Cynara scolymus phenol extract, preparation method thereof and application of cynara scolymus phenol extract in botanical herbicide

Abstract

The invention provides a cynara scolymus phenol extract and a preparation method thereof, and discloses a novel cynara scolymus phenol extract. The invention also provides an application of the cynara scolymus phenol extract in a botanical herbicide, the cynara scolymus phenol extract has an inhibition effect on other plants, and the efficient botanical herbicide based on the cynara scolymus phenol extract has the characteristics of being environment-friendly and safe to non-target organisms. Under 0.06 g / ml, the seedling length inhibition rate of the herbicide for weed seedlings is 27.9%-42.6%, and the root length inhibition rate of the herbicide for weed seedlings is 37.9%-45.3%, so that the chlorophyll content is reduced by 15.5%-46.9%, and the photosynthetic rate is reduced by 25.5%-36.2%. After 21 days of treatment, the chlorophyll content of weed seedlings is reduced by 39.5%-67% and the photosynthetic rate is reduced by 41.1%-74.8% under the condition that the concentration of the herbicide is 0.06 g / ml.

Description

Technical Field

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

[0002] In agricultural production, weeds compete with crops for resources, seriously affecting crop growth and yield. Traditional chemical herbicides have problems such as environmental pollution, increased weed resistance, and harm to non-target organisms. As people pay more and more attention to environmental protection and ecological security, the development of green, efficient, and safe botanical herbicides has become an important research direction in the agricultural field.

[0003] Echium vulgare has been found in previous studies to contain secondary metabolites with allelopathic effects, and its extracts have effects on the growth of a variety of 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 a 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 sibiricum described in the present invention contains a variety of phenolic compounds, such as phenolic acids (benzoic acid, protocatechuic acid, trans-ferulic acid, etc.), flavonoids (naringin, kaempferol, etc.) and flavonols (catechin, epicatechin). The extract synergizes with azadirachtin and has an inhibitory effect on the germination of common weed seeds and the growth of seedlings.

[0006] 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 concentrations of 247.7 ± 28.7 μg / g and 265.3 ± 1.6 μg / g were detected by HPLC. The concentrations of 247.7 ± 28.7 μg / g and 265.3 ± 1.6 μg / g were detected by HPLC. The concentrations of 247.7 ± 28.7 μg / g and 265.3 ± 1.6 μg / g were detected by HPLC.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), and 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 = 23.451min, m / z = 329.00, 583.5 ± 5.18 μg / g), hesperidin (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 ± Two flavonols, namely (+)-catechin (Rt = 9.189 min, m / z = 289.00, concentration was 377 ± 1.45 μg / g) and epicatechin (Rt = 13.795 min, m / z = 289.00, concentration was 18.56 ± 2.14 μg / g).

[0007] These different types of phenolic substances each have specific chemical structures and properties, and work synergistically in the allelopathic effects of plants, together giving the extract herbicidal activity, distinguishing it from other plant extracts. The extract synergizes with azadirachtin and has an inhibitory effect on the germination of common weed seeds and the growth of seedlings.

[0008] Select blue thistle plants in full bloom, wash them, dry them in an environment with a temperature of 20-25°C and a relative humidity of 50-60% until the moisture content is 10-15%, and then cut them into uniform pieces less than 2 cm; 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℃, the extraction time was 40-50 minutes, and the crude extract was obtained by filtration; 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 a refined echium phenolic extract is obtained by vacuum concentration.

[0009] The present invention discloses a highly effective botanical herbicide based on echium phenol extract, which is prepared from the following substances in proportion by mass: 100 parts of echium phenolic extract, 6-8 parts of azadirachtin, 3-4 parts of xanthan gum and sodium carboxymethyl cellulose (mixed in a ratio of 1:1); For spray formulations, add sodium dodecylbenzene sulfonate: 1.5 - 2.5 parts; For wettable powder formulations, add 35 - 45 parts of diatomaceous earth.

[0010] Azadirachtin is used as the synergist, which has a synergistic effect with the cyanidin phenol extract. The amount of azadirachtin added is 6-8% of the extract mass. Azadirachtin itself has multiple biological activities, which can interfere with the physiological processes of weeds. On the one hand, azadirachtin can affect the balance of weed hormones, such as inhibiting the synthesis or action of growth-promoting hormones such as auxin and gibberellin in weeds. Auxin and gibberellin play a key role in the growth and development of plants. They can promote cell elongation and division and regulate the growth direction and morphological construction of plants. When azadirachtin inhibits the action of these hormones, the growth of weeds will be hindered. On the other hand, azadirachtin can also destroy the membrane structure of weed cells, interfere with the transport and metabolism of substances in cells, and cause dysfunction of weed cells. The cell membrane is an important barrier for cells to exchange substances and transmit information with the external environment. Once the cell membrane structure is destroyed, the entry and exit of substances in cells will be affected, and the normal metabolic activities of cells will not be able to proceed.

[0011] Xanthan gum and sodium carboxymethyl cellulose can form a network structure in the solution, which can prevent the active 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.

[0012] The phenolic extract of Echium sibiricum itself has low toxicity to non-target organisms. After the optimized formula, the impact on non-target organisms such as beneficial insects, birds and soil microorganisms is further reduced. Phenolic substances mainly act on the physiological processes of weeds and have little effect on the physiological systems of non-target organisms. Azadirachtin has a repellent and inhibitory effect on pests, has minimal harm to beneficial organisms, does not destroy the structure and function of soil microbial communities, and is relatively selective for non-target organisms.

[0013] The positive effects of the present invention are: providing a blue thistle phenol extract; the blue thistle phenol extract has an inhibitory effect on other plants, and can be based on a highly efficient botanical herbicide of blue thistle phenol extract, which has the characteristics of being environmentally friendly and safe for non-target organisms. The herbicide of the present invention has a seedling length inhibition rate of 27.9% to 42.6% and a root length inhibition rate of 37.9% to 45.3% on weed seedlings at 0.06g / ml, resulting in a 15.5% to 46.9% decrease in chlorophyll content and a 25.5% to 36.2% decrease in photosynthetic rate. 21 days after treatment, the herbicide of the present invention at 0.06g / ml caused the chlorophyll content of weed seedlings to decrease by 39.5% to 67%, and the photosynthetic rate to decrease by 41.1% to 74.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .Schematic diagram of the plant structure of Echium in full bloom; Figure 2 .Flow chart of optimized preparation process of Echium extract; Figure 3 :The effect of blue thistle botanical herbicide on the germination rate of different weed seeds is a bar graph showing the comparison between the germination rate of weed seeds under different concentrations of blue thistle botanical herbicide and the control; Figure 4: A bar graph showing the effect of blue thistle botanical herbicide on the seedling length and root length of weed seedlings during the T2 period, showing the changes in the inhibitory or promoting effects of different concentrations of blue thistle botanical herbicide on seedling length and root length; Figure 5: A graph showing the effect of blue thistle botanical herbicide on the chlorophyll content of potted weeds, showing the inhibitory trend of different concentrations of blue thistle botanical herbicide on the chlorophyll content at different times; Figure 6: A chart showing the effect of blue thistle botanical herbicide on the photosynthetic rate of potted weeds, showing the inhibition trend of photosynthetic rate at different concentrations of blue thistle botanical herbicide at different times. DETAILED DESCRIPTION

[0015] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0016] Example 1, preparation of phenolic extract (specific process flow as shown in Figure 1 )

[0017] 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 ± 0.42min, 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 main analytes in the HPLC were 247.47 min, m / z = 163.00, with a concentration of 20.52 ± 7.34 μg / g), trans-ferulic acid (Rt = 18.744 min, m / z = 193.00, with a concentration of 323.4 ± 1.96 μg / cm³), rosmarinic acid (Rt = 22.209min, m / z = 359.00, with a concentration of 16.25 ± 0.98 μg / g), p-hydroxybenzoic acid (Rt = 23.754 min, m / z = 717.00, with a concentration of 29.29 ± 0.6 μg / g), and catechol (Rt = 23.754 min, m / z = 515.00, with a concentration of 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), and 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), hesperidin (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, namely (+)-catechin (Rt = 9.189 min, m / z = 289.00, concentration was 377 ± 1.45 μg / g) and epicatechin (Rt = 13.795 min, m / z = 289.00, concentration was 18.56 ± 2.14 μg / g).

[0018] These different types of phenolic substances of the present invention each have a specific chemical structure and properties, and work synergistically in the allelopathic effect of plants to jointly give the extract herbicidal activity, making it different from other plant extracts. The extract synergizes with azadirachtin and has an inhibitory effect on the germination of common weed seeds and the growth of seedlings. Example 2

[0019] By optimizing the extraction method, the extraction rate and purity of phenolic substances were improved, and the weed control effect was significantly enhanced. For common weeds such as barnyard grass (Echinochloa crusgalli), foxtail grass (Setaria viridis), and purslane (Portulaca oleracea), the seed germination and seedling growth can be significantly inhibited at a concentration of 0.01-0.06g / mL. Compared with traditional blue thistle extracts, under the same weed species and treatment time, the herbicide of the present invention increases the inhibition rate of weed germination rate by 15-25% ( Figure 3 Compared with the blank control, the inhibition of seedling growth at the experimental concentration reached 20~45% ( Figure 4 ).

[0020] Azadirachtin and phenolic extracts work synergistically to interfere with the physiological metabolic process of weeds more effectively. In terms of photosynthesis, it affects the light reaction and dark reaction stages. In the light reaction stage, azadirachtin and phenolic extracts may interfere with the function of chlorophyll and reduce its absorption, transmission and conversion efficiency of light energy. Chlorophyll is a key substance that absorbs light energy in photosynthesis. It can convert light energy into chemical energy to provide power for photosynthesis. When the function of chlorophyll is disturbed, the absorption, transmission and conversion of light energy will be hindered, thereby reducing the amount of ATP and NADPH produced in photosynthesis. In the dark reaction stage, azadirachtin may inhibit the activity of key enzymes involved in carbon fixation (such as RuBisCO), hindering the fixation of carbon dioxide and the synthesis of carbohydrates. Carbon fixation is the core process of the dark reaction in photosynthesis. Through the action of RuBisCO enzyme, carbon dioxide is fixed and converted into organic matter. When the activity of RuBisCO enzyme is inhibited, the fixation of carbon dioxide will be reduced, which will affect the growth and development of plants.

[0021] In terms of respiration, it interferes with the function of mitochondria in weed cells, inhibits the electron transfer process in the respiratory chain, reduces ATP generation, causes weed cells to lack energy supply, and affects their normal physiological activities. Mitochondria are the main place for cells to respire, and respiration is an important way for cells to obtain energy. When the function of mitochondria is disturbed and the electron transfer process is blocked, ATP generation will decrease, cells will lack energy, and will be unable to carry out various physiological activities normally, which will eventually lead to the stunted growth of weeds or even death.

[0022] The positive effects of the present invention are shown by the following experiments:

[0023] 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 0.1% potassium permanganate solution for 10-15 minutes, and then rinsed with distilled water to remove the potassium permanganate solution remaining on the surface of the seeds to avoid affecting seed germination.

[0024] The sterilized seeds were placed in culture dishes covered with two layers of filter paper, with 30 seeds placed in each culture dish to keep the seeds evenly distributed. Different concentrations (0.01g / mL, 0.02g / mL, 0.03g / mL, 0.04g / mL, 0.06g / mL) of the botanical herbicide treatment group of the present invention (spray type) and distilled water (0.00g / mL) control group were set up. For the treatment group, the herbicide solution of corresponding concentration was added in the morning and evening every day to ensure that the seeds were always immersed in the solution; the control group was added with an equal amount of distilled water.

[0025] 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 herbicides on seed germination was evaluated by comparing the germination of different concentration treatment groups and 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 rate of barnyard grass, foxtail grass, and purslane, which were reduced by 79%, 83.5%, and 80.5% respectively compared with the control (ck). The inhibition rates of traditional blue thistle extract at the same concentration were 55.7%, 58.1%, and 50.6%, respectively.

[0026] Pot experiment: The above weed seeds were cultivated to the seedling stage under suitable temperature, humidity and light conditions. Suitable conditions include temperature of 20-25℃, relative humidity of 60-70%, light intensity of 1000-2000 lux, and light time of 12-16 hours / day. When the seedlings grow to a certain height and the growth conditions are basically the same, the seedlings with the same growth are selected and transplanted into pots with different concentrations. Two plants are transplanted into each pot, and repeated three times to ensure the accuracy and reliability of the experimental results. For spray-type herbicides, spray treatment is carried out at a dosage of 10mL / pot. The control group is treated with an equal amount of distilled water. Within a period of time after treatment, the growth of weed seedlings is regularly observed and recorded, and indicators such as seedling length, root length, chlorophyll content, photosynthetic rate and stomatal conductance are measured. The measurement time points can be selected at T1 (7th day), T2 (14th day) and T3 (21st day) after treatment.

[0027] By comparing the changes in various indicators between different treatment groups and the control group, the inhibitory effect of blue thistle plant-based herbicide on the growth of weed seedlings was evaluated.

[0028] Conclusion: Reference Figure 4-6 , 14 days after treatment, the herbicide of the present invention at 0.06g / ml had a seedling length inhibition rate of 27.9%-42.6% and a root length inhibition rate of 37.9%-45.3% on weed seedlings, resulting in a 15.5%-46.9% decrease in chlorophyll content and a 25.5%-36.2% decrease in photosynthesis rate. 21 days after treatment, the herbicide of the present invention at 0.06g / ml resulted in a 39.5%-67% decrease in chlorophyll content and a 41.1%-74.8% decrease in photosynthesis rate of weed seedlings.

Claims

1. A echium phenol extract, characterized in that Contains 21 phenolic compounds: Among them, there were nine phenolic acids: including benzoic acid with a concentration of 2130 μg / g, protocatechuic acid with a concentration of 15.88 ± 0.54 μg / g, vanillic acid with a concentration of 71.01 ± 1.32 μg / g, chlorogenic acid with a concentration of 8.191 ± 0.36 μg / g, p-coumaric acid with a concentration of 20.52 ± 7.34 μg / g, trans-ferulic acid with a concentration of 323.4 ± 1.96 μg / cm³, rosmarinic acid with a concentration of 16.25 ± 0.98 μg / g, p-hydroxybenzoic acid with a concentration of 29.29 ± 0.6 μg / g, and catechol with a concentration of 228.1 ±1.75 μg / g; There were 10 kinds of flavonoids: naringin with a concentration of 2.507 ± 0.09 μg / g, kaempferol with a concentration of 535 ± 0.71 μg / g, rutin with a concentration of 4.781 ± 0.12 μg / g, apigenin-7-O-glucoside with a concentration of 4.02 ± 0.61 μg / g, quercetin with a concentration of 1523 ± 0.77 μg / g, naringenin with a concentration of 1.56 ± 0.12 μg / g, hesperidin with a concentration of 6.302 ± 1.60 μg / g, quercetin with a concentration of 583.5 ± 5.18 μg / g, hesperidin with a concentration of 7.427 ± 2.14 μg / g, and acacetin with a concentration of 69.5 ± 1.89 μg / g; There were two kinds of flavonols: (+)-catechin at a concentration of 377 ± 1.45 μg / g and epicatechin at a concentration of 18.56 ± 2.14 μg / g.

2. The method for extracting phenolic extracts from Echeveria oleifera according to claim 1, comprising the following steps: Select blue thistle plants in full bloom, wash them, dry them in an environment with a temperature of 20-25°C and a relative humidity of 50-60% until the moisture content is 10-15%, and then cut them into uniform pieces less than 2 cm; 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℃, the extraction time was 40-50 minutes, and the crude extract was obtained by filtration; 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 a refined echium phenolic extract is obtained by vacuum concentration.

3. Use of the echium phenolic extract according to claim 1 as an effective active ingredient in the preparation of a herbicide.

4. A highly effective botanical herbicide based on echium phenol extract, characterized in that It is mainly made of the following substances in proportion by mass: 100 parts of Echeveria phenolic extract, 6 - 8 parts of Azadirachtin, 3 - 4 parts of xanthan gum and sodium carboxymethyl cellulose (1:1 mixture).

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