Separation and purification method of abamectin

Abstract

The invention provides a separation and purification method of abamectin, and belongs to the technical field of biopesticide refining. According to the invention, high-speed counter-current chromatography (HSCCC) is adopted to separate an abamectin active compound (1%-99%), and a target product is obtained. Compared with the traditional process, the method has the advantages that the high-speed counter-current chromatography is simple to operate, short in production cycle, monitorable in process and good in repeatability, the used mobile phase can be recycled, industrial wastewater generated in the process of the traditional extraction method is greatly reduced, the later wastewater treatment cost is reduced, and the process is green, environment-friendly, non-toxic and harmless. The purity of abamectin B1a in the target product is greater than or equal to 99.5%, and the purity of obtained B1b is greater than or equal to 94.5%.

Description

Technical Field

[0001] The present invention relates to the technical field of biopesticide refining, in particular to a method for separating and purifying avermectin. Background Art

[0002] Abamectin is a low-toxic, highly effective, highly selective, green, and environmentally friendly biopesticide that completely eliminates the environmental pollution issues associated with traditional pesticides. Currently one of the world's most effective insecticides, abamectin boasts a domestic usage of approximately 2,500 tons, making it a leading pesticide.

[0003] Abamectin is composed of eight components with similar structures and properties, the basic structure of which is a 16-membered macrolide disoside. Component a (A1a, A2a, B1a, and B2a) is the major component, accounting for over 80%, while component b (A1b, A2b, B1b, and B2b) is the minor component, accounting for less than 20%. The differences in substituents and structures at positions C5, C22-C23, and C25 primarily distinguish the eight components. The distinction between A and B is based on the difference in the substituent at C5. The C5 substituent R1 of component A is CH3, while the C5 substituent R1 of component B is H. The distinction between 1 and 2 is based on the difference in XY at C22-C23. XY in component 1 is CH=CH, while XY in component 2 is CH2-CH(OH). The distinction between a and b is based on the difference in the substituent R2 at C25. The C25 substituent R2 of component a is C2H5, and the C25 substituent R2 of component b is CH3.

[0004] Among these eight components, B1a has the strongest insecticidal activity. Among B1, B1a is more active than B1b, so the focus of avermectin purification is also on how to obtain a high-purity B1a component. The molecular structure of avermectin B1a is very different from that of other components, which also makes their physical properties similar. This is a major difficulty in preparing a high-purity B1a component. In the current avermectin refining process, alcohols are mainly used for refined crystallization. The production steps generally include: heating and dissolving, filtration, concentration, cooling and crystallization, etc., and the purpose of improving the purity of the crystals is achieved through cyclic refining. The operation steps are cumbersome and the purity is not high enough. Summary of the Invention

[0005] The object of the present invention is to provide a method for separating and purifying avermectin, which is used to solve the technical problems of complicated steps and low purity of avermectin separation and purification methods in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for separating and purifying avermectin, comprising the following steps:

[0008] Step 1), taking the crude extract of the avermectin fermentation broth and mixing it with ethanol, then heating and filtering it in sequence, and removing the ash to obtain a solution;

[0009] Step 2), mixing the solution with water, allowing it to stand, and filtering it with suction to obtain a crude avermectin product;

[0010] Step 3) preparing a solvent system, and after standing, obtaining an upper phase and a lower phase, respectively, which are used as one of the mobile phase and the stationary phase;

[0011] Taking another stationary phase as a sample solvent, dissolving the crude avermectin in the sample solvent, and performing ultrasonic heating to dissolve the crude avermectin until saturation, thereby obtaining a sample solution;

[0012] Step 4) The mobile phase and the stationary phase are pumped into a high-speed countercurrent chromatograph respectively. After equilibrium, the sample is injected from the injection port, and the mobile phase is continuously pumped in for elution. The components are collected and then concentrated by rotary evaporation and dried to obtain avermectin B1a and avermectin B1b.

[0013] Furthermore, in step 1), the mass ratio of the avermectin fermentation broth to ethanol is 1:15.

[0014] Furthermore, in step 1), the heating temperature is 45-55°C.

[0015] Furthermore, the volume ratio of the ethanol to water is 2-5:1-4.

[0016] Furthermore, in step 2), the mixing is carried out while stirring, the stirring speed is 100 to 300 rpm, and the stirring time is until the system cools to room temperature;

[0017] The standing time is 10 to 15 hours.

[0018] Furthermore, in step 3), the solvent system is three or four of ethyl acetate, methanol, n-hexane, water, ethanol, dichloromethane and acetonitrile.

[0019] Furthermore, in step 3), when the solvent system is composed of four phases, the volume ratio of each phase is 1-5:1-3:1-5:1-3 in order of increasing polarity;

[0020] When the solvent system consists of three phases, the volume ratio of each phase is 1-5:1-3:1-5 in order of increasing polarity.

[0021] Furthermore, in step 3), the injection volume is 15 to 30 mL, and the elution flow rate is 5 to 15 mL / min;

[0022] The elution rotation speed is 700-900 rpm.

[0023] Furthermore, in step 3), the temperature of the ultrasonic heating is 40-60° C., and the frequency of the ultrasonic heating is 20 kHz-100 kHz.

[0024] Rotary evaporation enrichment is to remove the mobile phase and filter it, then dissolve the filtered part in an organic solvent and continue rotary evaporation.

[0025] Furthermore, in step 3), the drying is vacuum drying, and the drying temperature is 45-60°C.

[0026] Beneficial effects of the present invention:

[0027] The separation and purification method of the present invention has a purity of avermectin B1a of ≥99.5%, and a purity of B1b of ≥94.5%. The process is simple, the purification effect is good, and the yield is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a chromatogram of the separation of avermectin by HSCCC in Example 1, wherein the horizontal axis is in min, the horizontal axis unit length is 6, and the origin is 0; the vertical axis is in mV, the vertical axis unit length is 20, the origin is 0, and the first coordinate point on the vertical axis close to the origin is (0, 40 mV);

[0029] Figure 2 is the liquid chromatogram of avermectin B1a in Example 1;

[0030] Figure 3 This is the liquid chromatogram of Avermectin B1b in Example 1. DETAILED DESCRIPTION

[0031] The present invention provides a method for separating and purifying avermectin, comprising the following steps:

[0032] Step 1), taking the crude extract of the avermectin fermentation broth and mixing it with ethanol, then heating and filtering it in sequence, and removing the ash to obtain a solution;

[0033] Step 2), mixing the solution with water, allowing it to stand, and filtering it with suction to obtain a crude avermectin product;

[0034] Step 3) preparing a solvent system, and after standing, obtaining an upper phase and a lower phase, respectively, which are used as one of the mobile phase and the stationary phase;

[0035] Taking another stationary phase as a sample solvent, dissolving the crude avermectin in the sample solvent, and performing ultrasonic heating to dissolve the crude avermectin until saturation, thereby obtaining a sample solution;

[0036] Step 4) The mobile phase and the stationary phase are pumped into a high-speed countercurrent chromatograph respectively. After equilibrium, the sample is injected from the injection port, and the mobile phase is continuously pumped in for elution. The components are collected and then concentrated by rotary evaporation and dried to obtain avermectin B1a and avermectin B1b.

[0037] In the present invention, in step 1), the mass ratio of the avermectin fermentation broth to ethanol is 1:15.

[0038] In the present invention, in step 1), the heating temperature is 45-55°C, preferably 48-52°C, and more preferably 50°C.

[0039] In the present invention, the volume ratio of ethanol to water is 2-5:1-4, preferably 4:3.

[0040] In the present invention, in step 2), the mixing is carried out under stirring, the stirring speed is 100 to 300 rpm, preferably 200 rpm, and the stirring time is until the system cools to room temperature;

[0041] The standing time is 10 to 15 hours, preferably 12 to 14 hours, and more preferably 13 hours.

[0042] In the present invention, in step 3), the solvent system is preferably three or four of ethyl acetate, methanol, n-hexane, water, ethanol, dichloromethane and acetonitrile.

[0043] In the present invention, in step 3), when the solvent system is composed of four phases, the volume ratio of each phase is 1-5:1-3:1-5:1-3, preferably 2-4:2:2-4:2, in order from small to large polarity;

[0044] When the solvent system is composed of three phases, the volume ratio of each phase is 1-5:1-3:1-5, preferably 2-4:2:2-4, in order from small to large polarity.

[0045] In the present invention, the solvent system is allowed to stand and separated to obtain upper and lower phases, which are then ultrasonicated and cooled before being used as stationary phases, mobile phases or sample solvents.

[0046] In the present invention, the ultrasonic time is preferably 15 minutes; the ultrasonic frequency is preferably 20 kHz to 100 kHz, more preferably 50 kHz; and the cooling time is preferably 10 minutes.

[0047] In the present invention, in step 3), the injection volume is 15 to 30 mL, preferably 18 to 25 mL, and more preferably 20 mL; the elution flow rate is 5 to 15 mL / min, preferably 8 to 12 mL / min, and more preferably 10 mL / min;

[0048] The elution rotation speed is 700-900 rpm, preferably 800 rpm.

[0049] In the present invention, in step 3), the temperature of the ultrasonic heating is 40-60° C., preferably 50° C.; the frequency of the ultrasonic heating is 20 kHz-100 kHz, preferably 50 kHz.

[0050] In the present invention, the crude product is mixed with the stationary phase and ultrasonically heated to promote dissolution, which can reduce the injection volume and increase the sample loading amount.

[0051] Rotary evaporation enrichment is to filter the mobile phase after rotary extraction. Abamectin is insoluble in water, so the filtered portion is dissolved in an organic solvent and rotary evaporation is continued.

[0052] In the present invention, the organic solvent is preferably dichloromethane.

[0053] In the present invention, in step 3), the drying is vacuum drying, and the drying temperature is 45-60°C, preferably 50°C.

[0054] In the present invention, reagents such as avermectin technical, ethyl acetate, methanol, n-hexane, water, ethanol, dichloromethane, acetonitrile, and molecular sieves are all commercially available. The high-speed countercurrent chromatography equipment is a Doda HSCCC-TBE300C. The high-performance liquid chromatograph used in the present invention is an Agilent 1260. Liquid phase detection conditions are as follows: mobile phase: (methanol:acetonitrile:water) = 45:35:20, flow rate: 1 ml / min, column temperature: 25°C, detection wavelength: 245 nm, chromatographic column: Eclipse Plus C18.

[0055] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Weigh 20g of crude extract from the avermectin fermentation broth, add 5 times the mass of ethanol, and heat to 50°C to completely dissolve it. Then, filter and remove the ash to obtain a solution. Add distilled water to the solution to achieve a volume ratio of 4:3 between ethanol and distilled water. Cool the solution to room temperature while stirring at 200 rpm. Let it stand for 12 hours, then filter to obtain a crude avermectin product with a B1a content of ≥60%.

[0058] A solvent system was prepared with a volume ratio of 1:1:1:1 of n-hexane, ethyl acetate, methanol, and water. After standing and separating the liquids, the upper and lower phases were separated. The upper and lower phases were ultrasonically treated at a frequency of 50 kHz for 15 minutes and then cooled for 10 minutes. The upper phase was used as the stationary phase and the lower phase as the mobile phase. The upper phase was used as the sample solvent, and the crude avermectin was dissolved in the upper phase. Ultrasonic heating was performed at a frequency of 50 kHz and a temperature of 50°C until saturation, thereby obtaining a sample solution.

[0059] The mobile phase and the stationary phase were pumped into the high-speed countercurrent chromatograph respectively. After equilibrium, 20 mL of sample was injected from the injection port. The mobile phase was continuously pumped in. The elution was performed at a flow rate of 10 mL / min and a rotation speed of 800 r / min.

[0060] The fraction between 65 and 75 minutes was collected as the first sample, i.e., Abamectin B1b; the fraction between 105 and 130 minutes was collected as the second sample, i.e., Abamectin B1a. After the last peak, the sample was injected again.

[0061] The obtained samples were separately concentrated by rotary evaporation, the mobile phase was removed and filtered, the filtered portion was dissolved in dichloromethane and rotary evaporated again to remove residual moisture, vacuum dried at 50°C for 24 hours, and ground to obtain avermectin B1b with a purity of 95.01% and avermectin B1a with a purity of 99.95%.

[0062] Example 2

[0063] Different from Example 1, in this example, the solvent system has a volume ratio of n-hexane: ethyl acetate: methanol: water = 3:1:2:2.

[0064] Example 3

[0065] Different from Example 1, in this example, the solvent system has a volume ratio of dichloromethane:methanol:water=1:1:1.

[0066] Example 4

[0067] Different from Example 1, in this example, the solvent system has a volume ratio of ethyl acetate: n-butanol: water = 1:1:2.

[0068] Example 5

[0069] Different from Example 1, in this example, the solvent system has a volume ratio of ethyl acetate: n-butanol: water = 1:1:2.

[0070] Examples 5 to 8

[0071] Different from Example 2, in Examples 5 to 8, the rotation speed of the high-speed countercurrent chromatograph and the elution speed of the mobile phase are different.

[0072] The contents of avermectin B1a and B1b obtained in Examples 1 to 8 and the distinguishing extraction parameters are shown in Table 1. Table 1 The contents of avermectin B1a and B1b obtained in Examples 1 to 8 (area normalization method) and the distinguishing extraction parameters

[0073]

[0074] As can be seen from the above examples, the present invention provides a method for separating and purifying avermectin. As shown in Table 1, compared with conventional crystallization processes, the product produced using high-speed countercurrent chromatography has a higher avermectin B1a content and lower impurity content, while also producing high-purity B1b. The process is simple and can be continuously produced.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for separating and purifying avermectin, characterized in that: The following steps are involved: Step 1), taking the crude extract of the avermectin fermentation broth and mixing it with ethanol, then heating and filtering it in sequence, and removing the ash to obtain a solution; Step 2), mixing the solution with water, allowing it to stand, and filtering it with suction to obtain a crude avermectin product; Step 3) preparing a solvent system, and after standing, obtaining an upper phase and a lower phase, respectively, which are used as one of the mobile phase and the stationary phase; Taking another stationary phase as a sample solvent, dissolving the crude avermectin in the sample solvent, and performing ultrasonic heating to dissolve the crude avermectin until saturation, thereby obtaining a sample solution; Step 4) The mobile phase and the stationary phase are pumped into a high-speed countercurrent chromatograph respectively. After equilibrium, the sample is injected from the injection port, and the mobile phase is continuously pumped in for elution. The components are collected and then concentrated by rotary evaporation and dried to obtain avermectin B1a and avermectin B1b.

2. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 1), the mass ratio of the avermectin fermentation broth to ethanol is 1:

15.

3. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 1), the heating temperature is 45-55°C.

4. A method for separation and purification of avermectin according to claim 1, characterized in that, The volume ratio of the ethanol to the water is 2-5:1-4.

5. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 2), the mixing is carried out while stirring at a speed of 100 to 300 rpm, and the stirring time is until the system cools to room temperature; The standing time is 10 to 15 hours.

6. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 3), the solvent system is three or four of ethyl acetate, methanol, n-hexane, water, ethanol, dichloromethane and acetonitrile.

7. A method for separation and purification of avermectin according to claim 6, characterized in that, In step 3), when the solvent system is composed of four phases, the volume ratio of each phase is 1-5:1-3:1-5:1-3 in order from small to large polarity; When the solvent system consists of three phases, the volume ratio of each phase is 1-5:1-3:1-5 in order of increasing polarity.

8. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 3), the injection volume is 15-30 mL, and the elution flow rate is 5-15 mL / min; The elution speed is 700-900 rpm.

9. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 3), the temperature of the ultrasonic heating is 40-60° C., and the frequency of the ultrasonic heating is 20 kHz-100 kHz. Rotary evaporation enrichment is to remove the mobile phase and filter it, then dissolve the filtered part in an organic solvent and continue rotary evaporation.

10. A method for separation and purification of avermectin according to claim 1, characterized in that, In step 3), the drying is vacuum drying at a drying temperature of 45 to 60°C.

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