CN110981730B - Method for extracting trans-ethyl p-hydroxycinnamate from corn straws and application of trans-ethyl p-hydroxycinnamate as herbicide - Google Patents
Method for extracting trans-ethyl p-hydroxycinnamate from corn straws and application of trans-ethyl p-hydroxycinnamate as herbicide Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
Abstract
The invention discloses a method for extracting trans-ethyl p-hydroxycinnamate from corn straws and application of the trans-ethyl p-hydroxycinnamate as a herbicide, wherein the method comprises the following steps: A. extraction: weighing corn stalk powder, extracting with 60% ethanol for 7 days, collecting extractive solution, sequentially extracting for 2-4 times, and mixing extractive solutions to obtain first extract; B. and (3) extraction: dispersing the first extract by deionized water, extracting with 5 times of petroleum ether for 6 times, extracting the residue with 5 times of ethyl acetate for 6 times, mixing ethyl acetate extractive solutions, vacuum filtering, and rotary evaporating to obtain second extract; C. and (3) chromatographic column separation: and (3) sequentially carrying out three-stage chromatographic column separation on the second extract, collecting the high-activity E3-2-3 fraction, and purifying the high-activity E3-2-3 fraction by HPLC (high performance liquid chromatography) preparative chromatography to obtain the trans-ethyl p-hydroxycinnamate. The invention separates trans-ethyl p-hydroxycinnamate from corn straws for the first time, has excellent herbicidal activity, can be used for preventing and removing weeds, and has the advantage of environmental protection.
Description
Technical Field
The invention relates to the technical field of extraction of weeding active substances, in particular to a method for extracting trans-ethyl p-hydroxycinnamate from corn straws and application of the trans-ethyl p-hydroxycinnamate as a herbicide.
Background
The weeds have a wide growth range, and the growth of the weeds can cause serious harm to the normal growth of crops, so that the weeds in the field must be prevented and removed greatly. Due to long-term adaptation to local ecological environment and farming culture, weed communities with various characteristics of different crops are gradually formed, and even if the same crop is in different geographical positions and environmental conditions, the composition of the weed communities is greatly different. However, the harm of weeds to crops is basically the same, which can cause serious reduction of the yield and quality of agricultural products, even cause no grain harvest, and greatly increase the labor cost. Therefore, how to effectively control the farmland weeds becomes an important subject for the development of modern agricultural production.
Weed control refers to the act of artificially controlling weeds in the ecosystems of farmlands and forests. The method has various means, including methods of agricultural control, plant quarantine, biological control, chemical control and the like. The chemical control method is the best method for preventing and removing the weeds in the farmland due to the rapidness, high efficiency and obvious weed control effect, plays a great role in the development of agricultural economy, promotes the high-efficiency, rapid and healthy development of the agricultural economy, and is deeply trusted by farmers. However, the unreasonable use of chemical herbicides also brings a series of major problems such as pesticide residue, environmental pollution, pest drug resistance and the like. In addition, due to factors such as change of cultivation and farming systems, excessive dependence on herbicides, improper herbicide mixing, large-scale planting of transgenic crops and the like, weed communities are changed, and the development of chemical herbicides is seriously hindered.
Therefore, the development of novel herbicides which are highly effective, low in toxicity, low in residual and environmentally friendly is urgently needed. The plants are used as huge natural resources on the earth, contain abundant secondary metabolites based on phytochemical effect, and have the potential of being developed into herbicides. At present, trans-ethyl p-hydroxycinnamate is mainly applied to medicine and organic synthesis intermediates, and the herbicidal activity of the trans-ethyl p-hydroxycinnamate is not found before.
Disclosure of Invention
Aiming at the problems of pesticide residue, environmental pollution, pest resistance and the like of the existing chemical herbicide, the invention discovers trans-ethyl p-hydroxycinnamate with excellent herbicidal activity from corn straws for the first time and provides an extraction method thereof.
The invention provides the following technical scheme:
method for extracting trans-ethyl p-hydroxycinnamate from corn straws
A. Extraction: weighing corn straw powder, placing the corn straw powder in a sealed tank, adding 60% ethanol until the powder is soaked, placing the sealed tank in a constant temperature box at 25 ℃ for leaching for 7 days, collecting an extracting solution, stirring once every 7-9 hours, sequentially extracting for 2-4 times according to the method, combining all extracting solutions, performing vacuum filtration on the extracting solutions, and performing rotary evaporation to obtain a first extract;
B. and (3) extraction: dispersing the first extract by using deionized water, extracting the dispersion liquid by using petroleum ether with the volume 5 times of the dispersion liquid for 6 times, removing petroleum ether extract, extracting residues by using ethyl acetate with the volume 5 times of the dispersion liquid for 6 times, combining ethyl acetate extract, performing vacuum filtration, and evaporating the solvent by rotary evaporation to obtain a second extract;
C. and (3) chromatographic column separation:
performing primary column chromatography separation on the second extract: dissolving the second extract with acetone, adding silica gel, stirring, and adding into silica gel chromatographic column after acetone is volatilized; sequentially carrying out gradient elution by taking petroleum ether/acetone mixed solution with the volume ratio of 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1 and 0:1 and acetone/methanol mixed solution with the volume ratio of 50:1, 25:1, 10:1, 5:1, 1:1 and 0:1 as mobile phases, tracing and collecting all eluted components by thin-layer chromatography, merging the components with the same Rf value, then respectively carrying out rotary evaporation to remove the solvent, obtaining 16 fractions of E1-E16 according to the elution sequence, and collecting high-activity fraction E3;
fraction E3 was subjected to secondary column chromatography: sequentially carrying out gradient elution by taking petroleum ether/acetone mixed solution with the volume ratio of 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1 and 0:1 as a mobile phase, carrying out secondary column chromatographic separation, merging components with the same Rf value, respectively carrying out rotary evaporation to remove the solvent, separating 4 fractions E3-1, E3-2, E3-3 and E3-4 from fraction E3 according to the elution order, and collecting high-activity fraction E3-2;
fraction E3-2 was subjected to three-stage column chromatography: sequentially carrying out gradient elution by taking petroleum ether/acetone solutions with volume ratios of 10:1, 5:1, 3:1, 1:1 and 0:1 as mobile phases, carrying out three-stage column chromatographic separation, merging components with the same Rf value, then respectively carrying out rotary evaporation to remove a solvent, separating the components from an E3-2 fraction according to an elution sequence to obtain four fractions, namely E3-2-1, E3-2-2, E3-2-3 and E3-2-4, and collecting a high-activity fraction E3-2-3;
and (3) purification: e3-2-3 was further separated by HPLC preparative chromatography, and a fraction qn-6, i.e., ethyl trans-p-hydroxycinnamate, was isolated and purified from the E3-2-3 fraction.
Specifically, when E3-2-3 fraction was purified by HPLC preparative chromatography, YMC-ODS semi-preparative column (250 mm. times.10 mm) was used, and 47% by volume methanol-water solution was used as a mobile phase at a flow rate of 4mL/min, and fractions were collected and combined according to the peak of the compound appearing on the detector. Wherein qn-6 is the last compound to be isolated, and the purity of the compound is high, and the retention time tR is 36.3 min. The invention also provides application of the trans-ethyl p-hydroxycinnamate as a herbicide. Specifically, trans-ethyl p-hydroxycinnamate can be used as an active ingredient to be directly processed into a preparation for weed control, or used as a lead compound to form a compound with higher herbicidal activity through further structural modification or modification, and then applied to a herbicide.
In view of the fact that trans-ethyl p-hydroxycinnamate also has strong inhibitory activity and weak selectivity on crops such as wheat, corn and the like, the trans-ethyl p-hydroxycinnamate can be firstly used in non-cultivated land (such as leisure land, field side, roadside and the like) and crop fields insensitive to the trans-ethyl p-hydroxycinnamate in practical application. When the environment is applied, no specific requirements are imposed on the specific application method, and a conventional stem leaf spraying method can be adopted.
When the trans-ethyl p-hydroxycinnamate is applied to wheat fields and corn fields, the trans-ethyl p-hydroxycinnamate can be applied by a soil treatment method before seedlings are sowed, so that phytotoxicity to crops is avoided, and a weeding effect can be achieved. Wherein the soil treatment method comprises the following steps: before the crop is sown or before the emergence of seedlings after the crop is sown, the herbicide is applied to the soil to eliminate the weed seedlings.
The invention has the following beneficial effects:
the trans-ethyl p-hydroxycinnamate is separated from the corn straws for the first time, and is found to have excellent herbicidal activity, the inhibiting effect on dicotyledons is relatively higher than that of monocotyledons, and the trans-ethyl p-hydroxycinnamate can be used for preventing and removing weeds; the trans-ethyl p-hydroxycinnamate is a natural product in plants, and has the characteristics of easy degradation in the environment and environmental friendliness, so the discovery of the herbicidal activity of the compound is very in line with the development direction of pesticides. The extraction method provided by the invention can effectively separate the trans-ethyl p-hydroxycinnamate from the corn straws, turns waste into valuable and fully develops the utilization value of the corn straws.
Drawings
FIG. 1 is a graph showing the effect of preliminary extracts of corn stover in example 1 on the inhibition of radicles and hypocotyls of lettuce seedlings; the inhibition effect on the radicles of the vegetable seedlings is shown on the left side, and the inhibition effect on the radicles of the vegetable seedlings is shown on the right side;
FIG. 2 is a graph showing the effect of preliminary extracts of corn stover in example 1 on the inhibition of radicles and hypocotyls of wheat seedlings; the inhibition effect on the radicle of the wheat seedling is shown on the left side, and the inhibition effect on the radicle of the wheat seedling is shown on the right side;
FIG. 3 shows the inhibitory effect of different extracts of 60% ethanol extracts from corn stover on wheat seedling radicle and hypocotyl; the inhibition effect on the radicle of the wheat seedling is shown on the left side, and the inhibition effect on the radicle of the wheat seedling is shown on the right side;
FIG. 4 shows the inhibitory effect of different extractions of 60% ethanol extracts from corn stover on the radicles and hypocotyls of lettuce seedlings; the inhibition effect on radicles is shown on the left side, and the inhibition effect on radicles is shown on the right side;
FIG. 5 shows the yields of the fractions separated from ethyl acetate phase;
FIG. 6 is a graph of the inhibition of Amaranthus retroflexus and Echinochloa crusgalli seedlings by ethyl trans-p-hydroxycinnamate and pendimethalin; wherein the left test object is Amaranthus retroflexus, the right test object is Echinochloa crusgalli, wherein V is trans-ethyl p-hydroxycinnamate, and VI is pendimethalin.
Note: vertical lines in the graph indicate standard error; lower case english letters are used to indicate the analysis of the significance of the difference in growth inhibition of radicle or hypocotyl between solvent extracts, the difference in letters indicating a significant difference at the 5% level.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Test materials and activity tracking methods
1. Test plant material
The research takes the maize straws (variety: longevity and benefit) collected from Qingdao Jimo high-prosperity farms as research materials. The climate in the ground is suitable, the four seasons are clear, and the annual average temperature is 12 ℃. Washing corn stalks with deionized water, naturally air drying outdoors, crushing with a graded superfine continuous crusher, and storing in a low-temperature seed cabinet at 6 ℃ for later use.
2. Recipient plant seed
Lettuce (Lactuca sativa), wheat (Triticum aestivum), barnyard grass (Echinochloa crusgalli (L.) Beauv.) and Amaranthus retroflexus (Amaranthus Retroflexus L.) were used as recipient plants in this study. Wherein the wheat variety is Nicotiana No. 24, provided by Nicotiana agricultura; the lettuce variety is Yinongnian Gaila Hexixue lettuce purchased from Qingdao city Yang wholesale market seed station; amaranthus retroflexus and Echinochloa crusgalli were collected from the wastelands near Qingdao agricultural university where no herbicide was used. All the seeds of the test plants are stored in a seed cabinet at 6 ℃ for later use.
3. Activity tracking of extracts
In this study, the herbicidal (chemosensory) activity of each extract, extract and monomeric compound was measured by the agar method.
1) Treatment of test plant seeds
Soaking seeds of the plant to be tested in a 2% sodium hypochlorite solution for 10-15min, washing with distilled water for 5-6 times, and soaking in running water in a vessel for 6-8h to absorb water. Spreading two layers of kitchen paper in a square plate which is cleaned and disinfected by 75% alcohol, wetting the paper with distilled water, washing the plant seeds to be tested after water absorption with distilled water for a plurality of times, uniformly placing the seeds on the kitchen paper, covering the paper, placing the paper in a constant temperature climate incubator at 25 ℃ for accelerating germination, and keeping the radicles (seed roots) of the plant seedlings for later use when the radicles grow to 3-5 mm.
2) Preparation of extract-containing agar
Dissolving the extracts and monomer compounds in DMSO to obtain high-concentration mother solution. Then sucking a certain amount of mother liquor into 0.5% agar solution to prepare agar matrix containing extract. Agar medium containing only DMSO was used as a blank. The DMSO content remained consistent throughout the treatment.
3) Transplanting of test plant seedlings
Selecting receptor plant germinating seeds with basically consistent root length, firstly inserting 5 small holes on the surface of solidified agar culture medium by using sharp-nose tweezers, then gently inserting the radicle of the seed into 5 particles in each beaker, repeating for 3 times, placing the beaker in a paper box to shade light, and then culturing in a plant growth box for 3-4 days. The setting condition of the growth chamber is that the light is continuously circulated for 14h (25 ℃) and the dark is continuously circulated for 10h (20 ℃), and the relative humidity in the growth chamber is 60%.
4) Result measurement and data analysis
Each treated seedling was taken out of the beaker, and the length of its seed root (radicle) and coleoptile (hypocotyl) was measured with a vernier caliper to calculate the amount of growth. The data were analyzed using Excel software, the inhibition rates and standard errors for the radicle (radicle) and coleoptile (hypocotyl) were calculated, and the effective medium concentrations of each treatment were analyzed using SPSS software (EC 50).
Growth amount-treated radicle (or hypocotyl) length-untreated radicle (or hypocotyl) length
Inhibition (%) - (control growth amount-treated growth amount)/control growth amount × 100
EXAMPLE extraction separation of herbicidally active substances
1. Screening of extraction solvent
The activity of the extract is taken as an index, 60 percent methanol, 60 percent ethanol and water are taken as extraction solvents, and the solvent for extracting the weeding active substances in the corn straws is screened. Weighing 3 parts of 20g of corn straw powder in three conical flasks, extracting with 350mL of each of three solvents, sealing the opening of the conical flask with tinfoil paper, leaching in a constant-temperature shaking box at 25 ℃ for 3 days, filtering, and collecting the extract. Extracting for 3 times by the same method, mixing the filtrates of 3 times, and vacuum filtering. The extract of 60% methanol and 60% ethanol is rotary evaporated at 40 deg.C with rotary evaporator to obtain extract, and the water extract is subjected to water removal with freeze dryer, and then used for activity determination. The recipient plant is selected from wheat and lettuce, and represents Gramineae and broad-leaved plant respectively.
As shown in figure 1, each of the three solvent extracts showed significant inhibition of the growth of the radicle of the seedling of lettuce at 0.5 g.L-1The inhibition rates of the compounds are respectively 83.9 percent, 70.4 percent and 70.3 percent at the lowest treatment concentration, and are all obviously improved along with the increase of the treatment concentration, and the inhibition rates are 4 g.L-1The inhibition rate of the compound is more than 90% under the highest treatment concentration. But the inhibition effect on the growth of hypocotyl of lettuce seedlings is relatively low, and the difference between different extracts is large. Wherein the activity of the extract is relatively high at 0.5 g.L with 60% methanol and 60% ethanol-1～1g·L-1The inhibition rates under the treatment concentrations respectively reach 83.9-89.5 percent and 70.4-85.6 percent, and are respectively 4 g.L-1The time increases to 93.7% and 94.7%. The water extract is 0.5 g.L-1～1g·L-1The inhibition rate under the treatment concentration is less than 10 percent, and the inhibition activity is about 40 percent only under the high-concentration treatment.
As shown in figure 2, the extracts of three different solvents also showed strong inhibition effect on the growth of wheat seedlings, and the inhibition activity on the seed roots was higher than that on the coleoptiles. At 0.5 g.L-1～4.0g·L-1The inhibition rates of the 60% methanol, the 60% ethanol and the water extract on the wheat seedling seed roots (coleoptiles) are respectively 79.9-92.0% (33.2-92.0%) and 71.7-90.7% (2.3-65.7%) And 58.1 to 86.7% (23.9 to 51.2%).
The results show that the inhibitory activity of the methanol and ethanol extracts on the growth of lettuce and wheat seedlings is basically equivalent but higher than that of the water extracts under the same concentration. 60% ethanol was chosen as the extraction solvent, considering the high toxicity of methanol and the difficulty of extracting the fat-soluble active substance with aqueous extracts.
Effect of different extractions of 2.60% ethanol extract on wheat and lettuce seedling growth
(1) The experimental method comprises the following steps:
weighing 10kg of corn straw powder, placing the corn straw powder into a sealed tank, adding 60% ethanol until the powder is soaked, placing the powder into a constant temperature box at 25 ℃ for leaching for 7 days, stirring the powder once every 8 hours, extracting the powder for 3 times, combining the extracting solutions for 3 times, performing vacuum filtration (No. 2 filter paper, Whatman) and then evaporating the extracting solution to dryness by using a rotary evaporator at 40 ℃ to obtain an extract.
Taking a 500mL separating funnel, fixing, pouring an extract (40g) of a corn straw 60% ethanol solution extract dispersed by deionized water, adding a certain amount of petroleum ether for extraction, removing a petroleum ether phase after a certain time, adding new petroleum ether for secondary extraction, circulating the steps until no compound is detected under an ultraviolet lamp by a TLC chromatographic plate, and combining all petroleum ether extract liquor. The residue was then extracted with ethyl acetate and n-butanol in the same manner. The combined extracts were vacuum filtered (Whatman, No2.) and the solvent evaporated by rotary evaporator (40 ℃ C.) to obtain an extract and weighed. Wherein the aqueous phase extract is weighed after drying in a freeze dryer. The herbicidal (sensate) activity of each extract was measured.
(2) Results of the experiment
As shown in FIG. 3, the concentration of the compound is 0.25 g.L for wheat seedlings-1～2g·L-1At the treatment concentration of (3), the extract was at 0.25 g.L except for the n-butanol phase-1～0.5g·L-1Shows certain growth stimulating activity on the growth of coleoptile under low concentration, and the four solvent extracts show different degrees of inhibition on the growth of the seed root or the coleoptilePreparing the activity. Wherein, the inhibition effect of the ethyl acetate phase extract is the highest, and the growth inhibition rates of the seed roots and the coleoptiles in the tested concentration range respectively reach 52.1-94.6 percent and 22.2-84.4 percent; the petroleum ether phase extraction is the second, and the inhibition rates of the petroleum ether phase extraction and the petroleum ether phase extraction are respectively 28.9-73.6% and 11.1-48.7%. Whereas the inhibitory activity of the n-butanol and aqueous phase extracts was relatively low.
As shown in fig. 4, for the seedlings of the green vegetables, the ethyl acetate phase and the petroleum ether phase extract both have a significant growth inhibition effect on the growth of radicles and hypocotyls of the seedlings, but the inhibitory activity on the radicles is higher than that on the hypocotyls. They are in the range of 0.25 g.L-1～2g·L-1The inhibition rates on the growth of radicle (hypocotyl) reach 81.8-100% (35.2-93.7%) and 38.6-97.2% (23.1-79.0%), respectively. The n-butanol and aqueous extracts were relatively low in activity and showed some inhibitory activity against radicle growth over the tested concentration range, but showed some inhibitory activity against hypocotyl growth only in the high concentration treatment and slight growth stimulating activity in the low concentration treatment.
The above results indicate that the ethyl acetate phase extract is the most biologically active, the petroleum ether phase is the less active, and the n-butanol phase and the aqueous phase are relatively less active, and therefore, further separation of the active species in the ethyl acetate phase extract and the petroleum ether phase extract is emphasized.
3. Separation of ethyl trans-p-hydroxycinnamate from ethyl acetate phase extract
3.1 Ethyl acetate phase first-order column chromatography separation results and Activity measurement thereof
(1) The experimental method comprises the following steps: a12 cm X80 cm column was fixed to an iron support. 3000g of 200-mesh 300-mesh silica gel is weighed in a large beaker, petroleum ether is added until the silica gel is immersed, and stirring is carried out while adding, so as to remove bubbles. And filling the treated silica gel into a column by a wet method, and emptying the column by using petroleum ether for one day after filling the column. Weighing 40g of ethyl acetate phase extract, dissolving the ethyl acetate phase extract in a 500ml beaker by using a proper amount of acetone, adding 65g of silica gel (200 meshes and 300 meshes), continuously stirring and uniformly mixing, and loading the mixture into a column after the solvent is volatilized. Gradient elution was performed with petroleum ether/acetone mixed solution (1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1 and 0:1v/v) and acetone/methanol mixed solution (50:1, 25:1, 10:1, 5:1, 1:1 and 0:1v/v) as mobile phases. Tracking and collecting all components by TLC, combining the components with the same Rf value, respectively carrying out rotary evaporation to obtain n fractions of E1-En, weighing and calculating the yield of each component.
Wheat and lettuce were used as test plants and activity was measured according to the method described above. The fraction with the best activity and higher yield is selected for further column chromatographic separation, and the mobile phase composition used in the separation is found in the results and analysis part.
(2) The experimental results are as follows:
the separation result and the activity of the ethyl acetate phase first-stage column chromatography are confirmed by Thin Layer Chromatography (TLC) that petroleum ether/acetone and acetone/methanol with different proportions are gradient eluents for separating ethyl acetate phase substances. 40g of ethyl acetate extract phase is taken to be subjected to primary column chromatography separation and purification to obtain 16 components of E1-E16, and the yield of each component is shown in figure 5. It can be seen that the yield of the E13 fraction is the highest, which is 17.8%, the yield of the E3 fraction is 12.8%, and the yields of the other fractions are relatively low.
Furthermore, wheat and lettuce were used as recipient plants, and 0.5 g.L was measured-1The herbicidal (chemosensory) activity of each fraction at the treatment concentration is shown in table 1. It can be seen that the fractions showed different degrees of inhibitory activity on the growth of lettuce and wheat seedlings, except that the E1 fraction showed a slight stimulation on the growth of the radicles and hypocotyls of lettuce seedlings. The activity of E3 fraction is highest, and the inhibition rate of the E3 fraction on the radicle of lettuce (hypocotyl) and the root of wheat seed (coleoptile) is respectively as high as 100% (80.7%) and 80.5% (69.2%). The next ones of the E13 fractions were 90.3% (46.2%) and 87.7% (75.7%), respectively, while the lowest of the E1 fraction was only-9.7% (-7.3%) and 8.4% (3.1%). The activity of the other fractions is intermediate between them. Taking the yield and the biological activity of each component into comprehensive consideration, and selecting the high-activity fraction E3 for further separation and purification.
TABLE 10.5 g.L-1Effect of ethyl acetate phase fractions on lettuce and wheat seedling growth at concentration
Note: the data in the table are mean ± sd of 3 replicates. Lower case letters in the same column indicate significant differences between fractions at the 0.05 level.
3.2E3 fraction two-stage column chromatography separation results and Activity measurement thereof
4 fractions of E3-1, E3-2, E3-3 and E3-4 were obtained from the E3 fraction by two-stage column chromatography using petroleum ether/acetone mixed solutions (25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1 and 0:1v/v) in different ratios as eluents, and the yields were 17.1%, 31.5%, 25.6% and 25.8%, respectively. 4 fractions at 0.3 g.L-1The effect on wheat and lettuce seedling growth at concentration is shown in table 2. It can be seen that the E3-2 fraction has the highest inhibitory activity on the growth of seedlings of two recipient plants, and the inhibition rates on wheat seed roots (radicle) and lettuce radicle (hypocotyl) are 95.0% (74.5%) and 100% (100%), respectively, the E3-3 fractions are 77.5% (45.8%) and 77.1% (57.7%), respectively, the third fraction is E3-4, respectively, 36.8% (14.9%) and 51.9% (31.1%), and the E3-1 fraction is the lowest, and only 19.8% (8.2%) and 23.4% (11.3%), respectively. The E3-2 fraction is selected for further separation by comprehensively considering the yield and the biological activity of each component.
TABLE 20.3 g.L-1Effect of E3 fractions on the growth of lettuce and wheat seedlings at concentrations
Note: the data in the table are mean ± sd of 3 replicates. Lower case letters in the same column indicate significant differences between fractions at the 0.05 level.
3.3E3-2 fraction three-stage column chromatography separation results and Activity
By using petroleum ether/acetone solutions (10:1, 5:1, 3:1, 1:1 and 0:1v/v) in different ratios as eluents, four fractions, namely E3-2-1, E3-2-2, E3-2-3 and E3-2-4, are separated from the E3-2 fraction by three-stage column chromatography, and the yields of the four fractions are 21.5%, 23.6%, 17.8% and 37.1% respectively. The effect of the four fractions on lettuce and wheat seedling growth is shown in table 3. It was found that the concentration was 0.05 g.L-1At the concentration, only the E3-2-3 fraction showed higher inhibitory activity, the growth inhibition rates of the radicle (hypocotyl) and the seed root (coleoptile) of the two were 75.3% (46.6%) and 73.8% (32.8%), respectively, while the other three fractions showed lower activity except that the E3-2-1 fraction showed 70.8% inhibitory activity to the lettuce radicle. Thus, the E3-2-3 fraction was selected for further preparative chromatographic separation.
TABLE 30.05 g.L-1Effect of E3-2 fractions on lettuce and wheat seedling growth at concentration
Note: the data in the table are mean ± sd of 3 replicates. Lower case letters in the same column indicate significant differences between fractions at the 0.05 level. 3.4 further isolation and structural characterization of the active substances in the E3-2-3 fraction
The three fractions E3-2-3 were further separated by preparative chromatography to separate and purify 5 compounds, qn-1(13.1mg), qn-3-1(2.0mg), qn-4(50.0mg), qn-5-1(1.4mg) and qn-6(113.8mg), from the E3-2-3 fraction (421 mg).
The preparative chromatographic separation method comprises the following steps: separation and purification were carried out by HPLC preparative chromatography using YMC-ODS semi-preparative columns (250 mm. times.10 mm). A total of 5 compounds were separated from the E3-2-3 fraction, and the mobile phase was purified with 47% by volume of aqueous methanol at a flow rate of 4mL/min, and five compounds were eluted in the order of qn-1(tR ═ 7.2min), qn-3-1(tR ═ 17.7min), qn-4(tR ═ 19.5min), qn-5-1(tR ═ 30.7min) and qn-6(tR ═ 36.3min), and the final target compound qn-6 was isolated.
The compound is structurally identified by mass spectrum, 1H-NMR, 13C-NMR, DEPT spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectrum and other spectra, and the structure of the compound is further analyzed and clarified, namely p-hydroxybenzaldehyde (qn-1), cis-methyl p-hydroxycinnamate (qn-3-1, also called methyl p-hydroxycinnamate), trans-methyl p-hydroxycinnamate (qn-4, also called methyl p-hydroxycinnamate), cis-ethyl p-hydroxycinnamate (qn-5-1, also called ethyl p-hydroxycinnamate) and trans-ethyl p-hydroxycinnamate (qn-6, also called ethyl p-hydroxycinnamate). Their C, H signals are ascribed as follows:
p-hydroxybenzaldehyde (qn-1): 1H-NMR (500MHz, CD3OD) δ:9.71(1H, s, -CHO),7.73(2H, d, J ═ 8.6Hz, H-3,5),6.87(2H, d, J ═ 8.6Hz, H-2, 6); 13C-NMR (125MHz, CD3OD) δ:192.8(C ═ O),165.5(C-4),133.4(C-1),130.1(C-2,6),117.0 (C-1).
Cis-methyl p-hydroxycinnamate (qn-3-1): 1H-NMR (500MHz, CD3OD) δ:7.62(2H, d, J ═ 8.7Hz, H-2,6),6.85(1H, d, J ═ 12.8Hz, H-7),6.75(2H, d, J ═ 8.7Hz, H-3,5),5.77(1H, d, J ═ 12.8Hz, H-8),3.70(3H, s, -CH 3); 13C-NMR (125MHz, CD3OD) δ 168.8(C ═ O),160.0(C-4),145.0(C-7),133.6(C-2,6),127.5(C-1),116.3(C-8),115.8(C-3,5),51.7(-CH 3).
Trans-methyl p-hydroxycinnamate (qn-4): 1H-NMR (500MHz, CD3OD) δ:7.50(1H, d, J ═ 16.0Hz, H-7),7.33(2H, d, J ═ 8.6Hz, H-2,6),6.71(2H, d, J ═ 8.6Hz, H-3,5),6.20(1H, d, J ═ 16.0Hz, H-8),3.65(3H, s, OCH 3); 13C-NMR (125MHz, CD3OD) δ 169.7(C ═ O),161.3(C-4),146.5(C-7),131.1(C-2,6),127.0(C-1),116.8(C-3,5),114.8(C-8),52.0(-CH 3).
Cis-ethyl p-hydroxycinnamate (qn-5-1): 1H-NMR (500MHz, CD3OD) δ:7.60(2H, d, J ═ 8.6Hz, H-2,6),6.84(1H, d, J ═ 12.8Hz, H-7),6.74(2H, d, J ═ 8.6Hz, H-3,5),5.75(1H, d, J ═ 12.8Hz, H-8),4.16(2H, q, J ═ 7.2Hz, H-10)1.25(3H, t, J ═ 7.2Hz, H-11); 13C-NMR (125MHz, CD3OD) δ:168.4(C ═ O),160.0(C-4),144.7(C-7),133.5(C-2,6),127.7(C-1),116.9(C-8),115.8(C-3,5),61.2(C-10),14.5 (C-11).
Trans-ethyl p-hydroxycinnamate (qn-6): 1H-NMR (500MHz, CD3OD) δ:7.35(1H, d, J ═ 16.0Hz, H-7),7.18(2H, d, J ═ 8.6Hz, H-2,6),6.59(2H, d, J ═ 8.6Hz, H-3,5),6.04(1H, d, J ═ 16.0Hz, H-8),3.95(2H, q, J ═ 7.2Hz, H-10)1.05(3H, t, J ═ 7.2Hz, H-11); 13C-NMR (125MHz, CD3OD) δ 169.2(C ═ O),161.0(C-4),146.2(C-7),131.0(C-2,6),127.0(C-1),116.7(C-3,5),115.2(C-8),61.4(C-10),14.6 (C-11).
The structural formulas of the 5 compounds are as follows, wherein the compounds are p-hydroxybenzaldehyde (qn-1), cis-methyl p-hydroxycinnamate (qn-3-1), trans-methyl p-hydroxycinnamate (qn-4), cis-ethyl p-hydroxycinnamate (qn-5-1) and trans-ethyl p-hydroxycinnamate (qn-6) from left to right:
example herbicidal Activity assay of ethyl di-trans-p-hydroxycinnamate
1. Herbicidal activity of trans-ethyl p-hydroxycinnamate
The effect of trans-ethyl p-hydroxycinnamate on wheat, lettuce, redroot amaranth and barnyard grass is shown in table 4. As can be seen from the table, the trans-ethyl p-hydroxycinnamate at different concentrations significantly inhibited the growth of seedlings of 4 test plants at 0.025 g.L-1The inhibition rates for lettuce and redroot amaranth radicles and wheat and barnyard grass radicles were 39.4%, 85.7%, 22.0% and 61.3%, respectively, and for lettuce and redroot amaranth hypocotyls and wheat and barnyard grass coleoptiles were 13.3%, 53.8%, 0.77% and 19.6%, respectively, at the lowest treatment concentration of (b). And in the range of 0.1-0.2 g.L-1The inhibition rate of trans-ethyl p-hydroxycinnamate on germ and radicle of wheat, lettuce, redroot amaranth and barnyard grass basically reaches 90-100%. In general, the weed inhibiting effect of the herbicide is obviously better than that of crops, and the inhibiting effect of the herbicide on dicotyledonous plants is also relatively higher than that of monocotyledonous plants.
TABLE 4 Effect of trans-ethyl p-hydroxycinnamate on lettuce, Amaranthus retroflexus, wheat and barnyard grass seedling growth
Note: the data in the table are mean ± sd of 3 replicates. Lower case letters in the same column indicate significant differences between fractions at the 0.05 level.
2. Comparative test with conventional herbicides
To further verify the herbicidal activity of trans-ethyl p-hydroxycinnamate, a comparative test was conducted on pendimethalin, a common herbicide, and the results are shown in fig. 6 and table 5. Trans-ethyl p-hydroxycinnamate at 0.05 g.L-1At the concentration of 0.1 g.L for the recipient plant Amaranthus retroflexus-1Has obvious inhibiting effect on the growth of barnyard grass seedlings under the concentration. Wherein, the inhibitory activity (96.3 percent to 97.0 percent) of the trans-ethyl p-hydroxycinnamate on the growth of the radicle of the amaranthus retroflexus is higher than that of a contrast medicament pendimethalin (92.0 percent); the inhibition of barnyard grass seed root has no significant difference (98.7% -100%) with the control. These results indicate that the herbicidal activity of trans-ethyl p-hydroxycinnamate was higher than or equal to the control.
TABLE 5 comparison of herbicidal Activity of ethyl trans-p-hydroxycinnamate with pendimethalin
Note: the data in the table are mean ± sd of 3 replicates. Lower case letters in the same column indicate significant differences between fractions at the 0.05 level.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (4)
1. The method for extracting trans-ethyl p-hydroxycinnamate from corn straws is characterized by comprising the following steps:
A. extraction: weighing corn straw powder, placing the corn straw powder in a sealed tank, adding 60% ethanol until the powder is soaked, placing the sealed tank in a constant temperature box at 25 ℃ for leaching for 7 days, collecting an extracting solution, stirring once every 7-9 hours, sequentially extracting for 2-4 times according to the method, combining all extracting solutions, performing vacuum filtration on the extracting solutions, and performing rotary evaporation to obtain a first extract;
B. and (3) extraction: dispersing the first extract by using deionized water, extracting the dispersion liquid by using petroleum ether with the volume 5 times of the dispersion liquid for 6 times, removing petroleum ether extract, extracting residues by using ethyl acetate with the volume 5 times of the dispersion liquid for 6 times, combining ethyl acetate extract, performing vacuum filtration, and evaporating the solvent by rotary evaporation to obtain a second extract;
C. and (3) chromatographic column separation:
performing primary column chromatography separation on the second extract: dissolving the second extract with acetone, adding silica gel, stirring, and adding into silica gel chromatographic column after acetone is volatilized; carrying out gradient elution by sequentially taking petroleum ether/acetone mixed solution with the volume ratio of 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1 and 0:1 and acetone/methanol mixed solution with the volume ratio of 50:1, 25:1, 10:1, 5:1, 1:1 and 0:1 as mobile phases, tracking and collecting all eluted components by thin-layer chromatography, merging the components with the same Rf value, then respectively carrying out rotary evaporation to remove the solvent, obtaining 16 fractions of E1-E16 according to the elution sequence, and collecting high-activity fraction E3;
fraction E3 was subjected to secondary column chromatography: sequentially carrying out gradient elution by taking petroleum ether/acetone mixed solution with the volume ratio of 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1 and 0:1 as a mobile phase, carrying out secondary column chromatographic separation, merging components with the same Rf value, respectively carrying out rotary evaporation to remove the solvent, separating 4 fractions E3-1, E3-2, E3-3 and E3-4 from fraction E3 according to the elution order, and collecting high-activity fraction E3-2;
fraction E3-2 was subjected to three-stage column chromatography: sequentially carrying out gradient elution by taking petroleum ether/acetone solutions with volume ratios of 10:1, 5:1, 3:1, 1:1 and 0:1 as mobile phases, carrying out three-stage column chromatographic separation, merging components with the same Rf value, then respectively carrying out rotary evaporation to remove a solvent, separating the components from an E3-2 fraction according to an elution sequence to obtain four fractions, namely E3-2-1, E3-2-2, E3-2-3 and E3-2-4, and collecting a high-activity fraction E3-2-3;
and (3) purification: e3-2-3 was further separated by HPLC preparative chromatography, and a fraction qn-6, i.e., ethyl trans-p-hydroxycinnamate, was isolated and purified from the E3-2-3 fraction.
2. The method for extracting trans-ethyl p-hydroxycinnamate from corn stalk as claimed in claim 1 wherein 200-300 mesh silica gel is used in step C, and the specification of chromatographic column is 12cm x 80 cm.
3. The method for extracting trans-ethyl p-hydroxycinnamate from corn stover according to claim 1, wherein the separation and purification by HPLC preparative chromatography is carried out using YMC-ODS semi-preparative column with a specification of 250mm x 10 mm.
4. The method for extracting trans-ethyl p-hydroxycinnamate from corn stalks according to claim 1, wherein the method for separating the fraction qn-6 by HPLC preparative chromatography is specifically as follows: the mobile phase adopts 47 percent methanol-water solution by volume, the flow rate is 4mL/min, and the retention time tR of qn-6 is 36.3 min.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103450023A (en) * | 2013-08-27 | 2013-12-18 | 青岛农业大学 | Extraction method of ethyl gallate in peony leaves and new application thereof |
| CN103922935A (en) * | 2014-03-31 | 2014-07-16 | 曹庸 | Method for separating P-hydroxyl ethyl cinnamate from raspberry leaves and application of P-hydroxyl ethyl cinnamate |
| CN107927149A (en) * | 2017-11-30 | 2018-04-20 | 中国农业大学 | A kind of application of the p-Coumaric Acid ester in sterilizing anti-staling agent for fruit and vegetable and the sterilizing anti-staling agent for fruit and vegetable of the ester containing p-Coumaric Acid |
| CN108605931A (en) * | 2018-04-20 | 2018-10-02 | 中国热带农业科学院热带生物技术研究所 | Application of four kinds of compounds in preparing nematicidal drug |
| WO2018195422A1 (en) * | 2017-04-20 | 2018-10-25 | Spero Energy, Inc. | Extraction of natural ferulate and coumarate from biomass |
| CN108911985A (en) * | 2018-07-09 | 2018-11-30 | 集美大学 | The method and its application of p-Coumaric Acid ethyl ester are isolated and purified from camellia pollen |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104542609B (en) * | 2015-01-05 | 2016-08-24 | 乔建国 | A kind of herbicide preventing and kill off Semen Cuscutae |
| WO2019210929A1 (en) * | 2018-02-22 | 2019-11-07 | Université de Liège | Herbicidal composition comprising at least one phenolic active compound |
-
2019
- 2019-12-13 CN CN202111545024.1A patent/CN114190381B/en active Active
- 2019-12-13 CN CN201911287276.1A patent/CN110981730B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103450023A (en) * | 2013-08-27 | 2013-12-18 | 青岛农业大学 | Extraction method of ethyl gallate in peony leaves and new application thereof |
| CN103922935A (en) * | 2014-03-31 | 2014-07-16 | 曹庸 | Method for separating P-hydroxyl ethyl cinnamate from raspberry leaves and application of P-hydroxyl ethyl cinnamate |
| WO2018195422A1 (en) * | 2017-04-20 | 2018-10-25 | Spero Energy, Inc. | Extraction of natural ferulate and coumarate from biomass |
| CN107927149A (en) * | 2017-11-30 | 2018-04-20 | 中国农业大学 | A kind of application of the p-Coumaric Acid ester in sterilizing anti-staling agent for fruit and vegetable and the sterilizing anti-staling agent for fruit and vegetable of the ester containing p-Coumaric Acid |
| CN108605931A (en) * | 2018-04-20 | 2018-10-02 | 中国热带农业科学院热带生物技术研究所 | Application of four kinds of compounds in preparing nematicidal drug |
| CN108911985A (en) * | 2018-07-09 | 2018-11-30 | 集美大学 | The method and its application of p-Coumaric Acid ethyl ester are isolated and purified from camellia pollen |
Non-Patent Citations (2)
| Title |
|---|
| 17个玉米品种的不同器官对生菜幼苗生长的抑制作用;孙小雪 等;《杂草学报》;20171231;第35卷(第4期);第17-24页 * |
| 玉米秸秆对4种农田杂草生长的抑制作用;张丽娟 等;《植物保护》;20161231;第42卷(第6期);第63-66、109页 * |
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