CN111072483B - Method for extracting methyl p-hydroxycinnamate from corn straws and application of methyl p-hydroxycinnamate as herbicide - Google Patents
Method for extracting methyl p-hydroxycinnamate from corn straws and application of methyl p-hydroxycinnamate as herbicide Download PDFInfo
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Abstract
The invention discloses a method for extracting methyl p-hydroxycinnamate from corn straws and application of the methyl 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, mixing extractive solutions, and rotary evaporating 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 high-activity E13-2-1 fraction, and purifying the high-activity E13-2-1 fraction by HPLC (high performance liquid chromatography) preparative chromatography to obtain the methyl p-hydroxycinnamate. The method separates the methyl p-hydroxycinnamate from the corn straws for the first time, has excellent herbicidal activity, can be used for preventing and removing weeds, and has the advantage of environmental friendliness.
Description
Technical Field
The invention relates to the technical field of extraction of weeding active substances, in particular to a method for extracting methyl p-hydroxycinnamate from corn straws and application of methyl 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, methyl p-hydroxycinnamate is an important intermediate for organic synthesis, medicine and anti-epinephrine esmolol, is only used for industry, and experts in electronics and optical fibers extend the application of cis-methyl p-hydroxycinnamate to the field of electronics and optical fiber communication, so that methyl p-hydroxycinnamate becomes an excellent touch screen and high-end optical fiber material. Herbicidal activity has not been previously discovered.
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 the p-hydroxy methyl cinnamate with excellent herbicidal activity from the corn straws for the first time and provides the extraction method thereof.
The invention provides the following technical scheme:
a method for extracting methyl p-hydroxycinnamate from corn stalks comprises the following steps:
A. extracting, namely 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 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 extract: dissolving the second extract with acetone, adding silica gel, stirring, and adding into silica gel chromatographic column after acetone is volatilized; gradient elution is sequentially carried out 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, elution components are tracked and collected by thin-layer chromatography, components with the same Rf value are combined, then rotary evaporation is carried out to remove the solvent, 16 fractions E1-E16 are obtained according to the elution sequence, and high-activity fractions E3 and E13 are collected.
Fraction E13 was subjected to secondary column chromatography: carrying out gradient elution by using a petroleum ether/acetone mixed solution and an acetone/methanol mixed solution as eluents according to the volume ratio of 10:1, 5:1, 3:1, 2:1 and 0:1 in sequence, combining components with the same Rf value, respectively carrying out rotary evaporation to remove the solvent, separating from an E13 fraction according to the elution sequence to obtain 5 fractions including E13-1, E13-2, E13-3, E13-4 and E13-5, and collecting the E13-2 fraction with high activity.
Fraction E13-2 was subjected to three-stage column chromatography: sequentially taking petroleum ether/acetone solutions with volume ratios of 3:1, 2:1 and 0:1 and acetone/methanol solution mixed solutions with volume ratios of 3:1, 1:1 and 0:1 as eluents, separating by three-stage column chromatography, separating from E13-2 fraction to obtain three fractions of E13-2-1, E13-2-2 and E13-2-3, and collecting high-activity E13-2-1 fraction.
Preferably, 200-mesh 300-mesh silica gel is used in the step C, and the size of the chromatographic column used for the three-time column chromatography separation is 12cm multiplied by 80 cm.
And (3) purification: further separating and purifying the E13-2-1 fraction by HPLC preparative chromatography to obtain 2 compounds, namely qn-3-1, namely cis-methyl p-hydroxycinnamate; qn-4, namely trans-methyl p-hydroxycinnamate.
Preferably, on the basis, the step C further comprises the following steps:
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 on fraction E3, merging separated components with the same Rf value, removing the solvent through rotary evaporation, respectively obtaining 4 fractions E3-1, E3-2, E3-3 and E3-4 according to the elution sequence, and collecting 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 0:1v/v as mobile phases, separating by three-stage column chromatography, merging separated components with the same Rf value, removing a solvent by rotary evaporation, and separating 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;
wherein, the collection of each fraction is obtained by sampling eluent and carrying out thin-layer chromatography detection to obtain Rf values of all components, combining the components with the same Rf values, and respectively carrying out rotary evaporation on the components.
The high-activity fraction E3-2-3 is further purified by HPLC preparative chromatography, and fractions qn-3-1 and qn-4 are separated and purified from the E3-2-3 fraction and are cis-methyl p-hydroxycinnamate and trans-methyl p-hydroxycinnamate respectively.
Preferably, in the HPLC preparative chromatography, two highly active fractions E3-2-3 and E13-2-1 were separated and purified using YMC-ODS semi-preparative columns (250 mm. times.10 mm specification), and the fractions were collected and combined according to the peak of the compound appearing on the detector. Of these, qn-3-1(tR ═ 17.7min) and qn-4(tR ═ 19.5min) were separated in the second and third fractions, respectively, in the preparative separations of the E3-2-3 fraction, while only these two compounds were separated in the E13-2-1 fraction, after 31.0min and after 36.5min, respectively, and the purity of the compounds was high. However, due to the different mobile phases (47% methanol-water (v/v) in the former and 35% methanol-water (v/v) in the latter), the time for separating the two compounds was different at the same flow rate (4 mL/min).
And finally, respectively combining the cis-form methyl p-hydroxycinnamate and the trans-form methyl p-hydroxycinnamate obtained in the step C.
The invention also protects the application of p-hydroxy methyl cinnamate as herbicide. Specifically, the hydroxy-or trans-methyl p-hydroxycinnamate can be used as an active ingredient to be directly processed into a preparation for weed control, or can be used as a lead compound to be further subjected to structural modification or modification to form a compound with higher herbicidal activity, and then the compound is applied to herbicides.
In view of the fact that the trans-or trans-methyl p-hydroxycinnamate has strong inhibitory activity on crops such as wheat and corn and is weak in selectivity, the trans-or trans-methyl p-hydroxycinnamate can be used in non-arable land (such as leisure land, field side, roadside and the like) and crop fields insensitive to the trans-or trans-methyl 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 herbicide is applied to wheat fields and corn fields, the herbicide can be applied by a soil treatment method before seedlings are sown, so that the herbicide can not only avoid the phytotoxicity to crops, but also achieve the weeding effect. 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 also protects a herbicide, which comprises methyl p-hydroxycinnamate as an active ingredient, wherein the application concentration of the methyl p-hydroxycinnamate is 0.025-0.4 g.L < -1 >; the methyl p-hydroxycinnamate is at least one of cis methyl p-hydroxycinnamate and trans methyl p-hydroxycinnamate.
The invention has the following beneficial effects:
1. methyl p-hydroxycinnamate is separated from 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 methyl p-hydroxycinnamate can be used for preventing and removing weeds. The two components are natural products in plants, so that the pesticide composition has the characteristics of easy degradation in the environment and environmental friendliness, and is very in line with the development direction of pesticides.
2. The extraction method of the p-hydroxy methyl cinnamate has high purity of the separation product. The extraction method provided by the invention can effectively separate the methyl 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 Bacopa monniera and barnyard grass seedlings by methyl p-hydroxycinnamate and pendimethalin; wherein the left test object is amaranthus retroflexus, the right test object is barnyard grass, wherein II is cis-methyl p-hydroxycinnamate, III is trans-methyl 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%), 71.7% -90.7% (2.3% -65.7%) and 58.1% -86.7% (23.9% -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 a certain growth stimulating activity to the growth of the coleoptile at low concentration, and the four solvent extracts show different degrees of inhibitory activity to the growth of the seed root or the coleoptile. 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 activities of the n-butanol and water phase extracts were relatively low, and showed some inhibitory activity on the growth of radicles in the range of the concentrations tested, but showed some inhibitory activity on the growth of hypocotyls only in the high concentration treatment, and showed slight growth stimulation in the low concentration treatmentAnd (4) activity.
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 methyl 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. And (3) selecting two fractions E3 and E13 for further separation and purification by comprehensively considering the yield and the biological activity of each component.
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.
3.2 results of two-stage column chromatography of E3 and E13 fractions and determination of their Activity
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. As can be seen, the E3-2 fraction showed the highest inhibitory activity on the seedling growth of two recipient plantsThe inhibition rates for wheat seed root (radicle) and lettuce radicle (hypocotyl) were 95.0% (74.5%) and 100% (100%), respectively, the E3-3 fractions were inferior, 77.5% (45.8%) and 77.1% (57.7%), respectively, the third E3-4 fraction was 36.8% (14.9%) and 51.9% (31.1%), respectively, and the E3-1 fraction was the lowest, having 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.
5 fractions of E13-1, E13-2, E13-3, E13-4 and E13-5 are separated from the E13 fraction by two-stage column chromatography with petroleum ether/acetone (10:1, 5:1, 3:1, 2:1 and 0:1v/v) and acetone/methanol (10:1, 5:1, 3:1, 1:1 and 0:1v/v) as eluents at different ratios, wherein the yields of the fractions are respectively 25.7%, 16.5%, 14.0%, 13.2% and 30.6%. They are in the range of 0.3 g.L-1The effect on lettuce and wheat seedling growth at concentration is shown in table 3. As can be seen, the two fractions E13-1 and E13-2 both significantly inhibit the growth of seedlings of two kinds of recipient plants, and the inhibition rates of radicles (hypocotyls) and seed roots (coleoptiles) of the two kinds of recipient plants respectively reach 98.8% -100% (81.6% -84.8) and 87.0% -95.6% (78.7% -87.2%). The two fractions E13-3 and E13-4 showed relatively low inhibitory effect on the hypocotyls (coleoptiles) of 68.2-85.1% (62.0-76.7%) of the embryo roots (seed roots) but only 16.2-28.5% (31.7-49.6%) of the embryo roots (seed roots). The E13-5 fraction is less active and only acts on the roots of wheat seedsShows 52.2 percent higher inhibitory activity. The yield and the biological activity of each component are comprehensively considered, and two fractions E13-1 and E13-2 are selected for further separation. Wherein the E13-1 fraction is used directly for preparative chromatography.
TABLE 30.3 g.L-1Effect of E13 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.
3.3 results and Activity of E3-2 and E13-2 fractions on three-stage column chromatography
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 4. 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 40.05 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.
Oil in different proportionsThree fractions E13-2-1, E13-2-2 and E13-2-3 were separated from the E13-2 fraction by three-stage column chromatography using ether/acetone solutions (3:1, 2:1 and 0:1v/v) and acetone/methanol solutions (3:1, 1:1 and 0:1v/v) as eluents, and their yields were 31.9%, 42.7% and 25.4%, respectively. The effect of the four fractions on lettuce and wheat seedling growth is shown in table 5. It was found that the concentration was 0.05 g.L-1At the concentration, only the E13-2-1 fraction showed higher inhibitory activity, the growth inhibition rates of the radicle (hypocotyl) and the radicle (coleoptile) of the two were respectively 71.8% (32.8%) and 83.5% (72.7%), and the other three fractions showed lower activity except that the E13-2-3 fraction showed higher inhibitory activity of 69.3% to the lettuce radicle. Thus, E13-2-1 was selected for further preparative chromatographic separation.
TABLE 50.05 Effect of E13-2 fractions at g.L-1 concentration on lettuce and wheat 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.
3.4 further isolation and structural characterization of the active substances in the E3-2-3, E13-2-1 fractions
Further separating two fractions of E3-2-3 and E13-2-1 by preparative chromatography, and separating and purifying 5 compounds, namely qn-1(13.1mg), qn-3-1(2.0mg), qn-4(50.0mg), qn-5-1(1.4mg) and qn-6(113.8mg), from E3-2-3 fraction (421 mg); 2 compounds qn-3-1(2.0mg) and qn-4(5.8mgin) were separated and purified from the E13-2-1 fraction (57.9 mg). As can be seen from the above, qn-3-1 and qn-4 are present in the E3-2-3 fraction and E13-2-1 fraction, respectively; finally, qn-3-1 and qn-4 in the two fractions are combined respectively.
The preparative chromatographic separation method comprises the following steps: separation and purification were performed by HPLC preparative chromatography using YMC-ODS semi-preparative columns (250 mm. times.10 mm). Wherein 5 compounds were separated from the E3-2-3 fraction, and the mobile phase was washed with 47% aqueous methanol (v/v) at a flow rate of 4mL/min to obtain five compounds 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 target compounds qn-3-1 and qn-4 were separated in the second and third fractions, respectively.
When qn-3-1 and qn-4 were separated from the E13-2-1 fraction, a 35% aqueous methanol solution (v/v) was used as a mobile phase, a flow rate of 4mL/min was used, and elution was carried out for two compounds, qn-3-1(tR ═ 31.0min) and qn-4(tR ═ 36.5min), in this order.
The compounds are 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 structures of the compounds are further analyzed and identified, 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 methyl 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 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) are respectively arranged from left to right:
examples herbicidal Activity assay of methyl Bip-Hydroxycinnamate
To further determine the herbicidal activity of the extracted compounds, the effect of cis-methyl p-hydroxycinnamate, trans-methyl p-hydroxycinnamate on the growth of lettuce, wheat, redroot amaranth and barnyard grass seedlings, respectively, was determined.
1. Herbicidal activity of cis-methyl p-hydroxycinnamate
The results are shown in table 6, the medicament can obviously inhibit the growth of seedlings of lettuce, amaranthus retroflexus, wheat and barnyard grass under different concentrations of treatment, and the inhibition rate is improved along with the increase of the treatment concentration. Wherein, the concentration is 0.025 g.L-1～0.8g·L-1For lettuce, 0.0125 g.L under the concentration-1～0.2g·L-1The inhibition rates of the growth of the amaranthus retroflexus radicle (hypocotyl) under the concentration are respectively 58.5-100 percent (33.0-100 percent) and 74.4-100 percent (50.4-100 percent) and are respectively 0.025-0.8 g.L-1Concentration ofThe inhibition rates of the growth of wheat and barnyard grass seed roots (coleoptile) respectively reach 19.8-100% (11.4-78.3%) and 86.7-100% (45.4-80.9%), which shows that the extract has stronger inhibition activity on amaranthus retroflexus and barnyard grass weeds and two crops of wheat and lettuce, and the selectivity is poor.
TABLE 6 Effect of cis-methyl p-hydroxycinnamate on seedling growth of 4 test plants
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. Herbicidal activity of trans-methyl p-hydroxycinnamate
The effect of trans-methyl p-hydroxycinnamate on wheat, lettuce, redroot amaranth and barnyard grass is shown in table 7. As can be seen from the table, the trans-methyl p-hydroxycinnamate obviously inhibited the growth of seedlings of 4 test plants under different concentrations of treatment, and the effect is similar to that of cis-p-hydroxycinnamic acid, wherein the inhibition activity of the trans-methyl p-hydroxycinnamate on the seed root/radicle of the test plant is obviously higher than that of the coleoptile/hypocotyl at 0.025 g.L-1The inhibition rates for radicles of lettuce and redroot amaranth and for seed roots of wheat and barnyard grass were 60.5%, 82%, 17.25% and 56.2%, respectively, and the inhibition rates for hypocotyls of lettuce and redroot amaranth and for coleoptiles of wheat and barnyard grass were 35.9%, 55.3%, 4.92% and 24.9%, respectively. Overall, the inhibition of dicotyledonous plants is better than that of monocotyledonous plants, e.g. at 0.2 g.L-1The inhibition rates of radicles/hypocotyls of lettuce and Amaranthus retroflexus were 100% and all seedlings died, but the inhibition rates of the seed roots/coleoptiles of wheat and barnyard grass were relatively low, 92.7%/75.8% and 98.0%/73.6%, respectively.
TABLE 7 Effect of methyl trans-para-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.
3. Comparative test with conventional herbicides
To further verify the herbicidal activity against methyl hydroxycinnamate, a comparative test was conducted with pendimethalin, a common herbicide, and the results are shown in fig. 6 and table 8. It was found that the concentration was 0.05 g.L-1At the concentration, two methyl p-hydroxycinnamate pairs the acceptor plant Amaranthus retroflexus and 0.1 g.L-1Has obvious inhibiting effect on the growth of barnyard grass seedlings under the concentration. The inhibition activity (96.3-97.0%) of trans-methyl p-hydroxycinnamate and cis-methyl p-hydroxycinnamate on the growth of the radicle of the amaranthus retroflexus is higher than that of a contrast medicament pendimethalin (92.0%); the inhibitory activity of the two on the hypocotyl of the amaranthus retroflexus (67-74.3%)) is also higher than that of a control medicament pendimethalin (65.8%); the inhibition of barnyard grass seed roots is not obviously different from that of a control (98.9-100%); these results indicate that the two isolated compounds are higher than the control drug pendimethalin.
TABLE 8 comparison of the herbicidal Activity of methyl 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 (7)
1. A method for extracting methyl 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; 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 fractions E3 and E13;
fraction E13 was subjected to secondary column chromatography: sequentially carrying out gradient elution by using petroleum ether/acetone mixed solution and acetone/methanol mixed solution in the volume ratio of 10:1, 5:1, 3:1, 2:1 and 0:1 as eluent, merging components with the same Rf value, respectively carrying out rotary evaporation to remove the solvent, separating from E13 fraction according to the elution sequence to obtain 5 fractions including E13-1, E13-2, E13-3, E13-4 and E13-5, and collecting high-activity E13-2 fraction;
fraction E13-2 was subjected to three-stage column chromatography: sequentially taking petroleum ether/acetone solutions with volume ratios of 3:1, 2:1 and 0:1 and acetone/methanol solution mixed solutions with volume ratios of 3:1, 1:1 and 0:1 as mobile phases for gradient elution, carrying out three-stage column chromatographic separation, merging components with the same Rf values, respectively carrying out rotary evaporation to remove a solvent, separating from an E13-2 fraction according to an elution sequence to obtain three fractions of E13-2-1, E13-2-2 and E13-2-3, and collecting a high-activity E13-2-1 fraction;
and (3) purification: further purifying the E13-2-1 fraction by HPLC preparative chromatography to obtain 2 compounds qn-3-1, namely cis-methyl p-hydroxycinnamate; qn-4, namely trans-methyl p-hydroxycinnamate.
2. The method for extracting methyl p-hydroxycinnamate from corn stover according to claim 1, wherein the step C further comprises:
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 on fraction E3, merging separated components with the same Rf value, removing the solvent through rotary evaporation, respectively obtaining 4 fractions E3-1, E3-2, E3-3 and E3-4 according to the elution sequence, and collecting 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 0:1v/v as mobile phases, separating by three-stage column chromatography, and separating from an E3-2 fraction to obtain four fractions, namely E3-2-1, E3-2-2, E3-2-3 and E3-2-4;
further separating E3-2-3 by HPLC preparative chromatography, and separating and purifying fractions qn-3-1 and qn-4 from E3-2-3 fraction, which are cis-methyl p-hydroxycinnamate and trans-methyl p-hydroxycinnamate respectively;
and finally, respectively combining the cis-form methyl p-hydroxycinnamate and the trans-form methyl p-hydroxycinnamate obtained in the step C.
3. The method for extracting methyl p-hydroxycinnamate from corn stalk as claimed in claim 1, wherein 200-300 mesh silica gel is used in step C, and the size of the chromatographic column used in the third-time column chromatography is 12cm x 80 cm.
4. The method for extracting methyl p-hydroxycinnamate from corn stover according to claim 1, wherein YMC-ODS semi-preparative column with a size of 250mm x 10mm is used for separation and purification by HPLC preparative chromatography in step C.
5. The method for extracting the methyl p-hydroxycinnamate from the corn stalks according to the claim 1, wherein the method for separating qn-3-1, qn-4 from the E13-2-1 fraction by preparative chromatography is specifically as follows: the mobile phase was eluted with 35% by volume of methanol in water at a flow rate of 4mL/min, a retention time tR of qn-3-1 of 31.0min and a retention time tR of qn-4 of 36.5 min.
6. The method for extracting the methyl p-hydroxycinnamate from the corn stalks according to the claim 2, wherein the method for separating qn-3-1, qn-4 from the E3-2-3 fraction by preparative chromatography is specifically as follows: the mobile phase was eluted with 47% by volume methanol-water at a flow rate of 4mL/min, a retention time tR of qn-3-1 of 17.7min and a retention time tR of qn-4 of 19.5 min.
7. The use of methyl p-hydroxycinnamate as claimed in claim 1 as a herbicide in lettuce, wheat, redroot amaranth and barnyard grass.
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