阅读说明：本技术 一种稻曲菌素a纯品的制备方法 (Preparation method of pure ustiloxin A ) 是由 王刚 徐剑宏 史建荣 满惠子 于 2020-06-11 设计创作，主要内容包括：本申请公开了一种稻曲菌素A纯品的制备方法,涉及农产品安全检测技术领域,具体包括：取稻曲球粉末或稻绿核菌培养物粉末,加入提取液进行超声提取,过滤,得到稻曲菌素A粗提取液；调节稻曲菌素A粗提取液的pH,再采用大孔吸附树脂柱进行柱层析,收集洗脱液；将含有稻曲菌素A的洗脱液旋转蒸发,再用高速逆流色谱分离纯化,收集洗脱液；选择只含有稻曲菌素A的洗脱液进行合并后旋转蒸干,再溶于水中,冷冻干燥,即制得稻曲菌素A纯品。本申请解决了稻曲菌素A纯品分离纯化成本高的问题,提高了单次制备量,并降低了分离纯化成本。(The application discloses a preparation method of a pure oryzanol A product, relates to the technical field of agricultural product safety detection, and specifically comprises the following steps: taking rice koji ball powder or rice sclerotium rolfsii culture powder, adding the extracting solution for ultrasonic extraction, and filtering to obtain a crude ustilagin A extracting solution; adjusting the pH value of the crude extract of ustilaginoidea virens A, performing column chromatography by adopting a macroporous adsorption resin column, and collecting eluent; carrying out rotary evaporation on the eluant containing ustilaginoidin A, separating and purifying by using high-speed counter-current chromatography, and collecting the eluant; and combining the eluates containing only ustilaginoidea virens A, evaporating to dryness, dissolving in water, and freeze-drying to obtain the final product. The application solves the problem of high cost of the separation and purification of the ustilaginoidin A pure product, improves the single preparation amount and reduces the cost of the separation and purification.)

1. A preparation method of a pure product of ustiloxin A is characterized by comprising the following steps:

(1) taking rice koji ball powder or rice sclerotium rolfsii culture powder, adding the extracting solution for ultrasonic extraction, filtering, and collecting the filtrate to obtain a crude ustilagin A extracting solution;

(2) adjusting the pH value of the crude ustilaginoidin A extract, performing column chromatography on the crude ustilaginoidin A extract by using a macroporous adsorption resin column, and collecting eluent;

(3) carrying out rotary evaporation on the eluant containing ustilaginoidin A, separating and purifying by using high-speed counter-current chromatography, and collecting the eluant;

(4) rotary evaporating the eluent containing ustilaginoidea virens A to dryness, dissolving in water, and freeze drying to obtain ustilaginoidea virens A pure product.

2. The method for preparing a pure ustiloxin A according to claim 1, wherein the extract is an aqueous solution containing 1.0% by volume of formic acid and 0.1% by volume of Tween-20.

3. The method for preparing the pure ustilaginoidea virens A as claimed in claim 1, wherein the ultrasonic extraction is carried out at a ratio of 1/5-1/20.

4. The method for preparing a pure ustilaginoidea virens A according to claim 1, wherein the pH of the crude ustilaginoidea virens extract is adjusted to neutral in the step (2).

5. The method for preparing the pure ustilaginoidea virens A as claimed in claim 1, wherein the macroporous adsorbent resin column is a low-polarity macroporous adsorbent resin column HZ-801.

6. The method for preparing the pure ustilaginoidin A according to claim 1, wherein the step (2) is carried out by loading the pure ustilaginoidin A into a macroporous adsorbent resin column at a flow rate of 2-3 BV/h.

7. The method for preparing the pure ustilaginoidea virens A as claimed in claim 1, wherein the eluent used for the column chromatography is 20-30% volume fraction methanol aqueous solution.

8. The method for preparing the pure ustilaginoidea virens as claimed in claim 7, wherein the step (3) is carried out by rotary evaporation of the rice ustilaginoidea virens-containing eluent until no methanol remains.

9. The method for preparing the pure ustilaginoidea virens as claimed in claim 1, wherein in the steps (2) and (3), the content of the ustilaginoidea virens in the eluent is detected by TLC when the eluent is collected, and the developing solvent is a mixture of 1:1, and the color developing agent is an n-butyl alcohol solution containing ninhydrin with volume fraction of 1.5%.

10. The method for preparing the pure ustiloxin A as claimed in claim 1, wherein the high-speed counter-current chromatography is performed by using n-butanol: water: trifluoroacetic acid is 1:1:0.02, the upper layer solvent is a stationary phase, the lower layer solvent is a mobile phase, the flow rate of the mobile phase is 1.0-1.5 mL/min, the rotating speed of the instrument is 900-1200 rpm, and the operating temperature is 25-40 ℃.

Technical Field

The application relates to the technical field of agricultural product safety detection, in particular to a preparation method of a pure ustiloxin A product.

Background

False smut is a fungal disease often occurring on rice ears, and the pathogenic fungus is Rhizoctonia solani. The green smut pathogen infects rice ears to form yellow or black rice curly rice balls, and the disease can block the nutrition transportation of grains in the booting stage, prevent the grains from normally developing, cause the increase of the blight rate and the reduction of the thousand grain weight, and influence the rice yield. The rice green sclerotium can generate ustiloxin after infecting rice, so that rice crops are polluted by toxin; about 25% of rice crops worldwide are contaminated with ustiloxin every year, causing great economic loss and safety risks. It has been found that a total of 7 kinds of ustilaginoidins, namely ustilaginoidin A, B, C, D, E, F and ustilaginoidin G, share a common characteristic parent nucleus of a tridecylic depsipeptide formed by connecting through ether bonds, wherein ustilaginoidin A (Ustiloxin A, chemical structural formula 1) is the most main pollutant and accounts for more than 80% of all the ustilaginoidin pollutants.

Ustilagin A inhibits mitotic processes in eukaryotic cells, and the concentration of ustilagin A that inhibits mitotic processes in the majority (IC)₅₀) Is 0.7 mu mol/L, is equivalent to colchicine, so the ustiloxin A has strong cytotoxicity and phytotoxicity. Experiments show that the ustiloxin A has the inhibiting effect on the growth separation of human gastric, lung, mammary gland, colon and renal cell lines. When the domestic pigs are fed with rice contaminated with ustiloxin a, a toxic reaction will occur, which is expressed as: the growth of the pork pig is slowed down, and the weight gain rate is reduced; the pathological changes of various internal organs, such as liver, kidney and spleen, are obvious; influence the reproductive performance of the sows, and besides the ovary of the sows is congested and bled, the litter size, the litter weight for birth, the litter weight for weaning and the survival rate of piglets are all reduced, and dead fetus, dry dead fetus, malformed fetus and the like are produced. In addition, ustilagin A will also be a guideThe germination rate of the crop seeds is obviously reduced, and the yield of the succeeding crops is influenced.

In view of the universality and the hazard of the ustilaginoidea virens A pollution, the pollution risk assessment, toxicological research, control method development and the like aiming at the ustilaginoidea virens A pollution are widely concerned by the industry, and the demand on purified ustilaginoidea virens A toxin pure products is great. However, no commercial ustiloxin A standard product is sold in the world at present, and the common separation and purification method needs to use reversed phase silica gel filler and Sephadex Sephadex, so that the cost is high, and the wide application of the ustiloxin A standard product is severely limited. Therefore, the development of a large-scale, safe, efficient and cheap preparation and purification technology of ustiloxin A is urgently needed.

Disclosure of Invention

The embodiment of the application provides a preparation method of the pure ustilaginoidin A, solves the problem of high cost of separation and purification of the existing pure ustilaginoidin A, improves the single preparation amount of the pure ustilaginoidin A, and reduces the cost of separation and purification.

In order to achieve the above purpose, the present application mainly provides the following technical solutions:

the application provides a preparation method of a pure oryzanol A product, which comprises the following steps:

(1) taking rice koji ball powder or rice sclerotium rolfsii culture powder, adding the extracting solution for ultrasonic extraction, filtering, and collecting the filtrate to obtain a crude ustilagin A extracting solution;

(2) adjusting the pH value of the crude ustilaginoidin A extract, performing column chromatography on the crude ustilaginoidin A extract by using a macroporous adsorption resin column, and collecting eluent;

(3) carrying out rotary evaporation on the eluant containing ustilaginoidin A, separating and purifying by using high-speed counter-current chromatography, and collecting the eluant;

(4) rotary evaporating the eluent containing ustilaginoidea virens A to dryness, dissolving in water, and freeze drying to obtain ustilaginoidea virens A pure product.

Preferably, the extracting solution is an aqueous solution containing 1.0% by volume of formic acid and 0.1% by volume of tween-20.

Preferably, the material-liquid ratio adopted by the ultrasonic extraction is 1/5-1/20.

Preferably, the step (1) is performed by filtering with low-speed or medium-speed filter paper.

Preferably, the pH of the crude ustilaginoidin A extract is adjusted to neutral in the step (2).

Preferably, the macroporous adsorption resin column is a weak-polarity macroporous adsorption resin column HZ-801.

Preferably, when the column chromatography is carried out in the step (2), the sample is loaded to the macroporous absorption resin column at the flow rate of 2-3 BV/h.

Preferably, the eluent used for the column chromatography is 20-30% methanol water solution by volume fraction.

Preferably, the step (3) is that the eluent containing ustilaginoidin A is rotary evaporated until no methanol remains.

Preferably, in the steps (2) and (3), the content of the ustilagin A in the eluent is detected by TLC when the eluent is collected, and the volume ratio of the adopted developing solvent is 1:1, and the color developing agent is an n-butyl alcohol solution containing ninhydrin with volume fraction of 1.5%.

Preferably, in the step (4), the eluates containing only ustiloxin A are selected, combined, and then subjected to rotary evaporation to dryness.

Preferably, the high-speed countercurrent chromatography uses n-butanol: water: trifluoroacetic acid is 1:1:0.02, the upper layer solvent is a stationary phase, the lower layer solvent is a mobile phase, the flow rate of the mobile phase is 1.0-1.5 mL/min, the rotating speed of the instrument is 900-1200 rpm, and the operating temperature is 25-40 ℃.

One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

(1) by adopting the technology of combining macroporous adsorption resin column chromatography and high-speed counter-current chromatography, 1000mg of dry matter can be sampled at one time, a pure ustilaginoidin A product with the purity of more than 500mg and more than 95% can be prepared at one time, the sample loading amount is large, the separation and purification steps are simple, expensive precise instruments are not needed, the production cost is reduced, the yield of the pure ustilaginoidin A product is improved, and the preparation method has the advantages of large preparation amount, high recovery rate, low cost and strong practicability;

(2) according to the application, the ustilaginoidin A is purified by adopting a two-step method of macroporous adsorption resin column chromatography and high-speed counter-current chromatography, so that the raw material loss caused by multi-step preparation and purification is reduced;

(3) the embodiment of the application adopts a high-speed counter-current chromatography technology, and uses an organic solvent which is cheap and easy to obtain as a stationary phase, so that the preparation and purification cost of the ustilaginoidin A is greatly reduced while the recovery rate is improved.

Drawings

FIG. 1 is a liquid chromatography detection spectrum of a pure ustiloxin A prepared in the examples of the present application;

FIG. 2 is a high resolution mass spectrum of a pure ustiloxin A prepared in the examples of the present application;

FIG. 3 shows the pure ustilagin A prepared in the examples of this application¹H-NMR spectrum;

FIG. 4 shows the pure ustiloxin A prepared in the examples of the present application¹³C-NMR spectrum.

Detailed Description

The embodiment of the application provides a preparation method of the pure ustilaginoidin A, solves the problem of high cost of separation and purification of the existing pure ustilaginoidin A, improves the single preparation amount of the pure ustilaginoidin A, and reduces the cost of separation and purification.

Aiming at the problems, the method for extracting and purifying the ustilaginoidin A by using the on-line combination of the macroporous adsorption resin and the high-speed countercurrent chromatography (HSCCC) technology is designed and established, and has important application value. The macroporous adsorption resin has low cost, can be repeatedly regenerated and used, and has the cost advantage superior to that of silica gel matrix filler; the high-speed counter-current chromatography is a novel chromatographic device adopting a liquid-liquid distribution mechanism, can realize the automatic separation of a large number of samples, has high resolution and large sample carrying capacity, can be used together with other separation means, and is very suitable for the separation and purification of natural products.

In order to solve the above problems, the technical solution in the embodiment of the present application has the following specific ideas:

a preparation method of a pure product of the ustiloxin A comprises the following steps:

(1) taking rice koji ball powder or rice sclerotium rolfsii culture powder, adding the extracting solution for ultrasonic extraction, filtering, and collecting the filtrate to obtain a crude ustilagin A extracting solution;

(2) adjusting the pH value of the crude ustilaginoidin A extract, performing column chromatography on the crude ustilaginoidin A extract by using a macroporous adsorption resin column, and collecting eluent;

(3) carrying out rotary evaporation on the eluant containing ustilaginoidin A, separating and purifying by using high-speed counter-current chromatography, and collecting the eluant;

(4) rotary evaporating the eluent containing ustilaginoidea virens A to dryness, dissolving in water, and freeze drying to obtain ustilaginoidea virens A pure product.

According to the embodiment of the application, by adopting the technology of combining macroporous adsorption resin column chromatography and high-speed counter-current chromatography, 1000mg of dry matter can be sampled at one time, a pure ustilaginoidin A product with the purity of more than 500mg and the purity of more than 95% can be prepared at one time, the sample loading amount is large, the separation and purification steps are simple, expensive precise instruments are not needed, the yield of the pure ustilagin is improved while the production cost is reduced, and the preparation method has the advantages of large preparation amount, high recovery rate, low cost and strong practicability. According to the application, the two-step method of macroporous adsorption resin column chromatography and high-speed counter-current chromatography is adopted to purify the ustilaginoidin A, so that the raw material loss caused by multi-step preparation and purification is reduced.

The method for preparing the pure ustilaginoidin A product in the embodiment of the application is stable, and can be suitable for extracting and purifying the ustilaginoidin A in grain matrixes such as rice koji balls and rice. Wherein the rice koji ball powder can be prepared by collecting rice koji balls on rice, drying and crushing; the Rhizoctonia solani culture powder can be prepared by inoculating Rhizoctonia solani into grain culture medium such as rice, culturing, oven drying fermented grain, and pulverizing.

In the embodiment of the application, the aqueous solution containing 1.0% of formic acid and 0.1% of tween-20 is preferably used as the ultrasonic extracting solution, so that the content of formic acid is low, and the purification cost is obviously reduced; in the embodiment of the application, the material-liquid ratio adopted by the ultrasonic extraction is preferably 1/5-1/20, and the extraction time is 30 min; the ultrasonic extracting solution is preferably filtered by using low-speed or medium-speed filter paper to remove solid impurities, a centrifugal machine is not needed, and the operation is more convenient and faster compared with the conventional centrifugal method.

In the present embodiment, it is preferable that the pH of the crude extract of ustilaginoidin a in step (2) is adjusted to neutral, so that the ustilaginoidin a in the extract exists in a molecular form, thereby increasing the adsorption amount of the resin. When the crude extraction liquid of the ustilaginoidea virens A is subjected to column chromatography, a weak-polarity macroporous adsorption resin column HZ-801 is preferably adopted in the embodiment of the application, and the sample is loaded onto the macroporous adsorption resin column at the flow rate of 2-3 BV/h; preferably, 20-30% volume fraction methanol aqueous solution is used as eluent, gradient elution is preferably performed during elution, for example, 20% methanol aqueous solution is used for washing for 1BV, then 30% methanol aqueous solution is used for washing for 2BV, elution components are collected by a fraction collector, and then the content of the ustilagin A in each tube of eluent is detected. In the embodiment of the application, the content of the ustilagin A in each tube of eluent is preferably detected by TLC, and the volume ratio of the adopted developing solvent is 1:1, and the color developing agent is an n-butyl alcohol solution containing ninhydrin with volume fraction of 1.5%.

Before the separation and purification of the ustilaginoidin A-containing eluent after column chromatography is carried out by high-speed counter-current chromatography, the ustilaginoidin A-containing eluent is preferably subjected to rotary evaporation until no methanol remains in the eluent, so that the residual methanol is prevented from influencing the subsequent high-speed counter-current chromatography separation. In the embodiment of the application, the preferred high-speed countercurrent chromatography adopts n-butanol in a volume ratio: water: trifluoroacetic acid is 1:1:0.02 solvent system, the upper layer solvent is stationary phase, the lower layer solvent is mobile phase, the flow rate of the mobile phase is 1.0-1.5 mL/min, the rotating speed of the instrument is 900-1200 rpm, the operating temperature is 25-40 ℃, and the eluents of different time periods are quantitatively collected. In the embodiment of the application, TLC is preferably adopted to detect the content of the ustilaginoidin A in the eluent, and the adopted developing solvent is a mixture of the following components in a volume ratio of 1:1, and the color developing agent is an n-butyl alcohol solution containing ninhydrin with volume fraction of 1.5%.

In the preferred step (4) of the embodiment of the application, the eluates containing only ustiloxin A are selected, combined and then subjected to rotary evaporation to dryness.

For better understanding of the above technical solutions, the following detailed descriptions will be provided with reference to the drawings and specific embodiments of the specification, but the present invention is not limited thereto.