CN109867650B - Preparation method for extracting allelochemical Salcolin A from highland barley straws - Google Patents
Preparation method for extracting allelochemical Salcolin A from highland barley straws Download PDFInfo
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Abstract
The invention discloses a preparation method for extracting allelochemical Salcolin A from highland barley straws. Comprises the steps of material crushing, pure water ultrasonic extraction, enrichment and sugar removal, HPLC preparation and recrystallization. The pure water ultrasonic extraction method is adopted, so that the toxicity and pollution caused by the extraction of an organic solvent are avoided; the enriching and desugaring process adopts D101 macroporous resin on the extracted concentrated solution, firstly uses water to elute, then uses methanol to elute, and collects alcohol eluent to treat. The treatment can effectively remove impurities such as sugar macromolecules in the extract, improve separation efficiency and reduce raw material investment. The HPLC preparation can shorten the great separation time, has strong repeatability and reduces the investment of manpower and material resources. The Salcolin A obtained by the preparation method provided by the invention has the purity of over 95 percent, is simple to operate, and can greatly save the use amount of toxic solvents, labor and time.
Description
Technical Field
The invention belongs to the technical field of plant allelopathy active substances, and particularly relates to a preparation method for extracting allelopathy substance Salcolin A from highland barley straws.
Background
Farmland weeds are one of the important problems in agricultural production, compete with crops for resources such as light, water and fertilizer, become important factors influencing crop yield and quality, and cause huge loss to agricultural production. The harm of weeds is mainly controlled by adopting a chemical weeding method for a long time, the use of the herbicide not only pollutes the environment, but also the weeds are easy to generate drug resistance, and the adverse effects of food safety, public health, natural environment, weed drug resistance and the like are increasingly attracted by people. Allelochemical (Allelochemical) refers to an ecological phenomenon in which plants release specific chemical substances into the environment to affect the growth and development of other surrounding plants. By utilizing allelochemicals among plants, allelochemicals can be released into the environment and compete with weeds for living environment, so that the inhibition effect on the germination and growth of weed seeds is generated, and the control of weeds by utilizing allelochemicals does not bring about environmental problems such as pesticide residues and the like. In order to protect the environment for human survival and the sustainable development of agriculture, the development and use of chemical herbicides are strictly limited by the environment and ecology. The natural product herbicide has been highlighted by people, and phytochemicals are an important way for finding novel biological source herbicide lead compounds.
Highland barley (hull ssbarley) is a characteristic crop of Tibet plateau, a cereal crop of the genus barley of the family Gramineae, and is also called naked barley because of separation of inner and outer glumes and naked grains. The applicant firstly reports the allelopathy of the highland barley, the main allelopathy substance of the highland barley, the expression form of the allelopathy of the highland barley and the like at home and abroad through a large amount of research works in earlier period, simultaneously proves that Salcolin A (tricin4' -O- (thio-beta-guaiacylglycerol) ether) is the main highland barley grass-inhibiting allelopathy substance, and through a biological activity test, when the tested concentration is 100 mu g/mL, the growth sensation index of the highland barley roots of a receptor is 0.63. In addition, Salcolin A has inhibitory effect on growth of Microcystis aeruginosa with half inhibitory concentration (5d) of 6.02 × 10 - 5 mol l -1 . Salcolin A has novel structure, more isomers and various biological activities, and has wide prospect in the fields of scientific research and agricultural application. However, the chemical structure is complex, the chiral carbon is much, the chemical synthesis is difficult, and the source of the compound still depends on the separation from plants at present.
At present, the extraction and purification method of Salcolin A is very few, and the extraction and purification method taking highland barley as a raw material source is basically blank. The existing method is only micro extraction and separation in a laboratory, has no standard operation flow, and has the yield of more than ten milligrams without guarantee. The specific method comprises the following steps: crushing the materials, reflux extraction with 95% industrial methanol, concentration of the extract, extraction with organic solvent (or reflux extraction with organic solvent), silica gel column chromatography (normal phase column and reverse phase column), HPLC preparation and recrystallization. In the organic solvent extraction (or organic solvent reflux extraction) process, toxic organic solvents such as petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol and the like are usually used for extraction or extraction, in the silica gel column chromatography process, the wet column method is most commonly used for separation, namely, silica gel powder soaked by the organic solvent is used for filling a chromatography column, a sample is added to the upper part of the filled chromatography column, elution is carried out by using different organic solvents, eluent is collected in parts, the chromatography is repeated in such a way, and finally, HPLC preparation is carried out, and recrystallization is carried out until a pure product is obtained. The whole process is complicated and time-consuming, the contact time of preparation personnel and toxic solvents is long, the health of the preparation personnel is seriously damaged, a large amount of toxic and harmful organic solvents are generated, and the environmental hazard is serious.
The D101 macroporous adsorption resin is a synthetic polymer adsorbent with a porous spongy structure, and the purpose of separating and extracting organic substances with poor water solubility from an aqueous solution is achieved by physical adsorption through the huge specific surface area of the resin by virtue of van der Waals force between a resin framework and adsorbed molecules. The D101 resin is a non-polar adsorbent, is generally used for extracting and separating saponin substances in Chinese herbal medicines, and has the advantages of simple and convenient operation, low cost, repeated use of the resin and the like.
Disclosure of Invention
The invention aims to provide a more simple, convenient, efficient and environment-friendly preparation method for extracting and purifying Salcolin A (tricin4' -O- (threo-beta-guaiacylglycerol) ether) from highland barley straws, so as to solve the problems of low efficiency, time and material waste, long contact time between preparation personnel and toxic organic solvents, serious damage to the body health of the preparation personnel, environmental pollution and the like of the current method.
The invention is realized by the following technical scheme:
a preparation method for extracting allelochemicals Salcolin A from highland barley straws comprises the following steps:
1) crushing the material, namely removing water from fresh highland barley straws and crushing the highland barley straws into powder;
2) ultrasonic extracting with pure water, dissolving the powder of semen Avenae Nudae stalk in pure water, and ultrasonic filtering to obtain extractive solution;
3) enriching and desugarizing, subjecting the extractive solution to D101 macroporous adsorbent resin chromatography adsorption, sequentially eluting with water and methanol, collecting methanol eluate, and concentrating under reduced pressure to obtain paste extract;
4) HPLC preparation
Gradient elution, wherein the paste extract is fully dissolved in water and a small amount of methanol for sample loading, eluent is collected, and detection is carried out through thin layer chromatography, and components with the same Rf value as that of a Salcolin A standard product are obtained, so as to obtain crude components;
isocratic elution, fully dissolving the crude component, loading the sample, displaying 4 main chromatographic peaks in a chromatogram, collecting the component of the 4 th chromatographic peak within 18-24min of retention time, and comparing the component with the Rf value of a Salcolin A standard substance in silica gel thin layer chromatography to obtain a component with higher Salcolin A content;
fine purification, fully dissolving the components with higher Salcolin A content, loading, and mixing with a mobile phase: eluting with 70: 30(v/v) of A and B, and collecting the components with the maximum chromatographic peak after the retention time is longer than 20min to obtain high-purity Salcolin A eluate;
5) recrystallizing, concentrating the eluent at the same Rf value as Salcolin A standard by thin layer chromatography under reduced pressure, dissolving with methanol, standing the filtrate in a refrigerator at 4 deg.C, and filtering to obtain pure Salcolin A.
Further, the mass volume ratio of the highland barley straw powder to the pure water in the step (2) is 1: 2-5.
Further, the volume ratio of the extracting solution in the step (3) to the D101 macroporous adsorption resin is 2-5: 1.
Further, the gradient elution conditions in step (4) are as follows: phase A: h 2 O (0.1% TFA); phase B: acetonitrile; the flow rate is 25ml/min, and the chromatographic column is a C-18 preparative column (21.2mm multiplied by 150mm multiplied by 5 um); the ultraviolet detector has a wavelength of 254nm, the initial proportion of mobile phase B is 10%, the proportion of mobile phase B is increased to 30% in 0-10min, and the proportion of mobile phase B is increased to 30% in 10-10.2minThe sample is raised to 90% and kept for 3.3min, then the proportion of mobile phase B is reduced to 10% within 2min, the initial proportion is recovered, and the base line is kept stable.
Further, the isocratic elution conditions in the step (4) are as follows: phase A is H 2 O, 0.1% TFA; phase B: acetonitrile; the chromatographic column selects a C-18 preparation column (21.2mm multiplied by 150mm multiplied by 5 um); the ultraviolet detector is 254nm, the mobile phase A: B is 50: 50(v/v), the flow rate is 25mL/min, and the monitoring time is 30-35 min.
Further, the fine purification and de-conditioning of the step (4) are as follows: phase A is H 2 O, 0.1% TFA; phase B: acetonitrile; chromatographic column C-18 preparation column (21.2mm × 150mm × 5um) is selected; UV detector 254nm, mobile phase A: B70: 30 (v/v).
The invention has the beneficial effects that:
(1) firstly, according to the characteristic that a target substance Salcolin A (tricin4' -O- (threo-beta-guaiacylglycerol) ether) belongs to a chemosensory substance and can be obtained by water extraction, the method adopts a pure water ultrasonic extraction method in the extraction, and greatly avoids the problems of time consumption, expenditure, complex flow, long-time contact between preparation personnel and a toxic solvent, environmental pollution and the like caused by the extraction by adopting an organic solvent.
(2) Secondly, in the process of desugaring and enriching the extract, the volume ratio of the extract to the D101 macroporous adsorption resin is 2-5:1, the extract is subjected to chromatographic adsorption by the D101 macroporous adsorption resin, and is eluted by 2-4 column volumes of water and then 3-5 column volumes of methanol, the methanol eluent is collected and is concentrated under reduced pressure to be pasty to obtain the pasty extract. The treatment can effectively remove impurities such as sugar in the extract, enrich organic components such as Salcolin A (tricin4'-O- (thio-beta-guaiacylglycerol) ether) with poor water solubility in the extract, improve the bearing capacity and separation efficiency of silica gel in the column chromatography process, effectively enrich target component Salcolin A (tricin4' -O- (thio-beta-guaiacylglycerol) ether), avoid the use of toxic organic solvent in the organic solvent extraction or organic solvent reflux extraction process, and greatly shorten the time of the process flow.
(3) Thirdly, the method separates the target compound by preparative or semi-preparative liquid chromatography, adopts Salcolin A (tricin4' -O- (thio-beta-guaiacylglycerol) ether) as a guide, adopts thin layer chromatography to track and detect, increases mechanical intelligent operation compared with the prior method, has strong repeatability, can standardize the process flow, reduces the use of organic solvent and obviously improves the separation efficiency.
(4) Finally, the inventor proves through multiple experiments that high-purity Salcolin A (tricin4' -O- (thio-beta-guaiacylgyceryl) ether) can be extracted and purified from highland barley young plants or highland barley straws by adopting the method disclosed by the invention, the purity (detected by HPLC) can reach more than 95%, and the method can be used for various biological activity researches and has the advantages of simple preparation method, small using amount of toxic organic solvent in the preparation process, short contact time between preparation personnel and the toxic solvent, strong repeatability, standardized operation of process flow, environment protection and the like.
Drawings
FIG. 1 is an isocratic elution chromatogram of crude fractions from HPLC preparations.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood 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.
The preparation method for extracting allelochemicals Salcolin A from highland barley straws comprises the following steps:
firstly, crushing materials: drying fresh highland barley straw in the sun or at 45-55 deg.C in a drying oven, and pulverizing into powder with a shearing section of 1-3cm or a plant pulverizer;
secondly, ultrasonic extraction of pure water: putting a certain amount of powder into a reflux flask, adding pure water according to the mass volume ratio of the sample to the extraction solvent of 1:2-5, shaking up, pressing the sample under the liquid level, placing in an ultrasonic cleaner, performing ultrasonic treatment for 15min every 2h, and filtering after 1-3d to obtain an extracting solution;
thirdly, enriching and removing sugar: according to the volume ratio of the extracting solution to the D101 macroporous adsorption resin of 2-5:1, carrying out chromatographic adsorption on the extracting solution by using the D101 macroporous adsorption resin, eluting by using 2-4 column volumes of water, then eluting by using 3-5 column volumes of methanol, collecting methanol eluent, and concentrating under reduced pressure to obtain a paste extract;
fourthly, HPLC preparation: prepared in three steps
(1) Gradient elution, and dissolving the paste extract with water and a little methanol. Selecting A phase and H phase as mobile phase for preparing liquid phase 2 O (0.1% TFA); phase B: acetonitrile; the flow rate is 25ml/min (according to the capacity of the preparation column), and the chromatographic column is a C-18 preparation column (21.2mm multiplied by 150mm multiplied by 5 um); and when the ultraviolet detector is 254nm, the initial proportion of the mobile phase B is 10%, the proportion of the mobile phase B is increased to 30% in 0-10min, the proportion of the mobile phase B is increased to 90% in 10-10.2min, the mobile phase B is kept for 3.3min, the proportion of the mobile phase B is reduced to 10% in 2min, the initial proportion is recovered, and the base line is kept stable. Collecting 25-35 mL/part of eluent, detecting by thin layer chromatography, detecting each part of eluent by thin layer chromatography according to Rf value of Salcolin A standard substance in silica gel thin layer chromatography, and mixing components at the same position as Rf value of Salcolin A standard substance to obtain crude extract.
(2) Isocratic elution, dissolving the coarse component, loading, and selecting phase A and phase H as mobile phase for preparing liquid phase 2 O (0.1% TFA); phase B: acetonitrile; the chromatographic column selects a C-18 preparation column (21.2mm multiplied by 150mm multiplied by 5 um); uv detector 254nm, mobile phase: and (2) when the ratio of A to B is 50: 50(v/v), the flow rate is 25mL/min, the monitoring time is 30-35min, the chromatogram shows 4 main chromatographic peaks, the retention time is 18-24min, the component of the 4 th chromatographic peak is collected, and the thin-layer chromatography detection is compared with the Rf value of the Salcolin A standard product in silica gel thin-layer chromatography, so that the component with higher Salcolin A content is obtained.
(3) And (3) fine purification, namely fully dissolving the components with higher Salcolin A content, loading the components, and performing the following steps of (II) equipment conditions, wherein the mobile phase: eluting with A and B at 70: 30(v/v), and collecting the component with the largest chromatographic peak after the retention time is longer than 20min to obtain high-purity Salcolin A eluate.
Fifthly, recrystallization: and (3) performing thin-layer chromatography detection, concentrating the combined eluent at the same position of Rf value of the Salcolin A standard product under reduced pressure, fully dissolving the eluent by using methanol, filtering, standing the filtrate in a refrigerator at 4 ℃, and filtering out crystals after the crystals are separated out to obtain the pure Salcolin A (tricin4' -O- (threo-beta-guaiaacylglycerol) ether).
Wherein, the highland barley in the step (one) can be any one of highland barley green (green) seedlings or highland barley fresh straws.
If the eluent of high-purity Salcolin A cannot be obtained in the step (IV), fine purification can be carried out once by using the method (IV) (3).
Example 1
Taking 5Kg of fresh highland barley straws which are subjected to enzyme deactivation and airing for 2d, respectively placing the fresh highland barley straws into a 2L wide-mouth bottle at the position of about 3cm in a shearing section, adding 500mL of distilled water, pressing the straws below the leaf surface, sealing the opening with a sealing film, reserving a plurality of small ventilation holes, placing the straws into an ultrasonic cleaner, performing ultrasonic treatment for 4 times every 2 hours for 15min, and performing suction filtration by using double-layer filter paper after 3d to obtain an extracting solution, wherein each bottle contains about 380 plus 400mL of water. Taking 400mL of extract each time, passing through a chromatographic column (the column diameter is 7cm, the length is 100cm) filled with 1500g D101 macroporous resin, loading the sample at the flow rate of about 10mL/min, eluting with 2 column volumes of water, eluting with 4 column volumes of methanol, collecting methanol eluates, combining the eluates for 3 times, concentrating at 45 ℃ under reduced pressure to obtain paste, and obtaining 145g of paste in total. Liquid chromatography shimadzu LC-20AT, column: phenomenex LUNA C-18(21.2 mm. times.150 mm. times.5 um), flow rate: 25ml/min, mobile phase selected from phase A and phase H 2 O (0.1% TFA), phase B: acetonitrile; and when the ultraviolet detector is 254nm, the initial proportion of the mobile phase B is 10%, the proportion of the mobile phase B is increased to 30% in 0-10min, the proportion of the mobile phase B is increased to 90% in 10-10.2min, the mobile phase B is kept for 3.3min, the proportion of the mobile phase B is reduced to 10% in 2min, the initial proportion is recovered, and the baseline is kept stable (figure 1). Collecting 25 mL/part of eluate, collecting 12 fractions, numbering 1-12, detecting by thin layer chromatography, and detecting R in silica gel thin layer chromatography according to SalcolinA standard f Performing thin layer chromatography detection on each part of eluent, and mixing with SalcolinA standard substance R f And (4) taking the component with the same value and the component No. 7 as a crude component, and performing rotary evaporation to obtain 7.3g of extract. Fully dissolving the coarse components, loading, and selecting the mobile phase of the prepared liquid phasePhase A is selected from H 2 O (0.1% TFA); phase B: acetonitrile; the chromatographic column selects a C-18 preparation column (21.2mm multiplied by 150mm multiplied by 5 um); uv detector 254nm, mobile phase: a and B are 50: 50(v/v), the flow rate is 25mL/min, the monitoring time is 35min, 4 main chromatographic peaks are totally obtained, components of a 4 th chromatographic peak with the retention time of 18.1-18.6min are collected, and R measured in silica gel thin layer chromatography is detected by thin layer chromatography and SalcolinA standard substance f Comparing the values to obtain a component with higher SalcolinA content, and performing rotary evaporation to obtain 138mg of extract. Fine purification, fully dissolving the components with higher SalcolinA content, loading, keeping the conditions of the instrument unchanged, and taking the mobile phase: eluting with A and B at 70: 30(v/v), and collecting the components with maximum chromatographic peak retention time of 21.5-21.9min to obtain high-purity SalcolinA eluate. And (2) performing thin-layer chromatography detection, concentrating the combined eluent at the same position of Rf value of the Salcolin A standard product under reduced pressure, fully dissolving the eluent by using methanol, filtering, standing the filtrate in a refrigerator at 4 ℃, and filtering out crystals after the crystals are separated out, wherein the crystals are the pure Salcolin A (tricin4' -O- (threo-beta-guaiaacetylglucosyl) ether), weighing 108.7mg and detecting the purity by using liquid chromatography to be 96.57%.
The structural formula of the Salcolin A (tricin4' -O- (threo-beta-guaiacylgyceryl) ether) pure compound extracted from the highland barley straws is as follows:
example 2
Taking 5Kg of fresh highland barley straw, placing the fresh highland barley straw in a drying box, drying the highland barley straw at 45-55 ℃, crushing the highland barley straw into powder by a plant crusher, and weighing 3.2Kg of the highland barley straw. The extract is averagely divided into 10 parts, the 10 parts are placed in a 1000mL triangular flask, 600mL distilled water is added into each flask, the flask is fully soaked, a sealing film is used for sealing, a plurality of ventilation holes are reserved, the flask is placed in an ultrasonic cleaner for ultrasonic treatment for 15min every 2h for 4 times, and after 3 days, double-layer filter paper is used for suction filtration to obtain extract, wherein each flask is about 400 mL. Passing 400mL of extractive solution each time through a chromatographic column (column diameter 7cm, length 100cm) containing 1500g D101 macroporous resin at a flow rate of about 10mL/min, eluting with 2 column volumes of water, eluting with 4 column volumes of methanol, collecting methanolMixing the eluates, mixing 3 times of eluates, concentrating at 45 deg.C under reduced pressure to obtain paste, and collecting 181 g. Hanbang NS4000 liquid chromatography, column: phenomenex LUNA C-18(21.2 mm. times.150 mm. times.5 um), flow rate: 25ml/min, mobile phase selected from phase A and phase H 2 O (0.1% TFA), phase B: acetonitrile; and when the ultraviolet detector is 254nm, the initial proportion of the mobile phase B is 10%, the proportion of the mobile phase B is increased to 30% in 0-10min, the proportion of the mobile phase B is increased to 90% in 10-10.2min, the mobile phase B is kept for 3.3min, the proportion of the mobile phase B is reduced to 10% in 2min, the initial proportion is recovered, and the base line is kept stable. Collecting 25 mL/part of eluate, collecting and mixing 12 fractions, numbering 1-12, detecting by thin layer chromatography, and detecting R in silica gel thin layer chromatography according to SalcolinA standard f Performing thin layer chromatography detection on each part of eluent, and mixing with SalcolinA standard substance R f And (4) taking the component with the same value and the component No. 7 as a crude component, and performing rotary evaporation to obtain 8.9g of extract. Fully dissolving the coarse components, loading, and selecting phase A and phase H as mobile phase for preparing liquid phase 2 O (0.1% TFA); phase B: acetonitrile; the chromatographic column selects a C-18 preparation column (21.2mm multiplied by 150mm multiplied by 5 um); uv detector 254nm, mobile phase: a and B are 50: 50(v/v), the flow rate is 25mL/min, the monitoring time is 40min, 4 main chromatographic peaks are totally obtained, components of a 4 th chromatographic peak with the retention time of 18.7-19.3min are collected, and R measured in silica gel thin layer chromatography is detected by thin layer chromatography and SalcolinA standard substance f Comparing the values to obtain a component with higher SalcolinA content, and performing rotary evaporation to obtain an extract of 118 mg. Fine purification, fully dissolving the components with higher SalcolinA content, loading, keeping the conditions of the instrument unchanged, and taking the mobile phase: eluting with 70: 30(v/v) of A and B, collecting the components with maximum chromatographic peak with retention time of 22.5-23.1min, and obtaining high-purity SalcolinA eluate. And (2) performing thin-layer chromatography detection on the combined eluent at the point where the Rf value of the Salcolin A standard substance is the same, concentrating under reduced pressure, fully dissolving with methanol, filtering, standing the filtrate in a refrigerator at 4 ℃, after crystallization, filtering out crystals to obtain a pure Salcolin A (tricin4' -O- (threo-beta-guaiaacetylglucosyl) ether) product, weighing 88.4mg, and detecting the purity by liquid chromatography to be 97.05%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A preparation method for extracting allelochemicals Salcolin A from highland barley straws is characterized by comprising the following steps:
1) crushing the material, namely removing water from fresh highland barley straws and crushing the highland barley straws into powder;
2) ultrasonic extracting with pure water, dissolving the powder of semen Avenae Nudae stalk in pure water, and ultrasonic filtering to obtain extractive solution;
3) enriching and desugarizing, subjecting the extractive solution to D101 macroporous adsorbent resin chromatography adsorption, sequentially eluting with water and methanol, collecting methanol eluate, and concentrating under reduced pressure to obtain paste extract;
4) HPLC preparation
Gradient elution, dissolving the paste extract with water and a small amount of methanol, collecting the eluate, detecting by thin layer chromatography, and collecting the components appearing at the same point as the Rf value of Salcolin A standard product to obtain crude extract;
isocratic elution, fully dissolving the crude extract, loading, collecting the fraction with retention time of 18-24min, detecting by silica gel thin layer chromatography, and comparing with Salcolin A standard Rf value to obtain a fraction with high Salcolin A content;
fine purification, fully dissolving the components with higher Salcolin A content, loading, and performing mobile phase: eluting with 70: 30(v/v) A and B, and collecting the components with the maximum chromatographic peak after the retention time is more than 20min to obtain high-purity Salcolin A eluate;
5) recrystallizing, concentrating the high-purity Salcolin A eluate at the same Rf value as Salcolin A standard product by thin layer chromatography under reduced pressure, dissolving with methanol, standing the filtrate in a refrigerator at 4 deg.C, and filtering to obtain pure Salcolin A.
2. The preparation method of the allelochemical Salcolin A extracted from highland barley straw as claimed in claim 1, wherein the mass-to-volume ratio of highland barley straw powder to pure water in step (2) is (1: 2-5).
3. The method for preparing the allelochemicals Salcolin A extracted from highland barley straws as claimed in claim 1, wherein the volume ratio of the extract in step (3) to the D101 macroporous adsorbent resin is 2-5: 1.
4. The preparation method of the allelochemicals Salcolin A extracted from highland barley straw as claimed in claim 1, wherein the gradient elution condition in step (4) is as follows: phase A: h 2 O, 0.1% TFA; phase B: acetonitrile; the flow rate is 25ml/min, and the chromatographic column is a C-18 preparative column; and when the ultraviolet detector is 254nm, the initial proportion of the mobile phase B is 10%, the proportion of the mobile phase B is increased to 30% in 0-10min, the proportion of the mobile phase B is increased to 90% in 10-10.2min, the mobile phase B is kept for 3.3min, the proportion of the mobile phase B is reduced to 10% in 2min, the initial proportion is recovered, and the base line is kept stable.
5. The preparation method of the allelochemical Salcolin A extracted from highland barley straws as claimed in claim 1, wherein the isocratic elution conditions in step (4) are as follows: phase A is H 2 O, 0.1% TFA; phase B: acetonitrile; selecting a C-18 preparation column as a chromatographic column; and an ultraviolet detector with the wavelength of 254nm is used for eluting with mobile phase A: B of 50: 50(v/v), the flow rate is 25mL/min, and the monitoring time is 30-35 min.
6. The preparation method of the chemoattractant Salcolin A extracted from highland barley straw as claimed in claim 1, wherein the fine elution and purification conditions in step (4) are as follows: phase A is H 2 O, 0.1% TFA; phase B: acetonitrile; selecting a C-18 preparation column as a chromatographic column; UV detector 254nm, mobile phase A: B70: 30 (v/v).
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