阅读说明：本技术 3-苄基-6-甲基-2,5-哌嗪二酮的制备方法和应用 (Preparation method and application of 3-benzyl-6-methyl-2, 5-piperazinedione ) 是由 王磊 李镇标 郭燕锋 黄捷 姜仁旭 于 2021-08-31 设计创作，主要内容包括：本发明公开了一种3-苄基-6-甲基-2,5-哌嗪二酮的制备方法,将脱色芽孢杆菌进行发酵,将得到的发酵液进行固液分离,得到的上清液加入大孔树脂进行吸附,吸附后过滤得到树脂；加入无水乙醇进行洗脱,收集洗脱液,经旋转蒸发后以甲醇溶解,用滤膜除去不溶物,得到活性成分Ⅰ；将活性成分Ⅰ用硅胶柱进行进一步纯化,上样后先后用4种洗脱液进行洗脱,收集组分10,经旋转蒸发后以甲醇溶解,用滤膜除去不溶物；进一步用制备液相色谱分离：梯度洗脱后收集11～12min的洗脱液,经旋转蒸发后,进行冻干,得到3-苄基-6-甲基-2,5-哌嗪二酮。本发明制备方法简单易行,便于工业化生产,制备得到的3-苄基-6-甲基-2,5-哌嗪二酮可用于防治致病性尖孢镰刀菌导致的香蕉枯萎病。(The invention discloses a preparation method of 3-benzyl-6-methyl-2, 5-piperazinedione, which comprises the steps of fermenting bacillus decolorationis, carrying out solid-liquid separation on the obtained fermentation liquor, adding macroporous resin into the obtained supernatant for adsorption, and filtering to obtain resin after adsorption; adding absolute ethyl alcohol for elution, collecting eluent, dissolving the eluent by methanol after rotary evaporation, and removing insoluble substances by a filter membrane to obtain an active ingredient I; further purifying the active ingredient I with silica gel column, eluting with 4 kinds of eluents after loading, collecting component 10, dissolving with methanol after rotary evaporation, and removing insoluble substances with filter membrane; further separating by preparative liquid chromatography: and (3) after gradient elution, collecting the eluent for 11-12 min, and freeze-drying after rotary evaporation to obtain the 3-benzyl-6-methyl-2, 5-piperazinedione. The preparation method is simple and feasible, and is convenient for industrial production, and the prepared 3-benzyl-6-methyl-2, 5-piperazinedione can be used for preventing and treating banana wilt caused by pathogenic fusarium oxysporum.)

A method for preparing 3-benzyl-6-methyl-2, 5-piperazinedione is characterized by comprising the following steps:

(1) fermenting decolorizing bacillus, and performing solid-liquid separation on the obtained fermentation liquor, wherein the decolorizing bacillus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020498; and (3) after solid-liquid separation, adding the obtained supernatant into macroporous resin for adsorption for 6-24h, wherein the volume ratio of the macroporous resin to the supernatant is (1: 10-1): 30, adsorbing and filtering to obtain resin; collecting resin, adding absolute ethyl alcohol for elution, collecting eluent, dissolving the eluent by methanol after rotary evaporation, and removing insoluble substances by a filter membrane to obtain an active ingredient I;

(2) loading the active ingredient I into a column by using 200-300-mesh silica gel, and adding an eluent for elution: the eluent is (petroleum ether): ethyl acetate ═ 3: 1; ② petroleum ether: ethyl acetate ═ 1: 1; ③ Dichloromethane: methanol 3: 1; sequentially eluting according to the serial number sequence of the eluents, collecting the components eluted by the third eluent, dissolving the components in methanol after rotary evaporation, and removing insoluble substances by using a filter membrane;

(3) and (3) carrying out preparative liquid chromatography separation on the components after insoluble substances are removed in the step (2): wherein the mobile phase is acetonitrile containing 0.1% TFA and water containing 0.1% TFA, gradient elution is carried out at 21mL/min, eluent of 11-12 min is collected, and freeze-drying is carried out after rotary evaporation to obtain the 3-benzyl-6-methyl-2, 5-piperazinedione.

2. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the culture medium used for fermenting the bacillus decolorationis in the step (1) is an LB culture medium.

3. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the fermentation conditions in the step (1) are 30-37 ℃, 200-220 rpm/min and 36 hours.

4. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the solid-liquid separation mode in the step (1) is centrifugation under the conditions of 6000-.

5. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the dosage of the absolute ethyl alcohol in the step (1) is 1.5 to 2.5 times of the volume of the macroporous resin.

6. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the elution time of the absolute ethyl alcohol in the step (1) is 0.5-3 h.

7. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: in the step (2), each eluent is 400mL, each eluent is 100mL and is a collection unit, the eluent is numbered as components 1-12 according to the elution sequence, and the component 10 is collected for rotary evaporation.

8. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the filter membranes in the steps (1) and (2) are filter membranes with the pore diameter of 0.22 mu m.

9. The process for preparing 3-benzyl-6-methyl-2, 5-piperazinedione as claimed in claim 1, wherein: the column for preparing the liquid chromatogram in the step (3) is Aglilent Pursuit XRs C18.

10. The application of the 3-benzyl-6-methyl-2, 5-piperazinedione prepared by the method according to any one of claims 1 to 9 in inhibiting fusarium oxysporum and preventing and treating banana wilt.

Technical Field

The invention relates to the field of microbial metabolites and application thereof, in particular to a preparation method and application of 3-benzyl-6-methyl-2, 5-piperazinedione.

Background

3-benzyl-6-methyl-2, 5-piperazinedione is one of the piperazinediones, and has been reported to be isolated and identified from tubers of the medicinal plant pinellia ternata, and can also be obtained through a conventional chemical synthesis route. However, the production of 3-benzyl-6-methyl-2, 5-piperazinedione from the metabolite has not been reported.

Disclosure of Invention

The invention aims to provide a preparation method of 3-benzyl-6-methyl-2, 5-piperazinedione, which is prepared from a microorganism source for the first time, is simple and feasible, and is convenient for industrial production. .

In order to achieve the purpose, the invention adopts the following technical scheme:

the preparation method of the 3-benzyl-6-methyl-2, 5-piperazinedione is characterized by comprising the following steps:

(1) fermenting decolorizing bacillus, and performing solid-liquid separation on the obtained fermentation liquor, wherein the decolorizing bacillus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020498; and (3) after solid-liquid separation, adding the obtained supernatant into macroporous resin for adsorption for 6-24h, wherein the volume ratio of the macroporous resin to the supernatant is (1: 10-1): 30, adsorbing and filtering to obtain resin; collecting resin, adding absolute ethyl alcohol for elution, collecting eluent, dissolving the eluent by methanol after rotary evaporation, and removing insoluble substances by a filter membrane to obtain an active ingredient I;

(2) loading the active ingredient I into a column by using 200-300-mesh silica gel, and adding an eluent for elution: the eluent is (petroleum ether): ethyl acetate ═ 3: 1; ② petroleum ether: ethyl acetate ═ 1: 1; ③ Dichloromethane: methanol 3: 1; sequentially eluting according to the serial number sequence of the eluents, collecting the components eluted by the third eluent, dissolving the components in methanol after rotary evaporation, and removing insoluble substances by using a filter membrane;

(3) and (3) carrying out preparative liquid chromatography separation on the components after insoluble substances are removed in the step (2): wherein the mobile phase is acetonitrile containing 0.1% TFA and water containing 0.1% TFA, gradient elution is carried out at 21mL/min, eluent of 11-12 min is collected, and freeze-drying is carried out after rotary evaporation to obtain the 3-benzyl-6-methyl-2, 5-piperazinedione.

The 3-benzyl-6-methyl-2, 5-piperazinedione is prepared from microorganism sources (through steps of fermentation of bacillus decolorationis B1032 and the like) for the first time, the preparation method is not reported, the preparation method is simple, and the industrial production is facilitated; the prepared 3-benzyl-6-methyl-2, 5-piperazinedione has a strong inhibiting effect on fusarium oxysporum, provides a new choice for preparing a novel fusarium oxysporum biocontrol preparation, and can be used for preventing and treating banana wilt caused by pathogenic fusarium oxysporum.

3-benzyl-6-methyl-2, 5-piperazinedione (C)₁₁H₁₂N₂O₂) The structural formula of (A) is as follows:

the Bacillus decolorationis B1032 in the step (1) has a preservation number of CCTCC NO: M2020498, is preserved in China center for type culture Collection at Wuhan university in Wuhan, China at 9 month and 14 days of 2020, and is disclosed in China patent application CN 112175876A.

Further, the culture medium used for fermenting the bacillus decolorationis in the step (1) is LB culture medium. The LB culture medium is selected, and the components are simple and convenient, so that large-scale fermentation and preparation are easy, and the production is large-scale.

Further, the fermentation conditions in the step (1) are 30-37 ℃, 200-220 rpm/min and 36 hours. Under the fermentation condition, the OD600 of the decolorizing bacillus B1032 can reach more than 2.0, and the density of the bacteria reaches 1 × 10⁸More than cfu/ml. Due to high density of bacteria, the content of metabolites is high.

Further, the solid-liquid separation mode in the step (1) is centrifugation, and the conditions are 6000-12000rpm/min and 20 min.

Further, the dosage of the absolute ethyl alcohol in the step (1) is 1.5 to 2.5 times of the volume of the macroporous resin. At this level, the macroporous resin can be covered with absolute ethanol, and the substances in the macroporous resin can be eluted as completely as possible, and the eluent is not too dilute. If the dosage is too much, the content of eluted substances in the eluent is low; if the amount is too small, the material in the macroporous resin cannot be completely eluted.

Further, the elution time of the absolute ethyl alcohol in the step (1) is 0.5-3 h. Suitable elution times allow more ethanol to pass through the macroporous resin, eluting the adsorbed material, with efficiency.

Further, in the step (2), each eluent is 400mL, each 100mL is a collection unit, the elution units are numbered as components 1-12 according to the elution sequence, and the component 10 is collected for rotary evaporation. The components after eluting with the third eluent, i.e. 9-12, can be selected, and if component 10 is selected, the content of the target substance is higher.

Furthermore, the filter membranes in the steps (1) and (2) are filter membranes with the pore size of 0.22 μm. The use of a filter of this pore size is just as effective in intercepting the passage of microorganisms.

Further, the column for preparative liquid chromatography described in step (3) was an Aglilent Pursuit XRs C18.

The invention also provides application of the 3-benzyl-6-methyl-2, 5-piperazinedione prepared by the method in inhibiting fusarium oxysporum and preventing and treating banana wilt.

Banana wilt can cause devastating attacks on the banana industry, the main pathogenic bacterium is fusarium oxysporum Foc TR4, which is first discovered in taiwan area of china, erupted in malaysia and indonesia in the last 90 th century, rapidly spread to the world through the import and export trade of bananas, and then discovered in australia, south china, vietnam, laos, philippines, india, pakistan, israel, libamon, jodan, aman, and morsbick. The main pathogenic bacterium causing banana wilt is fusarium oxysporum.

Fusarium oxysporum is a plant fungus which can live in soil, can infect various plants, and causes harm to cucurbitaceae plants, bananas, cotton, tomatoes and other economic crops. The hypha and spore of fusarium oxysporum can survive in soil for several 10 years, and the hypha, spore and sclerotium can overwinter in soil and diseased plants to become an infection source and are spread in a long distance mainly through rainwater and other ways. After fusarium oxysporum invades the plant, cell wall degrading enzymes are secreted, so that pectin blocks the duct of the plant, the water transportation of a host is influenced, and the plant is withered and dead in severe cases. In addition, fusarium oxysporum can secrete fusaric acid (5-butyl-2-picolinic acid), and the pathogenic toxin can damage the cell membrane of the root system of the host, so that the permeability of the cell membrane of the root system is increased, and the host can be infected by pathogenic bacteria more easily; the toxin reduces the content of active oxygen in host mitochondria, prevents ATP synthesis, further hinders the water absorption of plants, and inhibits the growth of the plants; toxins also reduce the germination rate of plant seeds and affect the morphology of the seeds. Besides fusaric acid, fusarium oxysporum secretes toxins such as dehydrofusaric acid, fumonisin, enniatin, moniliformin, beauvericin, katoxin and the like, which are considered as main causes of plant injury caused by pathogenic bacteria. Since the pathogenic fusarium oxysporum is harmful to crops and the economic loss is particularly serious, a method for efficiently controlling fusarium oxysporum is required to be developed.

The 3-benzyl-6-methyl-2, 5-piperazinedione prepared by the invention has strong inhibition effect on fusarium oxysporum, provides a new choice for preparing a novel fusarium oxysporum biocontrol preparation, and can be used for preventing and treating banana wilt caused by pathogenic fusarium oxysporum.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Drawings

FIG. 1: gas-mass spectrum of 3-benzyl-6-methyl-2, 5-piperazinedione prepared in example 1.

FIG. 2: fourier infrared spectrum of 3-benzyl-6-methyl-2, 5-piperazinedione prepared in example 1.

FIG. 3: the structural formula of the 3-benzyl-6-methyl-2, 5-piperazinedione is shown in the specification.

FIG. 4: the effect of 3-benzyl-6-methyl-2, 5-piperazinedione on fusarium oxysporum (0.5mg/ml) prepared in example 1 is shown (note: P is the fermentation product 3-benzyl-6-methyl-2, 5-piperazinedione, CK is the negative control methanol).

Detailed Description

The present invention is illustrated below by way of specific examples, and it should be understood that the specific examples described herein are illustrative of the embodiments of the present invention only and are not limiting of the embodiments of the present invention. It should be further noted that, for the convenience of description, only some but not all of the features relating to the embodiments of the present invention are shown in the drawings. While various embodiments of the present invention will provide those skilled in the art with a more complete understanding of the present invention, the following embodiments may be modified in many different ways and are intended to be included within the scope of the present invention.

Bacterial strain and culture medium

Activation of fusarium oxysporum: transferring fusarium oxysporum stored at-25 ℃ into a PDA culture medium from an inclined plane, and placing the fusarium oxysporum in a biochemical incubator for culturing for 3-7 days at 28 ℃ until hyphae grow over the whole flat plate.

The PDA culture medium formula is as follows: 20% of potato leachate, 20g of glucose, 3g of monopotassium phosphate, 1.5g of magnesium sulfate, 12g of agar powder and 1000mL of distilled water, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 min. Note: weighing 200g of potatoes, cutting into small pieces, putting into 1000mL of boiling water, boiling for 20min, cooling, filtering with 400-mesh gauze to obtain potato leachate, adding the above reagents, and diluting to 1000 mL.

Example 1

The preparation method of 3-benzyl-6-methyl-2, 5-piperazinedione of the present example is as follows:

S1：

s11: activating decolorizing bacillus: marking and activating bacillus decolorationis B1032 stored at minus 80 ℃ on an LB flat plate, culturing overnight in an incubator at 37 ℃, selecting a single colony to be inoculated in 10mL of LB liquid culture medium, carrying out shaking culture at 37 ℃ and 220r/min for 12h to obtain a seed solution, inoculating the seed solution into 1000mL of LB liquid culture medium according to the inoculum size of 1% (v/v), carrying out shaking culture at 37 ℃ and 220r/min for 36h until OD600 reaches 2.0, centrifuging at 12000rpm/min for 20min to remove thalli, and collecting a supernatant.

Bacillus decolorationis B1032, CCTCC NO: M2020498, has been deposited in China center for type culture Collection at Wuhan university in Wuhan, China at 9/14/2020 and is disclosed in Chinese patent application CN 112175876A.

LB liquid medium: 10g of peptone Tryptone, 5g of Yeast powder Yeast extract, 7g of sodium chloride NaCl and 1000mL of distilled water, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 min.

S12: adding the supernatant obtained in the step S11 into a mixed solution according to the volume ratio of the feed liquid of 1: 30 adding macroporous resin HP20 for adsorption for 24h, and filtering to obtain resin after adsorption and extraction; filtering and collecting resin, adding anhydrous ethanol with the volume 2 times of that of the macroporous resin, eluting for 3h, collecting eluate, dissolving with methanol after rotary evaporation, and removing insoluble substances with filter membrane with pore diameter of 0.22 μm to obtain active ingredient I.

S2: loading the column with 200-300 mesh silica gel, wherein the height of the column is 30cm, the diameter of the column is 3cm, loading the active ingredient I, and adding an eluant for elution: the eluent is (petroleum ether): ethyl acetate ═ 3: 1; ② petroleum ether: ethyl acetate ═ 1: 1; ③ Dichloromethane: methanol 3: 1; 400mL and 100mL of each eluent are used as a collection unit, the eluent is numbered from 1 to 12 according to the elution sequence, and the 10 th component is collected; after rotary evaporation, the insoluble matter was removed by dissolving in methanol and filtering with a filter having a pore size of 0.22. mu.m.

S3: preparative liquid chromatography was performed on fraction 10 of example two S2: the method comprises the following steps of carrying out liquid chromatography on a column of aglent Pursuit XRs C18, carrying out gradient elution on the mobile phase A acetonitrile (containing 0.1% TFA) and B water (containing 0.1% TFA) at a rate of 21mL/min, collecting eluent for 11-12 min, carrying out rotary evaporation, and carrying out freeze-drying to obtain the 3-benzyl-6-methyl-2, 5-piperazinedione.

Specifically, the gradient elution conditions are shown in the following table:

TABLE 1 preparative liquid chromatography gradient elution conditions

Example 2:

the preparation method of 3-benzyl-6-methyl-2, 5-piperazinedione in this example is substantially the same as that in example 1, and only S11 is different from S12, specifically:

s11: activating decolorizing bacillus: marking and activating bacillus decolorationis B1032 stored at minus 80 ℃ on an LB flat plate, culturing overnight in an incubator at 30 ℃, selecting a single colony to be inoculated in 10mL of LB liquid culture medium, carrying out shaking culture at 30 ℃ and 200r/min for 12h to obtain a seed solution, inoculating the seed solution into 1000mL of LB liquid culture medium according to the inoculum size of 1% (v/v), carrying out shaking culture at 30 ℃ and 200r/min for 36h, centrifuging at 6000rpm/min for 20min to remove bacteria, and collecting a supernatant.

Bacillus decolorationis B1032, CCTCC NO: M2020498, has been deposited in China center for type culture Collection at Wuhan university in Wuhan, China at 9/14/2020 and is disclosed in Chinese patent application CN 112175876A.

LB liquid medium: 10g of peptone Tryptone, 5g of Yeast powder Yeast extract, 7g of sodium chloride NaCl and 1000mL of distilled water, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 min.

S12: adding the supernatant obtained in the step S11 into a mixed solution according to the volume ratio of the feed liquid of 1:10 adding macroporous resin HP20 for adsorption for 15h, and filtering to obtain resin after adsorption and extraction; filtering and collecting resin, adding anhydrous ethanol with the volume 1.5 times of that of the macroporous resin, eluting for 2h, collecting eluate, dissolving with methanol after rotary evaporation, and removing insoluble substances with filter membrane with pore diameter of 0.22 μm to obtain active ingredient I.

Example 3:

the preparation method of 3-benzyl-6-methyl-2, 5-piperazinedione in this example is substantially the same as that in example 1, and only S11 is different from S12, specifically:

s11: activating decolorizing bacillus: marking and activating bacillus decolorationis B1032 stored at the temperature of minus 80 ℃ on an LB flat plate, culturing overnight in an incubator at the temperature of 30 ℃, selecting a single colony to be inoculated in 10mL of LB liquid culture medium, carrying out shaking culture at the temperature of 34 ℃ and 210r/min for 12h to obtain a seed solution, inoculating the seed solution into 1000mL of LB liquid culture medium according to the inoculum size of 1% (v/v), carrying out shaking culture at the temperature of 34 ℃ and 210r/min for 36h, centrifuging at 10000rpm/min for 20min to remove bacteria, and collecting a supernatant.

Bacillus decolorationis B1032, CCTCC NO: M2020498, has been deposited in China center for type culture Collection at Wuhan university in Wuhan, China at 9/14/2020 and is disclosed in Chinese patent application CN 112175876A.

LB liquid medium: 10g of peptone Tryptone, 5g of Yeast powder Yeast extract, 7g of sodium chloride NaCl and 1000mL of distilled water, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 min.

S12: adding the supernatant obtained in the step S11 into a mixed solution according to the volume ratio of the feed liquid of 1: 20 adding macroporous resin HP20 for adsorption for 6h, and filtering to obtain resin; filtering and collecting resin, adding anhydrous ethanol for elution, wherein the use amount of the anhydrous ethanol is 2.5 times of the volume of the macroporous resin, the elution time is 0.5h, collecting eluent, dissolving the eluent with methanol after rotary evaporation, and removing insoluble substances by using a filter membrane with the pore diameter of 0.22 mu m to obtain the active ingredient I.

Example 4: GC-MS and FTIR detection of the product 3-benzyl-6-methyl-2, 5-piperazinedione of example 1

The product prepared in example 1 was dissolved in an appropriate amount of chromatographic methanol and filtered, followed by GC-MS (gas chromatography-mass spectrometry) separationAnd performing FTIR detection. FIG. 1 is a gas-mass spectrum of the product prepared in example 1, and FIG. 2 is a Fourier infrared spectrum of the product prepared in example 1. The results of GC-MS (see FIG. 1) and FTIR (see FIG. 2) indicate that the product is 3-benzyl-6-methyl-2, 5-piperazinedione having the formula C₁₁H₁₂N₂O₂The structural formula is shown in figure 3.

Example 5: activity detection

Dissolving the product 3-benzyl-6-methyl-2, 5-piperazinedione finally obtained in S3 of example 1 in methanol, adjusting the concentration to 0.5mg/mL, and detecting the bacteriostatic activity of fusarium oxysporum by taking the fusarium oxysporum as an indicator, wherein the specific method comprises the following steps:

1) taking cultured indicator bacteria, preparing spore suspension, and adjusting the concentration of the spore suspension to 1.0 × 10⁴cfu/mL；

2) Coating 100 mu L of spore suspension on a PDA plate;

3) placing sterilized filter paper sheets with the diameter of 6mm at 3 positions 2cm away from the center of the flat plate;

4) 10 mu L of 3-benzyl-6-methyl-2, 5-piperazinedione is added on the filter paper sheet, methanol is used as a negative control, 4 repeated groups are processed in each group, the culture is carried out at the constant temperature of 28 ℃ for 3d, and the diameter of the inhibition zone is measured.

The results show that: the 3-benzyl-6-methyl-2, 5-piperazinedione has a strong inhibition effect on fusarium oxysporum, and the diameter of a 0.5mg/mL inhibition zone is 9mm, which shows that the compound has a good inhibition effect (see fig. 4).

In the same way, the products obtained by the preparation methods described in the embodiments 2-3 are subjected to GC-MS and FTIR detection and bacteriostatic activity detection of Fusarium oxysporum, and the results also show that the products are 3-benzyl-6-methyl-2, 5-piperazinedione and have good bacteriostatic effect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.