CN107501199A - The extraction of fluorine thunder Rana monomeric compound and purification process - Google Patents

Abstract

The invention discloses a method for extracting and purifying a monomeric compound of Frellana, which comprises the following steps: 1) After the Bravecto preparation is pulverized into powder, sequentially use petroleum ether, methylene chloride and methanol for Soxhlet extraction until the reflux liquid is clarified, The dichloromethane extract or the three extracts were rotatably evaporated at low pressure to obtain the crude extract; 2) Weigh the silica gel with the same mass as the crude extract, dissolve it in petroleum ether and pour it into the chromatographic column, record it as 1 height unit, Then add 6 to 8 height units of silica gel; 3) Add the crude extract obtained in step 1) into the chromatography column; 4) Perform chromatographic separation with mixed solutions of petroleum ether-ethyl acetate in different volume ratios to obtain the target product. The method of the present invention can successfully extract the high-purity Frellaner compound from Bravecto, which satisfies the demand for the compound or the detection and testing problems.

Description

Extraction and purification method of frellaner monomer compound

technical field

The invention relates to the fields of medicinal chemistry and biomedicine, in particular to a method for extracting and purifying a novel isoxazole compound, Frellana, and its application in production and life.

Background technique

Isoxazoline compounds are a new class of insecticidal active substances obtained during the study of phthalic diamide and anthranilic diamide. At present, scientific research teams from various countries around the world, such as Japan, the United States, and Europe, have launched the research and development of this type of compound and its mechanism of action.

The novel isoxazole compound Freilaner is characterized in that: the research and development code is A1443, and the chemical name is 4,5-[(3,5-dichlorophenyl)-4,5-dihydro-5-trifluoro Methyl-3-isoxazolyl]-2-methyl-nitrogen-[2-oxo-2-[(2,2,2-trifluoroethyl)amino]ethyl]-benzamide, with p- Enantiomer structure, its chemical structural formula is:

The research and development process of Frellana is roughly as follows: In 2004, Japan’s Nissan Chemical Industry Co., Ltd. Mita and others successfully synthesized Frellana. In 2010, Ozoe et al. studied some of the biological and electrophysiological activities of Frellana in detail. In 2013, García-Reynaga et al. synthesized the radioligand [ ³ H] Frellaner, which was used to study the action site and mechanism of Frellaner at the γ-aminobutyric acid (GABA) receptor. In 2014, Zhao (the first inventor) et al comparatively studied the action sites of Frellana and its analogues on the GABA receptors of insects and mammals; Gassel et al. and Rohdich et al. The biological activity of the animal health innovation institute in Germany; the researchers of the German Animal Health Innovation Institute used Frellana to conduct a series of "clinical tests" on dogs, and tested the biological activity of Frellana on dog parasites and its toxicity to dogs; Lei Lana was approved by the US Food and Drug Administration (trade name Bravecto) to be sold in the European and American markets, with quarterly sales of 100 million US dollars.

Frelaner mainly works by interfering with GABA-gated chloride ion channels, which are similar to the targets of pesticides such as cyclopentadienes, phenylpyrazoles and macrolides. The successful research and development of Frellana has created a new research direction of GABA-gated chloride channel disruptors, which has attracted the attention and favor of veterinary medicine and pesticide scientists. Frellana is a broad-spectrum insecticide, which has good insecticidal activity against pests such as ticks, fleas, lice, hemiptera and diptera, and its toxicity is higher than or equal to that of commonly used insecticides. Insecticides are comparable. Frellana is more active against Ctenocephalides felis than traditional insecticides fipronil and imidacloprid; more active against copper fly (Lucilia cuprina) than fipronil, dieldrin, imidacloprid and deltamethrin Esters; the activity against tiny cattle ticks (Rhipicephalus microplus) is much higher than that of deltamethrin, and the insecticidal activity against fipronil-resistant white planthopper (Laodelphax striatellus) and two-spotted spider mite (Tetranychus urticae) is also better. Not only does Freilaner have no significant cross-resistance with existing insecticides, it even has good insecticidal activity against partially resistant pests. U.S. DuPont Company Lahm etc. have reported a series of isoxazoline compounds similar to Freylana structure, this class compound is to Lepidoptera, as Spodoptera frugiperda (Spodoptera frugiperda) and cotton bollworm (Helicoverpa zea); Pests of the order of Pteroptera, such as Empoasca fabae and of the order of Thysanoptera, such as Frankliniella occidentalis, have shown good insecticidal activity. In addition, Zhao (the first applicant) et al. found through radioligand competitive binding experiments that Freilaner had almost no effect on recombinantly expressed human GABA-gated chloride channels (β3 subunits) in vitro.

At present, Frellana has been commercialized as a veterinary drug and has been applied to the prevention and control of parasites. Since 2013, researchers at the German Institute for Animal Health Innovation have carried out a series of experimental studies on Frellana, using oral feeding methods to compare and study Frellana, Fipronil (Frontline) and bromide Control effect of permethrin (Scalibor) against dog parasites Ctencephalitus and Dermatitis variabilis. The results showed that Frellana had a very good killing effect on comb fleas and ticks. In 2014, Merck first commercialized Frellana as the veterinary drug Bravecto, which is used to kill dog parasites such as lice and fleas. A series of studies have shown that the drug can be quickly absorbed by dogs, and has the characteristics of quick effect, long half-life, long-lasting effect and low metabolic rate. At present, the drug has been approved by the FDA of the United States, and has begun to be sold and promoted in countries such as Europe and the United States. It is expected that its promotion in Asia and even China is just around the corner.

Fluralaner, as a new type of isoxazole insecticide, mainly acts by interfering with GABA-gated chloride ion channels, and interacts with the targets of cyclopentadienes, phenylpyrazoles and macrolides and other insecticides similar. However, current studies have found that fluralaner has a different mechanism of action from traditional GABA receptor-targeted insecticides such as fipronil, ethiprole, butylene fipronil, and abamectin. It plays an extremely important role and significance in insect resistance. The Fluralaner monomer compound is different from the Bravecto preparation, and has its specific research significance in experimental research. At present, fluralaner monomer compounds are in great demand in experimental research, but they are not yet available in the market, and the synthetic route is complex and technically difficult. Therefore, the acquisition of fluralaner has become an urgent problem to be solved.

Bravecto is a new drug with complex ingredients. The content of fluralaner is only 14%. Through HPLC-TOF-MS detection, it is found that there are impurities that are very close to the polarity of fluralaner. It is difficult to extract high-purity fluralaner from it. Therefore, how to Separation of impurities that are extremely close in polarity to fluralaner to obtain high-purity compound fluralaner to meet the needs of scientific research and other aspects such as detection and testing is an urgent problem to be solved at present.

Contents of the invention

The purpose of the present invention is mainly to provide a method for easily and efficiently extracting the monomeric compound of the effective active ingredient Frellana from Bravecto. This method can successfully extract the high-purity Frellana compound from Bravecto, which meets the demand for the compound or the detection and inspection problems.

The extraction and purification method of the Frellaner monomer compound provided by the invention comprises the following steps:

1) After the Bravecto preparation is pulverized into powder, Soxhlet extraction is performed sequentially with petroleum ether, dichloromethane, and methanol until the reflux liquid is clarified, and the dichloromethane extract or the three extracts are respectively evaporated at low pressure to obtain a crude extract;

2) Weigh the silica gel with the same quality as the crude extract, dissolve petroleum ether and pour it into the chromatography column. After the silica gel is completely precipitated, measure the height of the silica gel in the chromatography column, record it as 1 height unit, and then pour it into the chromatography column Add 6 to 8 height units of silica gel;

3) adding the crude extract obtained in step 1) into the chromatographic column;

4) The volume ratios are (7.8～8.2): 1, (5.8～6.2): 1, (3.8～4.2): 1, (2.8～3.2): 1, (1.8～2.2): 1, (0.8～ 1.2): 1, 2: (2.8～3.2) The petroleum ether-ethyl acetate mixed solution is subjected to chromatographic separation to obtain the target product.

In step 1) of the present invention, sherwood oil, dichloromethane, and methanol must be selected successively for Soxhlet extraction. Using other solvents and different extraction sequences to carry out Soxhlet extraction can not achieve a more complete extraction effect, and it is difficult to remove impurities with similar properties. separate. The inventor detected by HPLC-TOF-MS. In the present invention, most of the impurities in Bravecto, including impurities with similar polarity and a small amount of fluralaner, can be extracted by Soxhlet extraction of petroleum ether, and then Soxhlet extraction is performed with dichloromethane A small amount of impurities that do not include similar polarities and most of the fluralaner in Bravecto can be extracted. Finally, Soxhlet extraction with methanol can extract the remaining fluralaner and a small amount of impurities in Bravecto.

Preferably, in step 1) of the Soxhlet extraction process in the present invention, the extraction temperature of petroleum ether is 85-90°C; the extraction temperature of dichloromethane is 59-64°C; and the extraction temperature of methanol is 87-93°C. If it is lower than the above temperature range, the extract cannot be extracted. If it is higher than the above temperature range, the extract will volatilize and cause waste, or the extract will overflow from the condenser tube in large quantities, causing certain danger and waste. Preferably, the extraction temperature of petroleum ether is 88°C, the extraction temperature of dichloromethane is 61°C, and the extraction temperature of methanol is 90°C.

In step 2) of the present invention, silica gel with 7 to 9 height units is selected, and the fluralaner obtained by chromatography has the highest purity and the least impurity content. If the amount of silica gel is changed, the purity of the fluralaner obtained by chromatography will decrease, and the efficiency of washing with petroleum ether-ethyl acetate mixed solution will decrease. Preferably, silica gel with 8 height units is selected.

The preferred specific operation mode of step 3) of the present invention is: add the crude extract obtained in step 1) into dichloromethane to dissolve, then add silica gel with the same quality as the crude extract, dry and grind it into powder, and slowly add it to the chromatography column . In the present invention, the crude extract obtained in step 1) is added into dichloromethane to dissolve, and then silica gel with the same quality as the crude extract is added, so that the silica gel and the crude extract can be mixed more uniformly, and the silica gel and the crude extract are combined at the same time, so that Subsequent chromatographic substances with similar polarity will be chromatographed together with the buffer, so that Frellana and other impurities can be separated in different proportions of buffer washing.

Preferably, the drying temperature in step 3) of the present invention is 50-55°C. If the temperature is too low, the liquid cannot be removed by heating and evaporating; if the temperature is too high, as the heating progresses, when the substance in the watch glass tends to be viscous, it will splash a lot, causing the loss of the drug, and Possess certain dangers, such as splashing on the skin and scalding.

Preferably, the filtrate containing the target product obtained through chromatographic separation in step 4) of the present invention can be vacuum filtered, dried and ground to obtain the target product.

The silica gel described in the present invention is preferably 100-200 mesh.

In order to achieve the above object, the technical solution adopted in the present invention mainly includes the following steps:

(1) Pulverize Bravecto with a high-speed multifunctional pulverizer, extract it with petroleum ether in a water bath at 88°C until the petroleum ether extraction reflux liquid is clarified; continue to use dichloromethane for Soxhlet extraction in a water bath at 61°C, Until the dichloromethane extraction reflux is clarified; the medicinal powder continues to use methanol for Soxhlet extraction in a water bath at 90°C until the methanol extraction reflux is clarified;

(2) Dichloromethane or the extracts of dichloromethane, sherwood oil, and methanol are respectively evaporated to dryness at low pressure on a rotary evaporator to obtain the quality of the substance after rotary evaporation;

(3) Weigh 100-200 mesh silica gel with the same quality as the evaporated dry matter, dissolve it with petroleum ether and pour it into the chromatography column. After the silica gel is completely precipitated, use a scale to measure the height of the silica gel in the chromatography column, and record it as 1 height units, and then add 7 height units of silica gel to the chromatographic column;

(4) Add an appropriate amount of dichloromethane to the material after rotary evaporation to make it completely dissolve in the flask, pour it into an evaporating dish, add silica gel of equal mass, and heat and evaporate to dryness in a water bath at 50-55°C until ground Completely dry in the bowl, then fully grind to make it completely powdery, slowly add to the chromatography column;

(5) Prepare a petroleum ether-ethyl acetate mixed solution with a volume ratio of 8:1 for separation, rotate the liquid filtered in the chromatography column to dryness, then dissolve it with the solution, and detect it in an ultraviolet analyzer after spotting the plate And compared with the standard product, the chromatography has been carried out until no solid matter is precipitated after rotary evaporation;

(6) Prepare 6:1, 4:1, 3:1, 2:1, 1:1, 2:3 petroleum ether-ethyl acetate mixed solutions respectively, and add them to the chromatography column for washing; The liquid under the internal filter is evaporated to dryness under low pressure, and then dissolved with the corresponding mixed solution. After spotting the plate, it is detected in the ultraviolet analyzer and compared with the standard; the chromatography has been carried out until the rotary evaporation no longer has solid substances precipitated, and then replaced Next gradient solution chromatography;

(7) Pour the target product obtained by chromatography into a glass suction filter funnel for vacuum filtration, pour the obtained product into a glass petri dish, dry it for 0.5-1.5 h, and grind it into powder.

The product of the present invention can be detected by HPLC-TOF-MS, so as to determine its purity.

The invention extracts the high-purity Frellana compound from Bravecto for the first time, and the method is simple and fast, and has strong practical value and practical significance. Compared with the prior art, it has the following advantages:

1) Based on simple compound extraction methods such as Soxhlet extraction, low-pressure rotary evaporation, and chromatographic column separation, the effective active ingredient Frellana compound in Bravecto can be easily and quickly extracted; the extraction process only requires Soxhlet extraction, rotary evaporation, Chromatographic column separation, vacuum filtration, HPLC-TOF-MS detection and other operations are simple and fast;

2) The petroleum ether-ethyl acetate mixed solution is selected for chromatographic column filtration, which uses less solvent, has a high reuse rate, and is highly efficient and economical; in addition, the vacuum filtration operation is simple, the purification effect is good, and the obtained compound is high in purity;

3) The present invention carries out Soxhlet extraction sequentially according to the order of petroleum ether, dichloromethane and methanol: carrying out Soxhlet extraction with petroleum ether can extract most of the impurities in Bravecto, including impurities with similar polarity and a small amount of fluralaner, and then Soxhlet extraction with dichloromethane can extract a small amount of non-polar impurities and most of the fluralaner in Bravecto, and finally Soxhlet extraction with methanol can extract the remaining fluralaner and a small amount of impurities in Bravecto. Using other solvents and different extraction sequences to carry out the extraction shown can not achieve a more complete extraction effect, and HPLC-TOF-MS detection found that the impurities with similar properties cannot be separated;

4) In the method of the present invention, only the dichloromethane Soxhlet extraction part is further extracted and purified. The detection purity of the Frellana monomer compound obtained by mass spectrometry can reach more than 90%, and the detection purity by liquid chromatography is as high as 100%. 60% to 70%; if adding petroleum ether and methanol Soxhlet extraction, the extraction rate can reach about 80%.

5) The target object extracted by the present invention has excellent contact killing activity to sanitary pests and agricultural pests such as German cockroach and Chilo borer.

Description of drawings

Figure 1: Comparison chart with UV analyzer (254nm) after vacuum rotary evaporation of chromatography solution;

Figure 2: The mass spectrogram of the Frelaner compound detected by HPLC-TOF-MS;

Figure 3: The liquid chromatogram for the detection of Frellaner compounds;

Figure 4: The nucleomass ratio spectrum for the detection of Frellaner compounds;

Figure 5: Detection of the ultraviolet absorption peak spectrum of the Frelaner compound;

detailed description

In order to facilitate those skilled in the art to understand the technical solutions and beneficial effects of the present invention, specific implementation methods are described below in conjunction with the accompanying drawings. It should be pointed out that the following specific descriptions are all exemplary and are intended to provide further description of the present invention. Unless otherwise specified, all scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

Example 1: Extraction and Purification of Frellana

1. Main materials and reagents

Spotting capillary (0.5×100) was purchased from Jiangyan Kangda Experimental Equipment Factory; column chromatography silica gel (refined type) was purchased from Qingdao Ocean Chemical Factory Branch; chromatography column was purchased from Dongfang Scientific Instruments Import and Export Group Co., Ltd. Bravecto was purchased from Merck, Germany; petroleum ether was purchased from Sinopharm Chemical Reagent Co., Ltd.; dichloromethane, methanol and ethyl acetate were purchased from Guangdong Guanghua Technology Co., Ltd. The electric heating and constant temperature blast drying oven was purchased from Shanghai Tianheng Medical Equipment Co., Ltd.; the high-speed multifunctional pulverizer was purchased from Deqing Baijie Electric Co., Ltd.; the digital display constant temperature water bath was purchased from Jintan Ronghua Instrument Manufacturing Co., Ltd., Jiangsu Province; Extractor (150mL) and condenser tube (150mL) were purchased from Dongfang Scientific Instrument Import and Export Group Co., Ltd.; ZF-20D dark box ultraviolet analyzer and constant temperature water and oil bath were purchased from Gongyi Yuhua Instrument Co., Ltd.; The balance was purchased from Beijing Medical Balance Factory; the rotary evaporator was purchased from Shanghai Yuying Instrument Co., Ltd.; the circulating water multi-purpose vacuum pump was purchased from Hangzhou David Science and Education Instrument Co., Ltd.; the ultrasonic breaker was purchased from Kunshan Ultrasonic Instrument Co., Ltd.

2. Experimental steps

(1) Take 30g of Bravecto tablet, pulverize it into powder with a high-speed multifunctional pulverizer, and extract it with petroleum ether in a water bath at 88°C until the petroleum ether extract is clarified;

(2) Continue to use dichloromethane for Soxhlet extraction of the powdered medicinal powder in a water bath at 61°C until the dichloromethane extract is clarified;

(3) Continue to use methanol for Soxhlet extraction in a water bath at 90°C until the methanol extract is clarified;

(4) The crude extracts of petroleum ether, dichloromethane, and methanol were evaporated to dryness at low pressure on a rotary evaporator (the vacuum pump was kept on during the rotary evaporation process, the oil bath was adjusted to 49°C, and the flask was partially immersed in oil the water in the heating bath, and keep the condenser tube in the state of water). Weigh the mass of the material after evaporation with a tray balance, and obtain 12.5g, 10.25g, and 11.25g of the material respectively;

(5) Select a glass chromatography column with a suitable length, add an appropriate amount of chloroform after closing the valve, check whether there is water leakage, and select a chromatography column with a closed valve;

(6) Take by weighing 10.25g (dichloromethane partial substance) silica gel (100-200 mesh), pour in the chromatographic column after dissolving with sherwood oil, measure the silica gel in the chromatographic column with a scale after the silica gel is completely precipitated. The height, 2.5cm, is recorded as 1 height unit. Use the same method to add 7 height units of silica gel to the chromatography column, that is, 17.5cm, and the total height of silica gel in the chromatography column is 20cm;

(7) Add an appropriate amount of dichloromethane to some of the dichloromethane after rotary evaporation to completely dissolve it in the flask, pour it into a mortar, add silica gel of equal mass, and heat it on a water bath at 50-55°C Evaporate until it is completely dry in the mortar, and then grind it sufficiently to make it completely powdery;

(8) Slowly pour the powdered mixture into the chromatography column. After it is completely precipitated, cut a piece of filter paper slightly smaller than the inner diameter of the chromatography column, put it into the chromatography column, and make it flat on the upper layer of the mixture, and then Insert a ball of absorbent cotton into the column. Open the chromatographic column valve, let off most of the petroleum ether in the chromatographic column;

(9) Prepare a mixed solution of petroleum ether-ethyl acetate (8:1), add it to the chromatography column, and start washing (the liquid must always be kept in the chromatography column). The liquid filtered in the chromatography column was evaporated to dryness under low pressure, and then dissolved with the solution. After spotting the plate, it was detected in the ultraviolet analyzer and compared with the standard (Figure 1). Chromatography was carried out until no solid material was precipitated by rotary evaporation;

(10) Prepare 6:1, 4:1, 3:1, 2:1, 1:1, 2:3 petroleum ether-ethyl acetate mixed solutions respectively, and add them to the chromatography column for chromatography in sequence. The liquid filtered in the chromatography column was spun to dryness, then dissolved with the solution, spotted on a silica gel plate G254, detected at 254nm by an ultraviolet analyzer and compared with a standard (Figure 1). Chromatography has been carried out until no solid matter is precipitated by rotary evaporation, and then the next gradient solution is replaced for washing;

(11) Pour the target product obtained from chromatographic separation into a glass suction filter funnel for vacuum filtration, pour the obtained product into a glass petri dish, and dry (0.5-1.5h) to obtain 2.62g of solid, which is fluorine The relana compound is the object component of the present invention.

The extraction rate of the Frelaner compound extracted by this method is 62.38%, and its purity is >99% as detected by HPLC-TOF-MS (Fig. 2-Fig. 5).

Embodiment 2: Extraction and purification of Freilaner

1. The test equipment used and the reagent preparation are basically the same as Example 1;

2. Experimental method:

(1) Take 30g of Bravecto tablet, pulverize it into powder with a high-speed multifunctional pulverizer, and extract it with petroleum ether in a water bath at 88°C until the petroleum ether extract is clarified;

(2) Continue to use dichloromethane for Soxhlet extraction of the powdered medicinal powder in a water bath at 61°C until the dichloromethane extract is clarified;

(3) Continue to use methanol for Soxhlet extraction in a water bath at 90°C until the methanol extract is clarified;

(4) The crude extracts of petroleum ether, dichloromethane, and methanol were evaporated to dryness at low pressure on a rotary evaporator (the vacuum pump was kept on during the rotary evaporation process, the oil bath was adjusted to 49°C, and the flask was partially immersed in oil the water in the heating bath, and keep the condenser tube in the state of water). Weigh the mass of the material after evaporation with a tray balance, and obtain 12.5g, 10.25g, and 11.25g of the material respectively;

(5) Select a glass chromatography column with a suitable length, add an appropriate amount of chloroform after closing the valve, check whether there is water leakage, and select a chromatography column with a closed valve;

(6) Take by weighing 10.25g (dichloromethane partial substance) silica gel (100-200 mesh), pour in the chromatographic column after dissolving with sherwood oil, measure the silica gel in the chromatographic column with a scale after the silica gel is completely precipitated. The height, 2.5cm, is recorded as 1 height unit. Use the same method to add 7 height units of silica gel to the chromatography column, that is, 17.5cm, and the total height of silica gel in the chromatography column is 20cm;

(7) Add an appropriate amount of dichloromethane to some of the dichloromethane after rotary evaporation to completely dissolve it in the flask, pour it into a mortar, add silica gel of equal mass, and heat it on a water bath at 50-55°C Evaporate until it is completely dry in the mortar, and then grind it sufficiently to make it completely powdery;

(8) Slowly pour the powdered mixture into the chromatography column. After it is completely precipitated, cut a piece of filter paper slightly smaller than the inner diameter of the chromatography column, put it into the chromatography column, and make it flat on the upper layer of the mixture, and then Insert a ball of absorbent cotton into the column. Open the chromatographic column valve, let off most of the petroleum ether in the chromatographic column;

(9) Prepare a mixed solution of petroleum ether-ethyl acetate (8:1), add it to the chromatography column, and start washing (the liquid must always be kept in the chromatography column). The liquid filtered in the chromatography column was evaporated to dryness under low pressure, and then dissolved with the solution. After spotting the plate, it was detected in the ultraviolet analyzer and compared with the standard (Figure 1). Chromatography was carried out until no solid material was precipitated by rotary evaporation;

(10) Prepare 6:1, 4:1, 3:1, 2:1, 1:1, 2:3 petroleum ether-ethyl acetate mixed solutions respectively, and add them to the chromatography column for chromatography in sequence. Rotate the liquid filtered in the chromatography column to dryness, then dissolve it with the solution, spot the plate, detect it in the ultraviolet analyzer and compare it with the standard. Chromatography has been carried out until no solid matter is precipitated by rotary evaporation, and then the next gradient solution is replaced for washing;

(11) Adopt (6)-(10) identical method to come the 12.5g crude extract that obtains after petroleum ether Soxhlet extraction and the 11.25g crude extract that obtain after methanol Soxhlet extraction, separate the chromatographically obtained Pour the target product into a glass suction filter funnel for vacuum filtration, pour the obtained product into a glass petri dish, and dry (0.5-1.5h) to obtain 3.15g of solid, which is the Freilaner compound, which is the compound of the present invention. target component.

The extraction rate of the Frelaner compound extracted by this method is 75.00%, and the purity is >85%, wherein 2.20g has a purity >98%, 0.82g has a purity >91%, and 0.13g has a purity >85%.

activity test

One, the activity determination test of Frellana to German cockroach

1. Main materials and reagents

The German cockroach (Blattella germanica) indoor rearing strain was provided by our laboratory (Insect Physiology, Biochemistry and Molecular Biology Laboratory, School of Plant Protection, Nanjing Agricultural University), and was continuously reared with artificial diet without contacting any insecticides. Fipronil (92%), butene fipronil (92%), dichlorvos (93%), all provided by this laboratory; beta-cypermethrin (93%), propoxur (>99%), piperonyl butoxide ( 95%), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; acetone, purchased from Nanjing Agricultural University Logistics Group Company. Manual micro-droppers were purchased from Burkard; humidifiers, disposable plastic cups, rubber bands, cotton, vaseline, Petri dishes, square plastic cylinders, etc. were purchased from Nanjing Shoude Experimental Equipment Co., Ltd.; flour, milk powder, glucose, etc. , were purchased from supermarkets.

Breeding method: Mixed breeding in an indoor breeding tank regardless of age, 1/3 of the inner wall of the feeding tank mouth is coated with a 3-5cm wide Vaseline oil band to prevent escape. Place two petri dishes in the rearing tank, one petri dish is used to hold the mixed feed (flour:milk powder:glucose ratio is 6:3:1 mixed), the other petri dish is used to hold water and cotton, every 3-4 Feed and water were supplemented daily. Put multi-layer folded cardboard into the bottom of the separation cylinder as the habitat of Blattella germanica; the rearing cylinder is sealed with a single layer of gauze to prevent escape. Clean the breeding tank regularly to maintain a clean breeding environment. Observe the density of Blattella germanica in good time, 0.6-1.0/ ^cm2 is appropriate, which not only ensures the normal development of the insect body, but also makes full use of the rearing tank to raise the largest number of test insects. When the density is higher than this range, it should be reared in separate tanks . The breeding conditions are temperature (26±1)°C, humidity (60±5)%, light conditions (L:D is 16:8h).

2. Drug preparation

2.1 Preparation of Frellana solution

Prepare a 10,000mg/L stock solution of Frellana with acetone as a solvent, then dilute to 1.25mg/L, 2.5mg/L, 5.0mg/L, 7.5mg/L, 10.0mg/L, 12.5mg/L with acetone Six concentration gradients.

2.2 Preparation of commonly used pesticide solutions

Prepare 10,000mg/L mother liquors of fipronil, butylene-fipronil, dichlorvos, beta-permethrin, and propoxur with acetone as a solvent, and then dilute each agent to 5.0mg/L with acetone.

3. Determination of the activity of Frellana and commonly used insecticides against German cockroaches by spot method

30 male adults of Blattella germanica were selected and randomly divided into 3 groups with 10 adults in each group. After the test insects were anesthetized with CO ₂ , they were arranged on their backs in a petri dish, and were sequentially adjusted from low to high concentrations (0.16mg/L, 0.80mg/L, 4.0mg/L, 20.0mg/L, 100.0mg/L) with a manual micro-dropper. L) Freilaner. Drop 1.0 μL of the drug solution on the web between the second and third pair of foot bases of the test insect, then transfer it to a clean container, and feed it with mixed feed and water-soaked cotton balls. Acetone was set as a blank control, and the mortality rate was checked after 24h, 48h, 72h, 96h, and 120h. The standard of death is to turn the worm body upside down and not to crawl.

Use acetone to dilute the medicine into medicine solutions with different concentration gradients for future use. The test insects were anesthetized with CO ₂ , and 1.0 μL of the drug solution was dripped on the web between the second and third pair of foot bases of the test insects with a manual micro-dropper in order from low to high concentrations. 10 rats were dropped in each concentration level, and repeated 3 times. Acetone was used as blank control. After the medicine is dispensed, they are placed in disposable plastic cups containing feed and water-containing cotton for normal feeding, and the mouth of the cup is sealed with a single layer of gauze to prevent escape. Mortality was checked after 24h, 48h, 72h, 96h, and 120h. The standard of death is to turn the worm body upside down and not to crawl. Each drug was repeated more than 3 times.

4. Statistical analysis of data

Use POLO Plus, SPSS 17.0 and other software to obtain the regression equation of virulence y=a+bx, LC50, LD50, etc. The experimental data are shown in Table-1 and Table-2.

Table-1 Freilaner is to the virulence determination test result of German cockroach

Table-2 Freilaner and six commonly used medicaments to the sensitivity test results of German cockroaches

Note: * indicates significant difference, ** indicates extremely significant difference.

From the above data, it can be known that the LD ₅₀ values of Freilaner against Blattella germanica at 24h, 48h, 72h, 96h, and 120h were 0.0117, 0.0076, 0.0066, 0.0046, and 0.0045 μg/head, respectively. It can be seen that the virulence of Freilaner to the German cockroach increases with time in 0-96h, and the virulence after 96h tends to be stable. SPSS 17.0 software was used to analyze the significant difference between the toxicity of several conventional agents and Frellaner, and the results showed that 5mg/L butene fipronil, fipronil, dichlorvos, Frellaner, and beta-permethrin The lethality rates of esters and propoxur against German cockroaches were 80.00%, 80.00%, 58.89%, 51.11%, 42.22%, and 21.11%, respectively. It can be seen from Table 2 that the insecticidal activity of fipronil and butylene-fipronil is relatively good, higher than that of Freylana; followed by Dichlorvos, Freylana, and Beta-Permethrin, and the insecticidal activity of the three has no Significant difference; the insecticidal activity of propoxur was relatively the worst, and it was significantly lower than that of Frellana.

2. Indoor viability test of Freilaner against Chilo suppressalis

1 Main materials and reagents

For the strain of Chilo suppressalis grown indoors, 4th instar larvae of the same generation (9.00-11.00 mg/head) were selected from the Laboratory of Insect Physiology, Biochemistry and Molecular Biology, School of Plant Protection, Nanjing Agricultural University. Fipronil (92%) was provided by the Laboratory of Insect Physiology, Biochemistry and Molecular Biology, School of Plant Protection, Nanjing Agricultural University.

2 Toxicity determination method

The virulence determination method was improved by referring to the Capillary Titration Method of Technical Regulations for Monitoring the Resistance of Rice Chipotle Borer (NY/T 2058-2011), and it was suitable for the virulence determination of the 4th instar larvae of Chilo suppressalis. The original drug was weighed on an electronic balance, dissolved in acetone, and prepared into a mother liquor with a certain concentration. The dissolved mother liquor was divided into 2mL centrifuge tubes and stored at -20°C. Take a certain amount of mother liquor with a pipette, dilute it with acetone to 6-7 concentrations for later use, use acetone as a control, and each tube should not be less than 2mL.

Pick 9.00-11.00 mg of the 4th instar larvae of Chilo borer, and treat them according to the order of concentration of the test solution from low to high. Use a micro-dropper to draw the drug solution and drop it on the back of the chest of the test insects, drip 1 μL per test insect, and treat 30 insects at each concentration. Place the test insects after spotting in a large flat-bottomed test tube planted with rice seedlings, 10 in each test tube, and set 3 repetitions. The control group was dripped with an equal volume of acetone. Put the treated test worms back into the original breeding environment, observe the survival of the larvae at 24h and 48h, lightly touch the larvae with a brush, and the worms cannot coordinate movement as the death standard, and record the number of deaths.

3 Statistical analysis of data

The virulence regression equation was calculated using POLO PLUS (LeOra Software, 2003) software. Calculation parameters include b value, LC ₅₀ and its 95% confidence limit. The experimental data are shown in Table-3.

Table-3 Toxicity test results of Freilaner to indoor Chilo borer strains

It can be seen from the above data that the spot method was used to measure the activity of Freylana and fipronil on the strains of Chilo borer reared indoors. The results showed that the _LD50 values of Freylana for 24h and 48h were 0.022 and 0.003 respectively. μg/head. The LD ₅₀ values of fipronil treated for 24h and 48h were 0.025 and 0.004μg/head, respectively.

It can be seen that after 48 hours of treatment, the poisonous effects of Freilaner and fipronil on the 4th instar larvae of Chilo borer were significantly greater than 24 hours. After 48 hours of treatment, the LD ₅₀ values of Freilaner and Fipronil against C. borer were 0.003 and 0.004 μg/head, respectively, indicating that the activity of Freilaner against the 4th instar larvae of C. insecticidal effect.

Claims (8)

1. the method for extracting and purifying the monomeric compound of Freylana, is characterized in that, comprises the steps: 1) After the Bravecto preparation is pulverized into powder, Soxhlet extraction is performed sequentially with petroleum ether, dichloromethane, and methanol until the reflux liquid is clarified, and the dichloromethane extract or the three extracts are respectively evaporated at low pressure to obtain a crude extract; 2) Weigh the silica gel with the same quality as the crude extract, dissolve petroleum ether and pour it into the chromatography column. After the silica gel is completely precipitated, measure the height of the silica gel in the chromatography column, record it as 1 height unit, and then pour it into the chromatography column Add 6 to 8 height units of silica gel; 3) adding the crude extract obtained in step 1) into the chromatographic column; 4) The volume ratios are (7.8～8.2): 1, (5.8～6.2): 1, (3.8～4.2): 1, (2.8～3.2): 1, (1.8～2.2): 1, (0.8～ 1.2): 1, 2: (2.8～3.2) The petroleum ether-ethyl acetate mixed solution is subjected to chromatographic separation to obtain the target product. 2. the extraction and purification method of Freilaner monomer compound according to claim 1, is characterized in that, step 3) the specific mode of operation is: the crude extract that step 1) obtains is added dichloromethane to dissolve, Then add silica gel with the same quality as the crude extract, dry and grind it into powder, and slowly add it into the chromatography column. 3. the extraction and purification method of Frellana monomer compound according to claim 1, is characterized in that, step 1) in Soxhlet extraction process, sherwood oil extraction temperature is 85～90 ℃; Dichloromethane extraction temperature 59-64°C; methanol extraction temperature is 87-93°C. 4. the extraction and purification method of Frellana monomer compound according to claim 3, is characterized in that, step 1) in Soxhlet extraction process, sherwood oil extraction temperature is 88 ℃, and dichloromethane extraction temperature is 61 °C, the methanol extraction temperature is 90 °C. 5. the extraction and purification method of Frellana monomer compound according to claim 1, is characterized in that, step 2) in, takes by weighing the silica gel of equal quality with crude extract, pours into chromatography after sherwood oil dissolves After the silica gel is completely precipitated, measure the height of the silica gel in the chromatography column, record it as 1 height unit, and then add 7 height units of silica gel to the chromatography column. 6. The method for extracting and purifying the monomeric compound of Freylana according to claim 1, characterized in that the drying temperature in step 3) is 50-55°C. 7. the extraction and purification method of Frellana monomer compound according to claim 1, is characterized in that, step 4) middle chromatographic separation obtains the filtrate containing target product, vacuum suction filtration, drying and grinding get final product Obtain the target product. 8. The method for extracting and purifying the monomeric compound of Freylana according to claim 1, characterized in that, the silica gel is 100-200 mesh.