CN114933632B - Method for preparing 5 kinds of ustilaginoidea virens toxins through simultaneous separation and purification - Google Patents
Method for preparing 5 kinds of ustilaginoidea virens toxins through simultaneous separation and purification Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing 5 kinds of ustilaginoidea virens toxins by simultaneous separation and purification. The invention takes the whole rice false ball produced by rice false bacteria or the rice false ball spore powder produced by rice false bacteria as raw materials, water as extraction solvent, fine resin for column chromatography separation, solid phase extraction technology as enrichment purification method, semi-preparation type high performance liquid chromatography for preparation and collection, liquid chromatography-mass spectrometer for tracking and identification, and simultaneously, 5 kinds of false bacteria toxin monomer compounds are prepared by separation and purification. The method has the advantages of simple operation, high speed and efficiency, good repeatability, safety and no pollution, and the purity of the obtained 5 kinds of the ustilaginoidea virens toxins can reach more than 90%, so that the method can be used as a standard sample. The invention solves the key bottleneck problem of limiting the deep research of rice Qu Jundu element at present, and can provide a foundation for the research and formulation of the limit standard of rice Qu Jundu element in rice, wherein the limit standard of rice Qu Jundu element is related to the influence of rice on human health, the safety risk monitoring of rice quality and exposure evaluation.
Description
Technical Field
The invention relates to a method for preparing ustilaginoidea virens toxin by separation and purification, in particular to a technical method for preparing 5 ustilaginoidea toxins by simultaneous separation and purification.
Background
The ustilaginoidea virens toxin (Ustiloxins) is a mycotoxin produced by ustilaginoidea virens Ustilaginodea virens (Cooke) Takahashi and is a series of heterocyclic peptide compounds, and 7 ustilaginoidea virens toxins with similar structures, mainly comprising rice Qu Jundu elements Ustiloxins A, ustiloxins B, ustiloxins C, ustiloxins D, ustiloxins E, ustiloxins F and Ustiloxins G, have been separated at present. Since the content of ustilaginoidea virens Ustiloxins E and Ustiloxins G is very small, the researches on the ustilaginoidea virens mainly aim at Ustiloxins A, ustiloxins B, ustiloxins C, ustiloxins D and Ustiloxins F ustilaginoidea virens. False smut caused by false smut has been a major disease in rice production since the 80 s, and has been widely distributed throughout the world in regions of rice cultivation, with more serious countries including japan, china, united states, india, philippines, etc. The occurrence area of false smut in China is about 300 ten thousand hm 2, and the false smut presents a continuously aggravated trend, so that not only is the yield of rice in China seriously affected, but also the false smut becomes one of main influencing factors of rice quality safety, and the panic of vast growers is also caused due to the limitation of people on research and cognition of rice Qu Jundu at present.
With the increasing incidence of false smut in recent years, research on rice Qu Jundu is attracting more and more attention from students, and most of the research is focused on the fields of disciplines such as plant protection, crop cultivation, environmental science and genetic breeding. At present, the toxicity of the curculin to plants is widely accepted, and the curculin can inhibit the mitosis of plant cells so as to cause abnormal swelling of roots and germs of rice seedlings, and the main mechanism is that the curculin inhibits the polymerization of cell microtubulin, and interferes with the formation of cytoskeleton so as to cause the arrest of the mitosis process of the cells. However, the effect of the curculin on the human health is very little known, and only few reports on the early stage are made, and whether the curculin is ground into powder and mixed into feed for feeding animals (chickens, rabbits and rats) or the curculin water extract is injected intraperitoneally, even if the content is very low, the curculin water extract can cause chronic poisoning and visceral lesions of the animals, wherein bleeding points and nodules appear on the surfaces of hearts and lungs, severe necrosis appear on livers and kidneys, ulcers and severe corrosion appear on the stomach, even genetic toxicity exists, and the development of offspring is influenced. The researches mainly adopt whole rice false brims or crude extract of rice false brims, which is a very complex sample system, removes rice Qu Jundu element and a large amount of impurities, and the related documents have long years, cannot systematically reflect the toxicity of the rice false brims, and individual researches only relate to single compounds of the rice false brims A, so that the research on the influence of Guan Daoqu bacterial toxins on human health has not been greatly broken through and progressed.
"People take food as the day and take food as the first place. "food safety" is a matter of physical health and life safety for the masses of people. In recent years, the application of new technology and new method also brings serious challenges to food safety. With the popularization and application of new rice varieties, the cultivation mode, the fertilization dosage, the climate environment and other factors, false smut becomes a main disease in rice production and is increasingly serious, and the quality safety of rice is directly influenced while the yield of rice is influenced. The annual output of rice which is taken as the first grain production and consumption country in the world accounts for about 50 percent of the total output of grains in the country, and is the staple food of people in most areas in the country, and the quality and quality safety of the rice have great influence on the nutritional requirements and the physical health of consumers. However, the pollution condition of the rice sold on the market and the related products of the rice are not known. The incorporation of rice Qu Jundu into rice and product quality safety indicators has been delayed, and the pollution problem of ustilaginoidea virens in rice has been increasingly focused by the national government departments. The national agricultural rural department in 2020 recently goes out of the standards of the quality safety detection control method related to the false smut toxin, but the actual application of the method standard is greatly limited due to the lack of commercial supply of the standard sample of the false smut toxin, and a great number of rice and products cannot analyze and control the quality of the false smut toxin, so that the false smut toxin detection method has great potential safety hazard.
Whether the research progress of the rice Qu Jundu on the human health is slow or the practical application of the related quality safety detection control method standard of the ustilaginoidea virens toxin newly exported in China is limited, the root cause is mainly that the rice Qu Jundu element monomer compound is difficult to obtain. The branched chains of the ustilaginoidea virens toxin both contain carboxyl and amino groups, have the characteristics of high hydrophilicity and amphiprotic property, are used as an amphiprotic compound and are easy to ionize in a wider PH range, and have great difficulty by adopting a traditional purification method, so that the rice Qu Jundu element monomer compounds are very difficult to obtain. Referring to related documents, the related documents are mainly characterized in that a doctor of the university of tokyo in the early year Kobayashi and a teacher of the academy of agricultural sciences in Jiangsu province carry out the research of separating and purifying preparation of rice Qu Jundu, and the method of the doctor of Kobayashi mainly adopts the combination of reverse phase silica gel, resin and normal phase silica gel, and repeated purification is carried out for many times, so that the process is very complicated; the research of the separation and purification preparation of the rice Qu Jundu element by the sacrificial teacher only aims at single rice Qu Jundu element Ustiloxin A, and the separation and purification preparation methods of the rice Qu Jundu element A and the sacrificial teacher both use dichloromethane organic solvents, so that the pollution to the environment and the health of operators are greatly damaged. In addition, from the aspect of the synthesis angle, the synthetic process steps of the ustilaginoidea virens toxin are twenty or more steps due to the characteristic of a closed loop structure, and the yield is very low. There are various reasons for limitation, and various monomeric compounds of ustilaginoidea are not currently commercially available. The obtaining of each monomer compound of high-purity rice Qu Jundu is a precondition and basis for research and application of rice Qu Jundu, and is also the biggest bottleneck problem for restricting the deep research and application of rice Qu Jundu.
Disclosure of Invention
The invention aims at: aiming at the problems that no marketized rice Qu Jundu element monomer compound is supplied at present, the deep research of the ustilaginoidea virens toxin and the development of the risk control work of the quality safety of rice are limited, and the like, the invention provides a technical method for preparing 5 ustilaginoidea virens toxin by simultaneous separation and purification, which breaks through the key bottleneck problem of the current deep research of the ustilaginoidea virens toxin, and can provide necessary premise and foundation for the research and formulation of the human health influence, the rice quality safety risk monitoring, the exposure evaluation and the Qu Jundu element limit standard of rice in the future by Guan Daoqu element.
The specific technical scheme adopted by the invention is as follows:
A method for preparing 5 kinds of ustilaginoidea virens toxins through simultaneous separation and purification comprises the following steps:
s1, taking whole rice false smut balls or rice false smut ball spore powder as a raw material, taking water as an extraction solution to carry out homogenate crushing, vibration extraction and centrifugal separation on the raw material, and carrying out vacuum freeze drying on the obtained supernatant to obtain a crude extract of rice Qu Jundu;
s2, filling a fine resin HP20SS serving as a column chromatography filler into an organic medium pressure resistant chromatography column according to a wet column packing mode, mounting the organic medium pressure resistant chromatography column at a chromatographic column position of a high performance liquid chromatograph, and adopting the high performance liquid chromatograph as an infusion system to press the chromatography column;
s3, dissolving the crude extract with water, centrifuging, filtering the obtained supernatant to remove impurities by a membrane, and separating by column chromatography on a chromatographic column;
s4, performing elution of the ustilaginoidea virens toxin target compound by using a high performance liquid chromatograph as an infusion system, wherein an elution solution is a methanol-water system, the elution mode is gradient elution, the proportion of a mobile phase in the gradient elution is gradually increased from 5% to 25%, and the eluent is collected by using a full-automatic part collector tube by tube;
S5, tracking and identifying types of rice Qu Jundu in the collected eluates, merging eluates belonging to the same class of ustilaginous toxins, and respectively carrying out vacuum centrifugal concentration on the 5 eluates after merging to obtain column chromatography separation extracts of Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 ustiloxins;
S6, carrying out enrichment purification on column chromatography separation extracts of the 5 kinds of ustilaginoxin by a solid phase extraction technology, and then further purifying to finally obtain purified substances of the 5 kinds of Ustiloxin A, the 5 kinds of Ustiloxin B, the 5 kinds of Ustiloxin C, the 5 kinds of Ustiloxin D and the 5 kinds of Ustiloxin F.
Preferably, in the step S6, the enrichment and purification are performed by: and respectively carrying out solid phase extraction on column chromatography separation extracts of the 5 kinds of the Ustiloxin by a solid phase extraction column to obtain enrichment purified matters of the Ustiloxin A, the Ustiloxin B, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F5 kinds of the Ustiloxin.
Preferably, in S6, the purification is performed by: the enriched and purified matters of the 5 kinds of ustilaginous toxins obtained through enrichment and purification are further separated and prepared through preparative or semi-preparative high performance liquid chromatography respectively, a liquid chromatography fraction collector is used for automatically collecting the enriched and purified matters according to the retention time setting of target compounds, and the collected matters are subjected to vacuum freeze drying to finally obtain purified matters of 5 kinds of Ustiloxin monomer compounds Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F, wherein the content of the purified matters is more than 90% respectively.
Preferably, in S5, the collected eluent is identified by tracing by using a high performance liquid chromatography-mass spectrometer.
Preferably, in the high performance liquid chromatography-mass spectrometer, an ESI source anion mode is adopted, the chromatographic column is a C18 column, and a 0.1% formic acid water-methanol solution system is used as a mobile phase for gradient elution.
Preferably, the method for obtaining the rice false smut ball comprises the following steps: picking dark green or yellowish green spherical objects, namely rice false smut, from rice ears at the later stage of rice growth when the false smut occurs, and storing in a refrigerator at the temperature of-20 ℃ for later use; the method for obtaining the rice false ball spore powder comprises the following steps: and in the later stage of rice growth, picking dark green or yellowish green spherical objects, namely, rice false smut, from rice ears, airing, and collecting powder wrapped by the outer layer of the rice false smut, namely, rice false smut spore powder.
Preferably, in the step S1, the raw materials are soaked in water with the material ratio of 1:5, fully crushed and homogenized by a high-speed homogenizer, ultrasonically assisted by an ultrasonic extractor for 30min, then placed in a constant-temperature water bath oscillator for oscillation extraction at 35 ℃ for more than 8 hours, and centrifuged at a high speed at a speed of not less than 10000r/min, and the supernatant is subjected to vacuum freeze drying for 72 hours after being filtered by a warp cloth, so as to obtain the crude extract of the rice Qu Jundu.
Preferably, the aspect ratio of the organic-tolerant medium-pressure chromatographic column is 1:32, and in the gradient elution process, 5 mobile phase proportion gradients are sequentially arranged, wherein the elution volumes of the mobile phase proportion gradients are respectively 5%, 10%, 15%, 20% and 25%, the elution volume of each mobile phase proportion gradient is one column volume, and the flow rate of the eluent is 1.2mL/min.
Preferably, when the solid phase extraction column is used for carrying out solid phase extraction on column chromatography separation extracts of 5 Ustiloxin, the Ustiloxin A, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F4 Ustiloxin are subjected to enrichment and purification treatment by adopting a mixed cation exchange column (PCX), and the Ustiloxin B is subjected to enrichment and purification treatment by adopting a mixed cation exchange column (PCX) and a mixed anion exchange column (PAX) in series connection.
Preferably, in the purification process, a semi-preparative high performance liquid chromatograph is combined with an automatic fraction collector to perform isocratic elution with a methanol-water solution according to the retention time of the target compound, and the target compound is automatically collected according to the characteristic absorption wavelength of each of the 5 ustilaginoidea virens toxins.
Compared with the prior art, the invention has the following advantages:
1) The invention adopts fine resin filler for the first time and simultaneously carries out one-time complete separation on Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 ustilaginous toxins.
2) In the research of separation, extraction and purification preparation processes, the conventional ideas of carrying out integral extraction and separation, purifying again, separating and preparing by column chromatography and the like are broken through, after crude extract is obtained by extraction and separation, column chromatography separation of 5 kinds of ustilaginoidea toxin monomer compounds is carried out first, and after the completely separated 5 kinds of ustilaginoidea toxin compounds are obtained, enrichment and purification treatment are respectively carried out by adopting a solid phase extraction technology, so that the pre-purification process conditions can be simplified, meanwhile, the mutual compromise and interference among 5 kinds of toxins are avoided, the purification cost is saved, the loss of various toxins can be well reduced, and the yield is improved.
3) According to the respective characteristics of 5 kinds of ustilaginoidea virens toxins and the process characteristics of the method, different rice Qu Jundu kinds of ustilaginoidea virens toxins are subjected to enrichment and purification treatment by adopting different solid-phase extraction columns, and the enriched and purified rice Qu Jundu kinds of ustilaginoidea virens toxins have single solid-phase extraction columns and serial solid-phase extraction columns, and simultaneously have manage rates.
4) Compared with the existing repeated separation and purification method, the method has the advantages of simple process flow, high speed, high efficiency, good repeatability, no use of organic solvents such as methylene dichloride, safety and no pollution.
5) The invention creatively combines the high-performance liquid chromatograph with the column chromatography device, breaks through the traditional method of adopting the peristaltic pump as the infusion system in the column chromatography separation method, uses the high-performance liquid chromatograph instead of the peristaltic pump as the infusion system to be applied to the filling and eluting separation of column chromatography packing, avoids the problems of easy gas production, fault, large front-back flow velocity change, repeated filling and the like in the experimental process, has stable experimental result and good reproducibility, and has less problems of manually preparing eluting solution in the experimental process, saves time and labor, and establishes a set of perfect and effective column chromatography separation system.
The invention can be used for preparing 5 kinds of ustilaginoidea virens toxin monomer compounds by separation and purification, breaks through the key bottleneck problems of lack of ustilaginoidea virens toxin standard samples in the aspects of deep research for limiting the influence of ustilaginoidea virens toxin on human health, risk monitoring, risk assessment, limit standard research and the like for many years, and the obtained 5 kinds of ustilaginoidea virens toxin monomer compounds can provide basic preconditions and guarantee for deep research and application of ustilaginoidea virens toxin in the future and accelerate the cognition and development process of ustilaginoidea virens toxin by people.
Drawings
FIG. 1 is a graph showing column chromatographic separation of 5 kinds of ustilaginoidea virens toxins.
FIG. 2 is a high performance liquid chromatogram of a rice Qu Jundu extract Ustiloxin A purification.
FIG. 3 is a high performance liquid chromatogram of rice Qu Jundu extract Ustiloxin B purification.
FIG. 4 is a high performance liquid chromatogram of rice Qu Jundu extract Ustiloxin C purified product.
FIG. 5 is a high performance liquid chromatogram of a rice Qu Jundu extract Ustiloxin D purification.
FIG. 6 is a high performance liquid chromatogram of rice Qu Jundu extract Ustiloxin F purified.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.
In the invention, a method for preparing 5 kinds of ustilaginoidea virens toxins through simultaneous separation and purification is provided, which comprises the following steps:
S1, taking whole rice false smut balls or rice false smut ball spore powder as a raw material, taking water as an extraction solution to carry out homogenate crushing, vibration extraction and centrifugal separation on the raw material, and carrying out vacuum freeze drying on the obtained supernatant to obtain the crude extract of the rice false smut toxin.
In a preferred embodiment of the present invention, whole rice false ball or rice false ball spore powder can be used as the raw material of the present invention, and both can be obtained as follows:
The method for obtaining the rice false smut ball comprises the following steps: picking dark green or yellowish green spherical objects, namely rice false smut, from rice ears at the later stage of rice growth when the false smut occurs, and storing in a refrigerator at the temperature of-20 ℃ for later use;
the method for obtaining the rice false ball spore powder comprises the following steps: and in the later stage of rice growth, picking dark green or yellowish green spherical objects, namely, rice false smut, from rice ears, airing, and collecting powder wrapped by the outer layer of the rice false smut, namely, rice false smut spore powder.
As a preferred embodiment of the present invention, the specific process parameters for extracting the crude extract may be optimized according to the actual process, and the optimized process is as follows: soaking the raw materials in water with the material ratio of 1:5, fully crushing and homogenizing by a high-speed homogenizer, carrying out ultrasonic dissolution assistance by an ultrasonic extractor for 30min, then placing in a constant-temperature water bath oscillator for oscillation extraction at 35 ℃ for more than 8 hours, then centrifuging at a high speed at a rotating speed of not less than 10000r/min, filtering supernatant warp cloth, and carrying out vacuum freeze drying for 72 hours to obtain the crude extract of rice Qu Jundu. In this case, water is used as an extraction solution, and the extraction efficiency is higher than that of the method of extraction with methanol.
S2, filling the fine resin HP20SS serving as column chromatography filler into an organic medium pressure resistant chromatography column according to a wet column packing mode, installing the organic medium pressure resistant chromatography column at the position of a chromatographic column of a high performance liquid chromatograph, and adopting the high performance liquid chromatograph as an infusion system to press the chromatographic column.
The fine resin HP20SS is a commercially available product having a particle size distribution of 60 to 150. Mu.m. The organic-tolerant medium-pressure column may be a commercially available medium-pressure column, and its specific type is not limited as long as it can tolerate an organic phase in the eluent. The fine resin HP20SS is activated and suspended with a corresponding mobile phase during column packing, in order to fill the column, which can be achieved in particular according to the methods provided by the manufacturer.
As a preferred embodiment of the present invention, the fine resin HP20SS may be previously activated with methanol and then suspended in a methanol-water system having a methanol content of 20%. The suspension was then injected into the column according to the wet column loading protocol to gradually fill the organic medium pressure resistant column with fine resin HP20 SS. And then installing the organic medium pressure resistant chromatographic column in a high performance liquid chromatograph to replace the original chromatographic column, continuously using the 20% methanol-water solution system as eluent to press the organic medium pressure resistant chromatographic column, wherein the pressure of the pressure column is derived from a pump of the high performance liquid chromatograph, and the eluent is automatically pressed into the chromatographic column by the high performance liquid chromatograph. The column is pressed for 24 hours generally, and the chromatographic column to be loaded can be obtained.
In addition, the aspect ratio during column loading described above affects subsequent elution characteristics, and in a preferred embodiment of the present invention, the aspect ratio is preferably set to a 1:32 ratio.
The fine resin HP20SS is selected filler types after a large number of screening on the basis of researching the separation characteristics of Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 kinds of Ustiloxin, and can well realize the one-time complete separation of 5 kinds of Ustiloxin.
S3, dissolving the crude extract with water, centrifuging, filtering the obtained supernatant to remove impurities by a membrane, and then separating by column chromatography by a chromatographic column.
The basic implementation steps of column chromatography separation in the invention belong to the prior art, and specific process parameters thereof need to be optimized and adjusted according to actual practice. As a preferred embodiment of the present invention, the sample amount on the column can be calculated in advance according to the filling amount of the chromatographic column; then dissolving the crude extract in 50ml centrifuge tube with a small amount of water, ultrasonically dissolving for 30min with an ultrasonic extractor, centrifuging to obtain supernatant, filtering with 0.22 μm filter membrane, and separating with chromatography column filled with fine resin HP20 SS.
S4, eluting the ustilaginoidea virens toxin target compound by using a high performance liquid chromatograph as an infusion system, wherein an eluting solution is a methanol-water system, the eluting mode is gradient elution, the proportion of a mobile phase in the gradient elution is gradually increased from 5% to 25%, and the eluent is collected by using a full-automatic part collector tube by tube.
In the invention, the high performance liquid chromatograph is used as the transfusion system, so that the elution solution only needs to be placed in the high performance liquid chromatograph with the A-phase methanol and the B-phase water, and the gradient proportion is set, and the high performance liquid chromatograph can automatically perform gradient adjustment according to the corresponding proportion.
The specific flow phase gradient of the gradient elution needs to be optimally adjusted according to the actual test. As a preferred embodiment of the invention, in the gradient elution process of the organic medium pressure resistant chromatographic column with the aspect ratio of 1:32 of the packing, 5 mobile phase proportion gradients are sequentially arranged, wherein the elution volume of each mobile phase proportion gradient is one column volume (about 400 mL) and the eluent flow rate is 1.2mL/min, and the mobile phase proportion gradients are respectively 5%, 10%, 15%, 20% and 25%. The 5 Ustiloxin elution sequences were Ustiloxin B, ustiloxin F, ustiloxin a, ustiloxin C and Ustiloxin D, and in order to be able to separate 5 different rice Qu Jundu toxins, a fully automatic partial collector with a computer was used for collection, in this example a collection volume of 4 ml/tube.
S5, tracking and identifying types of oryzanol Qu Jundu in the collected eluates, merging eluates belonging to the same class of ustilaginous toxins, and respectively carrying out vacuum centrifugal concentration on the 5 eluates after merging to obtain column chromatography separation extracts of Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 ustiloxins.
In a preferred embodiment of the invention, the collected eluate can be identified by high performance liquid chromatography-mass spectrometry (LC-MS/MS). And when LC-MS/MS is used for tracking and identifying, the LC-MS/MS chromatographic-mass spectrometry conditions including chromatographic column, column temperature, sample injection amount, mobile phase and gradient elution program, ion source, scanning mode, scanning range, mass spectrum resolution, cracking mode, sheath gas (N2) pressure, auxiliary gas (N2) pressure, capillary temperature, capillary voltage and the like are determined according to the full-scanning mass spectrogram of the ustilago sativa toxin mass spectrum, the accurate mass number of parent ions, the isotope abundance ratio and the secondary cracking rule, so that the efficient and accurate analysis and confirmation method for the content of the 5 ustilago sativa toxins is obtained. As a preferred embodiment of the invention, in the high performance liquid chromatography-mass spectrometer, ESI source anion mode can be adopted, the chromatographic column is a C18 column, and 0.1% formic acid water-methanol solution system is used as a mobile phase for gradient elution.
S6, carrying out enrichment purification on column chromatography separation extracts of the 5 kinds of ustilaginoxin by a solid phase extraction technology, and then further purifying to finally obtain purified substances of the 5 kinds of Ustiloxin A, the 5 kinds of Ustiloxin B, the 5 kinds of Ustiloxin C, the 5 kinds of Ustiloxin D and the 5 kinds of Ustiloxin F.
It should be noted that the specific practice of the above enrichment purification and purification is not limited, and it is possible to perform enrichment and impurity removal purification and further purification of rice Qu Jundu element by the enrichment purification.
As a preferred embodiment of the invention, the enrichment and purification modes are as follows: and respectively carrying out solid phase extraction on column chromatography separation extracts of the 5 kinds of the Ustiloxin by a solid phase extraction column to obtain enrichment purified matters of the Ustiloxin A, the Ustiloxin B, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F5 kinds of the Ustiloxin.
The solid phase extraction columns required for different ustilaginoidea virens toxins are different. Further, in a preferred embodiment, when the solid phase extraction column is used to extract the column chromatography separation extracts of 5 kinds of ustilaginoxin, rice Qu Jundu elements Ustiloxin A, ustiloxin C, ustiloxin D and Ustiloxin F4 kinds of Ustiloxin are subjected to enrichment and purification treatment by using a mixed cation exchange column (PCX), and rice Qu Jundu element Ustiloxin B is subjected to enrichment and purification treatment by using a mixed cation exchange column (PCX) and a mixed anion exchange column (PAX) in series.
As a preferred embodiment of the present invention, the purification is performed in the following manner: the enriched and purified matters of the 5 kinds of ustilaginous toxins obtained through enrichment and purification are further separated and prepared through preparative or semi-preparative high performance liquid chromatography respectively, a liquid chromatography fraction collector is used for automatically collecting the enriched and purified matters according to the retention time setting of target compounds, and the collected matters are subjected to vacuum freeze drying to finally obtain purified matters of 5 kinds of Ustiloxin monomer compounds Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F, wherein the content of the purified matters is more than 90% respectively.
Further, in the purification process, a semi-preparative high performance liquid chromatograph is preferably used, wherein the semi-preparative high performance liquid chromatograph and an automatic fraction collector are combined to perform isocratic elution with a methanol-water solution according to the retention time of the target compound, and the automatic collection is performed according to the characteristic absorption wavelength of each of the 5 ustilaginoidea virens. In a preferred embodiment, the elution flow rate in a semi-preparative high performance liquid chromatograph is set to 1.5mL/min, the collector mode is set to time-based, and the mobile phase is as follows: the Ustiloxin A, ustiloxin C and Ustiloxin D3 kinds of Ustiloxin are eluted with 10% methanol-water solution isocratically, and the Ustiloxin B and Ustiloxin F2 kinds of Ustiloxin B are eluted with 5% methanol-water solution isocratically. The detection wavelength is determined to be 254nm and 290nm according to the characteristic absorption of different ustilaginoxin A, ustiloxin B and Ustiloxin C, and the 254nm wavelength is mainly used, and the 290nm wavelength is used for collecting references; ustiloxin D and Ustiloxin F detection wavelengths are 227nm and 287nm, with 227nm being the dominant wavelength and 287nm being the wavelength at which the reference is collected.
The technical scheme of the invention is further specifically described by the following specific examples.
Examples:
the materials and reagents, instruments and equipment, experimental methods, etc. used in this example are as follows:
1. Materials and reagents
1.1 Obtaining of rice false smut balls: at the later stage of rice growth, dark green or yellowish green spherical objects, namely rice false smut, are picked from the rice ears, and are stored in a refrigerator at the temperature of minus 20 ℃ for later use.
1.2 Reagents and consumables: the chromatographic column is a 2.5cm×100cm organic medium pressure resistant chromatographic column (Beijing Ruida Henghui Co., ltd.); HP20SS fine resin with particle size of 60-150 μm (Beijing green hundred grass technology development Co., ltd.); chromatographic pure methanol (merck chemical technologies (Shanghai); mass spectrometry grade methanol (merck chemical technologies (Shanghai); chromatographic pure formic acid (Beijing carboline technologies Co., ltd.); chromatographic pure ammonia (Shanghai Ala Biochemical technologies Co., ltd.); ultrapure water (homemade); 0.22 μm filter (Shanghai Annotation laboratory science and technology Co., ltd.); bond Elut Plexa PCX,500mg,6ml (Agilent technologies Co., ltd.); bond Elut Plexa PAX,500mg,6ml (Agilent technologies Co., ltd.); bond Elut C18, 500mg,6ml (Agilent technologies Co., ltd., U.S.); chromatography column AGILENT ECLIPSE Plus C18,2.1 mm. Times.50 mm,1.8 μm (Agilent technologies Co., ltd.); chromatography column AGILENT ECLIPSE XDB C18,9.4mm X250 mm,5 μm (Agilent technologies Co., ltd.).
2 Instruments and apparatus
A low temperature vacuum freeze dryer (LABCONCO, usa); low temperature centrifugal concentrator (LABCONCO, usa); high speed centrifuge (japanese field 3740); a constant temperature water bath oscillator (manufactured by Zhejiang Jin Huashi nabor instruments Co., ltd.); high speed refiners (Shanghai Froude fluid machinery Co., ltd.); ultrasonic cleaners (Shanghai department ultrasonic instruments Co., ltd.); high performance liquid chromatograph 1260 (agilent technologies, inc., usa); high performance liquid chromatography fraction collector type G1364C (agilent technologies, inc., usa); high performance liquid chromatography-mass spectrometer 6470 (Agilent technologies Co., ltd.); healForce ultra pure water system (hong Kong Likang biomedical technology control group of China); computer full-automatic partial collector DBS-100 type (Shanghai Qinghai Shanghai instruments and West China).
3 Experimental concrete method
3.1 Preparation of aqueous extract and crude extract freeze-dried powder of Magnaporthe grisea
Weighing 100g of whole rice false smut balls as raw materials, soaking the whole rice false smut balls in water with the material ratio of 1:5, fully crushing and homogenizing the whole rice false smut balls by using a high-speed homogenizer, ultrasonically assisting the whole rice false smut balls to dissolve for 30min by using an ultrasonic extractor, placing the whole rice false smut balls in a constant-temperature water bath oscillator, performing oscillation extraction for more than 8 hours at 35 ℃, then performing high-speed centrifugation at the rotation speed of 12000r/min, taking supernatant after gauze, and performing vacuum freeze drying for 72 hours to obtain rough extract freeze-dried powder I of the rice false smut balls.
3.2 Column chromatography separation of ustilaginoidea virens toxin
3.2.1 Column packing: the fine resin HP20SS is adopted as a filler for column chromatography, the particle size is 60-150 mu m, the chromatography column is a medium-pressure organic-resistant chromatography column, and the column size is 2.5cm multiplied by 100cm. The fine resin HP20SS is activated by methanol in advance, then a methanol-water system containing 20% of methanol is used for preparing a suspension, and the suspension is injected into a column according to the specific practice of wet column packing, so that the fine resin HP20SS is gradually filled into an organic medium pressure resistant chromatographic column, and is packed at an aspect ratio of 1:32, and the packing height is 80cm. And then installing the organic medium pressure resistant chromatographic column filled with the filler in a high performance liquid chromatograph to replace the original chromatographic column, adopting the high performance liquid chromatograph as an infusion system to press the column by using a methanol-water system of 20% methanol as a eluent, and loading the chromatographic column after 24 hours.
3.2.2 Column chromatography separation: 5g of the crude extract freeze-dried powder I of the rice false smut ball is weighed, dissolved in a 50mL centrifuge tube by 20mL of water, ultrasonically assisted by an ultrasonic extractor for 30min, centrifuged to obtain supernatant, filtered by a 0.22 mu m filter membrane and then separated by a chromatographic column. Gradient elution was performed at a flow rate of 1.2mL/min with a 5% -25% methanol-water elution system, with each proportional elution volume being one column volume, approximately 400mL. The 5 kinds of ustilaginoxin eluted in the order of Ustiloxin B, ustiloxin F, ustiloxin A, ustiloxin C and Ustiloxin D were collected by a computer fully automatic partial collector with a collection volume of 4 ml/tube.
In this example, as shown in FIG. 1, the column chromatography separation graph of 5 kinds of Ustiloxin is that the fine resin HP20SS can well realize one-time complete separation of Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 kinds of Ustiloxin, and the respective peaks hardly overlap.
3.2.3 LC-MS/MS tracking and identifying method of ustilaginoidea virens toxin
The types of the oryzanol Qu Jundu in the eluent collected by each tube are tracked and identified by a high performance liquid chromatography-mass spectrometer (LC-MS/MS), the eluents belonging to the same type of the oryzanol are combined, and the 5 eluents after combination are respectively subjected to vacuum centrifugal concentration to obtain extracts II of the column chromatography separation of the oryzanol A, the Ustiloxin B, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F5.
In the rice Qu Jundu element separation and purification process, a high performance liquid chromatography-mass spectrometer is adopted for tracking and identification, and the chromatographic mass spectrometry conditions are as follows: agilent EclipsePlus C18 (2.1 mm. Times.50 mm,1.8 μm); column temperature: 25 ℃; sample injection amount is 1.0 mu L; mobile phase: phase a is 0.1% aqueous formic acid; the phase B is methanol; flow rate: 0.2. Mu.L/min; gradient elution procedure: 0-2.00 min,90% A; 2.00-10.00 min, 90-20% of A; 10.00-12.00 min,90% A; ion source: an ESI source negative ion mode; scanning mode: full scanning; scanning range: m/z is 200-1 000; mass spectrum resolution: 100000; cleavage mode: collision Induced Dissociation (CID); sheath gas (N2) pressure: 241.33kPa; auxiliary gas (N2) pressure: 34.48kPa; the capillary temperature was 350 ℃.
3.3 Enrichment and purification of ustilaginoidea virens toxin
In order to verify the enrichment and purification effects of different solid phase extraction columns, 3 solid phase extraction columns with different purification principles of C18, mixed anion exchange columns and mixed cation exchange columns are respectively adopted to purify the chromatographic separation extracts of the 5 ustilaginoidea virens toxin columns, specifically C18, PAX and PCX. Comparing the enrichment and purification results of the three, the C18 adsorption capacity is strongest, almost all toxins are adsorbed, and elution is difficult; PAX and PCX have a certain degree of enrichment and purification effects, and because the structural formulas of the 5 kinds of ustilaginoidea virens toxins all contain a plurality of imino groups, the sample is purified by using two different treatment methods of adsorbing target substances and impurities and adopting the characteristic of being positively charged under acidic conditions, and finally, the PCX mixed cation exchange column is selected as the target substance adsorption mode. Since rice Qu Jundu B is eluted firstly when separated by column chromatography, the impurity content is higher, and the ideal effect can not be achieved by only adopting PCX enrichment and purification, the enrichment and purification are carried out by adopting a mode of connecting two solid phase extraction columns of PCX-PAX and PCX-PCX in series. The content and the confirmation result of the high-efficiency liquid phase are found that the enrichment and purification effect is better when PCX-PAX are connected in series, so that the rice Qu Jundu Utilioxin B is finally determined to be subjected to the series enrichment and purification by the PCX-PAX in the embodiment, and other Utilioxin A, utilioxin C, utilioxin D and Utilioxin F are directly subjected to the enrichment and purification by the PCX, so that Utilioxin A, utilioxin B, utilioxin C, utilioxin D and Utilioxin F5 Utilioxin enrichment and purification III are respectively obtained.
3.4 High performance liquid chromatography preparation and collection of rice Qu Jundu element
The 5 kinds of ustilaginoidea virens toxin enriched purified matters III are further purified and prepared through semi-preparation type high performance liquid chromatography respectively, and are collected according to the retention time of target compounds by adopting a semi-preparation type high performance liquid chromatography combined with an automatic fraction collector. Wherein, the flow rate is 1.5mL/min, the collector mode is set to be based on time, the mobile phases are Ustiloxin A, ustiloxin C and Ustiloxin D3 kinds of Ustiloxin B and Ustiloxin F2 kinds of Ustiloxin are eluted with 10% methanol-water solution isocratically, and the Ustiloxin B and Ustiloxin F2 kinds of Ustiloxin B are eluted with 5% methanol-water solution isocratically. The detection wavelength is determined to be 254nm and 290nm according to the characteristic absorption of different ustilaginoxin A, ustiloxin B and Ustiloxin C, and the 254nm wavelength is mainly used, and the 290nm wavelength is used for collecting references; ustiloxin D and Ustiloxin F detection wavelengths are 227nm and 287nm, with 227nm being the dominant wavelength and 287nm being the wavelength at which the reference is collected. The collected liquid is subjected to vacuum freeze drying to obtain purified matters IV of 5 Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F with the content of more than 90 percent.
Finally, fig. 2 to 6 respectively show the high performance liquid chromatograms of the purified substances of the rice Qu Jundu elements Ustiloxin a to F in this example, which indicates that 5 kinds of Ustiloxin are prepared by simultaneous separation and purification in this example, and the purity is higher without obvious impurities. The purity of the obtained 5 Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F can reach more than 90%, and the obtained 5 Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F can be used as standard samples.
In conclusion, the invention uses water as an extraction solvent, adopts fine resin to carry out column chromatography separation, adopts a solid phase extraction technology as an enrichment purification method, adopts semi-preparative high performance liquid chromatography to carry out preparation and collection, and adopts a liquid chromatography-mass spectrometer for tracking and identification, so that 5 Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F monomer compounds can be prepared by simultaneous separation and purification. The method has the advantages of simple operation, high speed, high efficiency, good repeatability, safety, no pollution and high purity.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (3)
1. The method for preparing 5 kinds of ustilaginoidea virens toxins by simultaneous separation and purification is characterized by comprising the following steps:
s1, taking whole rice false smut balls or rice false smut ball spore powder as a raw material, taking water as an extraction solution to carry out homogenate crushing, vibration extraction and centrifugal separation on the raw material, and carrying out vacuum freeze drying on the obtained supernatant to obtain a crude extract of rice Qu Jundu;
s2, filling a fine resin HP20SS serving as a column chromatography filler into an organic medium pressure resistant chromatography column according to a wet column packing mode, mounting the organic medium pressure resistant chromatography column at a chromatographic column position of a high performance liquid chromatograph, and adopting the high performance liquid chromatograph as an infusion system to press the chromatography column;
s3, dissolving the crude extract with water, centrifuging, filtering the obtained supernatant to remove impurities by a membrane, and separating by column chromatography on a chromatographic column;
S4, performing elution of the ustilaginoidea virens toxin target compound by using a high performance liquid chromatograph as an infusion system, wherein an elution solution is a methanol-water system, the elution mode is gradient elution, the proportion of a mobile phase in the gradient elution is gradually increased from 5% to 25%, and the eluent is collected by using a full-automatic part collector tube by tube;
S5, tracking and identifying types of rice Qu Jundu in the collected eluates, merging eluates belonging to the same class of ustilaginous toxins, and respectively carrying out vacuum centrifugal concentration on the 5 eluates after merging to obtain column chromatography separation extracts of Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F5 ustiloxins;
S6, carrying out enrichment purification on column chromatography separation extracts of the 5 kinds of ustilaginoxin by a solid phase extraction technology, and then further purifying to finally obtain purified substances of the 5 kinds of Ustiloxin A, the 5 kinds of Ustiloxin B, the 5 kinds of Ustiloxin C, the 5 kinds of Ustiloxin D and the 5 kinds of Ustiloxin F;
In the step S6, the enrichment and purification method is as follows: subjecting the column chromatography separation extracts of the 5 kinds of ustilaginoxin to solid phase extraction by a solid phase extraction column respectively to obtain enrichment purified matters of the Ustiloxin A, the Ustiloxin B, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F5 kinds of Ustiloxin;
In the step S6, the purification is performed in the following manner: respectively adopting a semi-preparative high performance liquid chromatograph and an automatic fraction collector to perform isocratic elution on the enriched and purified matters of the 5 kinds of ustilaginous toxins according to target compound retention time, performing automatic collection according to the characteristic absorption wavelengths of the 5 kinds of ustilaginous toxins, and performing vacuum freeze drying on the collected liquid to finally obtain purified matters of the 5 kinds of ustilaginous toxins monomer compounds Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D and Ustiloxin F, wherein the content of the Ustiloxin A, ustiloxin B, ustiloxin C, ustiloxin D is more than 90%; wherein the elution flow rate in the semi-preparative high performance liquid chromatograph was set to 1.5mL/min, the collector mode was set to time-based, and the mobile phase was as follows: isocratic elution of Ustiloxin A, ustiloxin C and Ustiloxin D3 types of Ustiloxin D with 10% methanol-water solution and isocratic elution of Ustiloxin B and Ustiloxin F2 types of Ustiloxin 5% methanol-water solution, determining Ustiloxin A, ustiloxin B and Ustiloxin C as 254nm and 290nm according to characteristic absorption of different Ustiloxin, and collecting reference with 254nm wavelength and 290nm wavelength; ustiloxin D and Ustiloxin F detection wavelengths are 227nm and 287nm, the reference is collected at 227nm, and 287 nm;
In the step S5, tracking and identifying the collected eluent by using a high performance liquid chromatography-mass spectrometer; in the high performance liquid chromatography-mass spectrometer, an ESI source negative ion mode is adopted, a chromatographic column is a C18 column, and a 0.1% formic acid water-methanol solution system is used as a mobile phase for gradient elution;
the diameter-to-height ratio of the organic medium pressure resistant chromatographic column is 1:32, in the gradient elution process, 5 mobile phase proportion gradients are sequentially arranged and respectively 5%, 10%, 15%, 20% and 25%, the elution volume of each mobile phase proportion gradient is one time of the column volume, and the flow rate of eluent is 1.2mL/min;
When the solid phase extraction column is used for carrying out solid phase extraction on column chromatography separation extracts of 5 Ustiloxin, the Ustiloxin A, the Ustiloxin C, the Ustiloxin D and the Ustiloxin F4 Ustiloxin are subjected to enrichment and purification treatment by adopting a mixed cation exchange column (PCX), and the Ustiloxin B of the rice Qu Jundu is subjected to enrichment and purification treatment by adopting a mixed cation exchange column (PCX) and a mixed anion exchange column (PAX) in series connection.
2. The method for preparing 5 kinds of ustilago toxins by simultaneous separation and purification according to claim 1, wherein the method for obtaining the ustilago balls is as follows: picking dark green or yellowish green spherical objects, namely rice false smut, from rice ears at the later stage of rice growth when the false smut occurs, and storing in a refrigerator at the temperature of-20 ℃ for later use; the method for obtaining the rice false ball spore powder comprises the following steps: and in the later stage of rice growth, picking dark green or yellowish green spherical objects, namely, rice false smut, from rice ears, airing, and collecting powder wrapped by the outer layer of the rice false smut, namely, rice false smut spore powder.
3. The method for preparing 5 kinds of ustilago toxins by simultaneous separation and purification according to claim 1, which is characterized in that: in the step S1, the raw materials are soaked in water with the material ratio of 1:5, fully crushed and homogenized by a high-speed homogenizer, ultrasonically assisted by an ultrasonic extractor for 30min, then placed in a constant-temperature water bath oscillator for oscillation extraction at 35 ℃ for more than 8 hours, and centrifuged at a high speed at a rotating speed of not less than 10000 r/min, and the supernatant is subjected to vacuum freeze drying for 72 hours after warp cloth filtration, so that the crude extract of rice Qu Jundu is obtained.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584941A (en) * | 2012-03-06 | 2012-07-18 | 江苏省农业科学院 | Paddy rice Ustiloxin A extracting and purifying method |
| CN104569416A (en) * | 2015-01-22 | 2015-04-29 | 中国农业大学 | Method for detecting ustiloxin B and special enzyme-linked immunosorbent kit for method |
| CN106279359A (en) * | 2016-08-11 | 2017-01-04 | 中国水稻研究所 | A kind of method preparing five kinds of ustilaginoidea virens toxin |
| CN111763241A (en) * | 2020-06-11 | 2020-10-13 | 江苏省农业科学院 | Preparation method of pure ustiloxin A |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584941A (en) * | 2012-03-06 | 2012-07-18 | 江苏省农业科学院 | Paddy rice Ustiloxin A extracting and purifying method |
| CN104569416A (en) * | 2015-01-22 | 2015-04-29 | 中国农业大学 | Method for detecting ustiloxin B and special enzyme-linked immunosorbent kit for method |
| CN106279359A (en) * | 2016-08-11 | 2017-01-04 | 中国水稻研究所 | A kind of method preparing five kinds of ustilaginoidea virens toxin |
| CN111763241A (en) * | 2020-06-11 | 2020-10-13 | 江苏省农业科学院 | Preparation method of pure ustiloxin A |
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