extract comprises the following steps: Select blue thistle plants in full bloom, wash them, and air-dry them at a temperature of 20-25°C and a relative humidity of 50-60% until the moisture content is 10-15%. Then cut them into uniform pieces less than 2 cm in size. Ultrasonic extraction of Echium plants was performed using a water / ethanol composite solvent system: the volume ratio of water to ethanol was 4:1-5:1; the solid-liquid ratio was 1:10-1:12; the ultrasonic power was 300-400W, the temperature was 35-40°C, the extraction time was 40-50 minutes, and the crude extract was filtered. The crude extract is subjected to adsorption separation using a macroporous adsorption resin; the crude extract is passed through a chromatography column packed with the macroporous adsorption resin at a flow rate of 1-1.5 BV / h, and is eluted with an ethanol aqueous solution with a volume fraction of 35-45%, and the eluate containing phenolic substances is collected and concentrated under reduced pressure to obtain a refined echium phenolic extract.

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