CN106167815A - The Preparation method and use of it dimension streptozotocin derivative - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of an asvermectin derivative, including 1) preparing a Saccharopolyspora rubrum seed liquid; The method of the invention has high conversion rate for preparing asvermectin derivatives and is economical and feasible. The asvermectin derivatives prepared by the invention are used to prepare medicines for preventing and controlling agricultural and forestry pests or harmful mites, and the medicine has excellent effects in preventing and controlling agricultural and forestry pests or harmful mites.

Description

Preparation method and application of asvermectin derivatives

technical field

The invention belongs to the field of biotechnology, and in particular relates to 4″-O-glucosyl tenvermectins A/B and its preparation method through microbial transformation and its application in the preparation of medicines for preventing and controlling agricultural and forestry pests and pest mites.

Background technique

Tenvermectin (Tenvermectin) is a new type of 16-membered macrolide antibiotic, which has the characteristics of broad spectrum, high efficiency, low toxicity, small dose, and safer use. The chemical structure formula of Tenvermectin A/B is as follows:

After activity determination, compared with abamectin, ivermectin and milbemycin, it has a better effect on aphids, mites, lepidopteran pests and other pests that harm crops and livestock intestinal parasites. It is a new generation of environmentally friendly insecticide with very promising prospects.

Microbial transformation is to modify the structure of complex substrates through microbial cells, that is, to use a certain enzyme or a series of enzymes produced in the metabolic process of microorganisms to catalyze specific parts (groups) of substrates to make the molecular structure of substrates Change to another similar compound. Microbial transformation has the advantages of reaction orientation, single product, less side reactions, fast biomass accumulation, short transformation time, high expression efficiency of invertase, mild reaction conditions, safety and environmental protection, etc. It is a convenient method for structural modification of natural active compounds. means. The conversion of asvermectin A/B by microorganisms to obtain new active compounds with similar structures is of great significance for the development and utilization of a new generation of asvermectin macrolide drugs.

Contents of the invention

The invention provides a preparation method and application of 4″-O-glucosyl tenvermectins A/B. The invention obtains a new tenvermectin derivative through microbial transformation of tenvermectins, which has good insecticidal and acaricidal effects, and has Potential to be developed into a new generation of environmentally friendly insecticides.

The technical scheme of the present invention is to provide a kind of preparation method of 4 "-O-glucosyl tenvermectins A/B, comprise the steps:

1) Preparation of Saccharopolyspora erythraea seed liquid: take the Saccharopolyspora erythraea strain Saccharopolyspora erythraea with the preservation number ATCC 11635, inoculate it on the YMS solid medium by streaking, and cultivate it in a constant temperature incubator at 20-28°C for 5- After 7 days, the activated strains were transferred to the liquid medium, placed in a horizontal shaker at 20-28°C and 250 rpm for 40 hours, and the seed liquid was obtained;

2) Preparation of 4″-O-glucosyl tenvermectins A/B: transfer the seed solution to the transformation medium, and after culturing for 24 hours, add the transformation substrate tenvermectins A/B dissolved in an organic solvent, and then add the transformation substrate tenvermectins A/B dissolved in an organic solvent, and then transfer the seed liquid to the transformation medium, and then add the transformation substrate tenvermectins A/B dissolved in an organic solvent, at 20-28 ℃, 150 hours under the condition of 250rpm, the fermentation broth was obtained; the extractant was used to extract the fermentation broth and the extractant was recovered, purified and separated to obtain pure 4″-O-glucosyl tenvermectins A/B; the 4″-O- The chemical structure of glucosyl tenvermectins A/B is characterized by:

The preparation method of 4 "-O-glucosyl tenvermectins A/B provided by the present invention, the method is by fermenting and cultivating red sugar polyspora Saccharopolyspora erythraea (ATCC 11635), then adding substrate tenvermectins A/B ( tenvermectins A/B) and then continue to cultivate for 150h, and finally obtain sixteen-membered macrolide compound 4″-O-glucosyl tenvermectins A/B through extraction and separation.

The strain of Saccharopolyspora rubrum is light yellow-brown or gray-yellow on YMS solid medium, and the spores are spherical, oval, and smooth. Saccharopolyspora rubrum is a Gram-positive bacterium G(+) with hyphal and spore-differentiating, producing short spore chains consisting of spores with spiny surfaces. The way to produce spores is that the end of the vegetative aerial mycelium begins to curl, and then the hyphae will produce a septum, divide the mycelium into several sections, then gradually thicken the septum to form a wall, and finally form a circular spore to release. The fungus was purchased from the "American Standard Biological Collection" with a preservation number of ATCC 11635.

As a preferred technical scheme, the preparation method of the liquid medium in step 1) is as follows: take 10 g of glucose, 2 g of yeast extract, 2 g of beef extract and 3 g of peptone, set the volume to 1000 ml with distilled water, and then use 1 mol/ 1 of NaOH to adjust the pH to 7.0, use 250ml Erlenmeyer flasks to pack 40ml each, and sterilize at 121°C and 0.15MPa for 20min.

As a preferred technical solution, the transformation medium in step 2) includes M-1 medium, M-2 medium, M-3 medium and M-4 medium with different carbon-nitrogen ratios, wherein,

The components of M-1 medium are: glucose 5g, brown sugar 10g, tryptone 5g, yeast extract 2.5g, sodium edetate 0.036g, betaine 1.2g and sodium propionate 0.11g;

The components of the M-2 medium are: industrial glucose 44g, beef extract 5.0g, peptone 6.0g, yeast extract 4g, casein peptone 3.0g, hot fried soybean cake powder 11.0g, NaCl 1.5g and K ₂ HPO ₄ 0.5g;

The components of the M-3 medium are: glucose 50g, soybean cake powder 5g, yeast extract 5g, peptone 4g, NaCl 5g, K ₂ HPO ₄ 2g and KH ₂ PO ₄ 1g;

The components of the M-4 medium are: industrial glucose 50g, NaCl 5g, (NH ₄ ) ₂ SO ₄ 2g, CaCO ₃ 6g and soybean powder 30g;

After the above components were adjusted to 1000ml with distilled water, the pH was adjusted to 7.0-7.2 with 1mol/l NaOH, and each 250ml Erlenmeyer flask was filled with 40ml, and sterilized at 121°C and 0.15MPa for 20min.

The present invention also relates to a method for improving the conversion rate of the sixteen-membered macrolide compound 4″-O-glucosyl tenvermectins A/B by changing the transformation medium components. The seed solution is transferred to the above four different transformation cultures In the medium (M-1 medium, M-2 medium, M-3 medium and M-4 medium), the carbon source of these four transformation mediums is based on glucose, and the nitrogen source is diversified, and With the necessary trace elements, under the condition that other transformation conditions remain unchanged, compare the conversion rate of astramectin A/B to 4″-O-glucosyl tenvermectins A/B by microorganisms under four different medium conditions high and low. The result shows that the conversion rate of conversion medium M-1 is the lowest, and the conversion rate of conversion medium M-4 (the carbon source is a single glucose, and the carbon-nitrogen ratio is about 1.5: 1) is the highest (detailed situation sees embodiment), so when Glucose is selected as the carbon source of the transformation medium, and when the carbon-to-nitrogen ratio is about 1.5:1, it is beneficial for the microbial transformation of asvermectin A/B into 4″-O-glucosyltenvermectins A/B.

As a preferred technical solution, the organic solvent in step 2) includes one or more of methanol, ethanol, and DMSO.

As a preferred technical solution, the extractant in step 2) includes one or more of ethyl acetate, n-butanol, isobutyl acetate, ether, petroleum ether, dichloromethane or chloroform.

As a preferred technical solution, the purification in step 2) includes reverse-phase high-performance liquid chromatography.

As a preferred technical scheme, the purification in step 2) also includes silica gel column chromatography.

A use of 4″-O-glucosyl tenvermectins A/B. The 4″-O-glucosyl tenvermectins A/B is used for preparing medicines for preventing and controlling agricultural and forestry pests or pest mites.

As a preferred technical solution, the agricultural and forestry pests include Lepidoptera Plutellaidae, Lepidoptera Noctuidae, Lepidoptera Satyridae, Lepidoptera Botryidae, Coleoptera Pyrethidae, Thyropidae Slippery One or more of the superfamily; said pests include spider mites.

As a preferred technical solution, the 4″-O-glucosyl tenvermectins A/B include a single 4″-O-glucosyl tenvermectin A, a single 4″-O-glucosyl tenvermectin B or a mixture of both.

The preparation method of 4"-O-glucosyl tenvermectins A/B of the present invention can prepare 4"-O-glucosyl tenvermectins A/B with high conversion rate. The 4″-O-glucosyl tenvermectins A/B has excellent effects in controlling agricultural and forestry pests or harmful mites.

detailed description

The present invention will be described below with specific examples. It should be understood that examples are used to illustrate the present invention rather than to limit the present invention, and the scope and core content of the present invention are determined according to the claims.

Example 1

1) Take the red Saccharopolyspora strain with the preservation number ATCC 11635, inoculate it on the YMS solid medium, culture it in a constant temperature incubator at 28°C for 7 days, and transfer the activated strain to the liquid culture cultured in a horizontal shaker at 28°C and 250rpm for 40 hours to obtain a seed solution, and transferred the seed solution to the transformation medium with a 5% inoculation amount, and added 500 U to each bottle of transformation medium after culturing for 24 hours. The transformation substrate asvermectin A/B mixture dissolved in methanol was continuously transformed and cultured at 28° C. and 250 rpm for 150 hours to obtain a fermentation broth.

2) Ferment and cultivate according to the above method to obtain 3L of fermented liquid, soak the fermented liquid with 6L of industrial ethanol overnight, and filter to obtain ethanol extract. The resulting extract was concentrated under reduced pressure at 50°C to remove the ethanol phase, and then extracted three times with an equal volume of ethyl acetate to obtain an ethyl acetate extract, which was concentrated to dryness at 50°C under reduced pressure to obtain 5 g of an oily substance.

Put the obtained oily substance on a silica gel column (particle size 100-200 mesh) for column chromatography, use chloroform:methanol=100:0-60:40 (V/V) for gradient elution, and identify by thin layer (TLC) After detection, 2 components were obtained. Fraction 2 was subjected to gel (Sephadex LH-20) column chromatography to obtain fraction 2-1, and then further purified by semi-preparative column chromatography to obtain pure 4″-O-glucosyl tenvermectin A and 4″-O-glucosyl Tenvermectin B.

Among them, the TLC identification method: the converted sample and the blank control point are placed on the silica gel G thin-layer plate, developed under the developer condition of chloroform:methanol (9:1), take it out and dry it after running, and observe under the ultraviolet lamp 254nm first , and then use concentrated sulfuric acid to develop color and check the conversion results.

Semi-preparative column chromatography conditions: mobile phase: methanol-acetonitrile-water (45:45:10); column C18, 9.4*250mm; detection wavelength: 244nm; flow rate: 1.5ml/min; column temperature: 24.4°C; sample injection Quantity: 60ul. The peaks with retention times of 16 min and 19.7 min were collected to obtain 4″-O-glucosyl tenvermectin A and 4″-O-glucosyl tenvermectin B. Configuration of samples and standards: Weigh an appropriate amount of samples and standards, use the mobile phase as a solvent, and configure them into a solution.

3) Structural identification of 4″-O-glucosyl tenvermectins A and B

The structure of 4″-O-glucosyl tenvermectins A/B was determined by 1D and 2D NMR, MS and other spectral analysis as follows:

The physical and chemical properties of 4″-O-glucosyl tenvermectin A are as follows:

Appearance: White powder substance

Solubility: easily soluble in chloroform, acetone, methanol, insoluble in water

Molecular formula: C ₅₁ H ₇₈ O ₁₉

ESI-MS m/z:993.23[MH] ^-

UVλ _max (EtOH)nm(logε):245(4.67), 239(4.63)

IR v _max cm ^-1 : 3387, 2930, 2875, 1721, 1646, 1450, 1383, 1340, 1305, 1270, 1170, 1062, 989

The physical and chemical properties of 4″-O-glucosyl tenvermectin B are as follows:

Appearance: White powder substance

Solubility: easily soluble in chloroform, acetone, methanol, insoluble in water

Molecular formula: C ₅₂ H ₈₀ O ₁₉

ESI-MS m/z:1007.22[MH] ^-

UVλ _max (EtOH)nm(logε):245(4.50), 239(4.46)

IR v _max cm ^-1 : 3369, 2928, 2873, 1716, 1637, 1452, 1382, 1340, 1304, 1268, 1168, 1118, 1064, 985

See Table 1 for ¹ H NMR (CDCl ₃ , 400 MHz) and ¹³ C NMR (CDCl ₃ , 100 MHz).

Table 1 NMR data of 4″-O-glucosyl tenvermectins A and B in CDCl ₃

(H spectrum, 400MHz; Carbon spectrum, 100MHz)

Example 2

Take the red Saccharopolyspora strain with the preservation number ATCC 11635, inoculate it on the YMS solid medium, culture it in a constant temperature incubator at 28°C for 7 days, transfer the activated strain into the liquid medium, Placed at 28°C, cultivated in a horizontal shaker at 250rpm for 40 hours to obtain a seed liquid, and transferred the seed liquid to the transformation medium (M-1) with a 5% inoculum size, and put it in each bottle of transformation medium after 24 hours of cultivation Add the transformation substrate asvermectin A/B mixture dissolved in methanol, continue the transformation culture for 150 hours at 28°C and 250rpm, extract the fermentation broth with ethyl acetate, and then detect 4″-O-glucosyltenvermectins A and B conversions were 5.77% and 7.69%, respectively.

Example 3

Take the red Saccharopolyspora strain with the preservation number ATCC 11635, inoculate it on the YMS solid medium, culture it in a constant temperature incubator at 28°C for 7 days, transfer the activated strain into the liquid medium, Placed at 28°C, cultivated in a horizontal shaker at 250rpm for 40 hours to obtain a seed solution, and transferred the seed solution to the transformation medium (M-2) with a 5% inoculum size, and put it in each bottle of transformation medium after culturing for 24 hours Add the transformation substrate asvermectin A/B mixture dissolved in ethanol, continue the transformation culture at 28°C and 250rpm for 150 hours, extract the fermentation broth with ether, and detect the transformation of 4″-O-glucosyltenvermectins A and B by HPLC The rates were 4.20% and 11.15%, respectively.

Example 4

Take the red Saccharopolyspora strain with the preservation number ATCC 11635, inoculate it on the YMS solid medium, culture it in a constant temperature incubator at 28°C for 7 days, transfer the activated strain into the liquid medium, Placed at 28°C, cultivated in a horizontal shaker at 250rpm for 40 hours to obtain a seed liquid, and transferred the seed liquid to the transformation medium (M-3) with a 5% inoculum size, and put it in each bottle of transformation medium after culturing for 24 hours Add the DMSO-dissolved transformation substrate asvermectin A/B mixture, continue the transformation culture at 28°C and 250rpm for 150 hours, extract the fermentation broth with petroleum ether, and then detect 4″-O-glucosyltenvermectins A and B by HPLC The conversions were 7.78% and 14.24%, respectively.

Example 5

Take the red Saccharopolyspora strain with the preservation number ATCC 11635, inoculate it on the YMS solid medium, culture it in a constant temperature incubator at 28°C for 7 days, transfer the activated strain into the liquid medium, Placed at 28°C and cultivated in a horizontal shaker at 250rpm for 40 hours to obtain a seed solution, which was transferred to the transformation medium (M-4) with a 5% inoculum size, and was placed in each bottle of transformation medium after 24 hours of cultivation. Add the transformation substrate asvermectin A/B mixture dissolved in methanol, continue the transformation culture for 150 hours at 28°C and 250rpm, extract the fermentation broth with dichloromethane, and then detect 4″-O-glucosyltenvermectins A and B conversions were 9.74% and 19.8%, respectively.

Example 6

Biological Activity of 4″-O-glucosyl tenvermectins A and B against Tetranychus cinnabarinus

Test agent: 98% (w/w) 4″-O-glucosyl tenvermectins A, B, 98% (w/w) tenvermectin A: Weigh 1g of 98% 4″-O-glucosyl tenvermectins A and B respectively into the beaker , and add 93g of methanol and 6g of surfactant nonylphenol ethoxylate to make a preparation with a concentration of 10000mg/L, and dilute it with water to provide a drug solution with a concentration of 0.005, 0.01, 0.02, 0.04, and 0.08mg/L. try.

Organisms to be tested: Tetranychus cinnabarinus: Inoculated on faba bean seedlings under the conditions of artificial climate chamber [(26±1)°C, RH (70±5)%, H/D14].

Experimental method: Leaf disc dipping method: select adult mites that are reared indoors and have the same physiological state. Select broad bean leaves with consistent growth, use a puncher to make leaf discs with a diameter of 2 cm, and place the leaves on the absorbent cotton in the center of a plastic dish with the back of the leaves facing up. Three leaf butterflies are picked in each dish, and the adult mites are picked and inoculated into the leaf discs with a small brush On top, 30 discs per leaf, add appropriate amount of water, and place in a cultivation room at (26±1)°C, light intensity 3000-4500lx, 14h/d, RH 50%-75%. After 2 hours, the number of adult mites was checked under a stereo microscope, and the number of mites on each leaf dish was not less than 20. The prepared medicines with mass concentrations of 0.005, 0.01, 0.02, 0.04, and 0.08mg/L were placed in beakers, and the leaves were clamped with tweezers to soak in order from low concentration to high concentration for 5 seconds. Adult mites, each mass concentration is a treatment, and each treatment is repeated 3 times. After the chemicals on the leaves are dried, the treated leaf discs are placed in an artificial climate chamber at (26±1)°C and a photoperiod of 14 hours for 24 hours, and a small amount of water is added to the culture dish to moisturize. The mites are very active after soaking the medicine, and they start to slow down their activities 5-8 hours after treatment, and the mites are still after 12-24 hours. Criteria for judging the death: lightly touch the mite body with a brush during the inspection, and those who do not move at all are judged as dead. The indoor activities of 4″-O-glucosyltenvermectins A and B were measured with Tetranychus cinnabarinus as the test insect, and the activities of 4″-O-glucosyl tenvermectins A and B and tenvermectin A were compared with tenvermectin A as the control agent .

Conclusion: See Table 2 for the experimental results. The LC50 of tenvermectin A against Tetranychus cinnabarinus is 0.0084mg/L, and the LC50 of the transformed product 4″-O-glucosyl tenvermectins A/B against Tetranychus cinnabarinus are 0.0156mg/L and 0.0113mg respectively /L, there was no significant difference in the activity of the transformed product 4″-O-glucosyl tenvermectins A/B and tenvermectin A against Tetranychus cinnabarinus.

Table 2: Activity of 4″-O-glucosyl tenvermectins A/B against Tetranychus cinnabarinus

Example 7

Activity determination of 4″-O-glucosyl tenvermectins A and B against pine xylophilus

Test agents: 4″-O-glucosyl tenvermectins A/B are from the above examples, and tenvermectin A is from Zhejiang Hisun Pharmaceutical Co., Ltd.

Test organisms: pine xylophilus as the test insect

Test method: use immersion method. Five mass concentrations of 1, 2, 5, 10, and 20 mg/L were established for each drug, and each concentration was repeated 3 times, and the experimental results were counted within 24 hours.

Test results: See Table 3 for the toxicity of various pesticides to pine xylophilus. The LC50 of tenvermectin A to pine wood nematode is 2.4391mg/L, and the converted product 4″-O-glucosyl tenvermectins A/B to pine wood nematode LC50 are 6.7984mg/L, 5.7980mg/L, and the transformed product is 4″ - O-glucosyl tenvermectins A/B activity against B. xylophilus compared with tenvermectin A, there was no significant difference.

Table 3: Activity of 4″-O-glucosyl tenvermectins A/B against pine xylophilus

Example 8

Activity Determination of 4″-O-glucosyl tenvermectins A and B against Plutella xylostella

Test agents: 4″-O-glucosyl tenvermectins A/B are from the above examples, and tenvermectin A is from Zhejiang Hisun Pharmaceutical Co., Ltd.

Test organisms: Plutella xylostella as test insect

Test method: the drug film method is used. Five mass concentrations of 25, 50, 100, 200 and 250 mg/L were established for each drug, each concentration was repeated 3 times, and the experimental results were counted within 24 hours.

Test results: See Table 4 for the toxicity of various pesticides to diamondback moth. The corrected mortality of tenvermectin A to Plutella xylostella at 250mg/L is 79.62%, and the corrected mortality of the transformed product 4″-O-glucosyl tenvermectins A/B to Plutella xylostella at 250mg/L is 70.21%, 74.87%, respectively. Compared with tenvermectin A, the latter product 4″-O-glucosyltenvermectins A/B has no significant difference in activity against diamondback moth.

Table 4: Activity of 4″-O-glucosyl tenvermectins A/B against diamondback moth

Example 9

Determination of the activity of 4″-O-glucosyl tenvermectins A and B against the bamboo borer

Test agents: 4″-O-glucosyl tenvermectins A/B are from the above examples, and tenvermectin A is from Zhejiang Hisun Pharmaceutical Co., Ltd.

Test organisms: Take bamboo borer as the test insect

Test method: using dipping method. Five mass concentrations of 1, 2, 5, 10 and 20 mg/L were established for each drug, each concentration was repeated 3 times, and the experimental results were counted within 24 hours.

Test results: Table 5 shows the toxicity of various pesticides to bamboo borer. The corrected mortality of tenvermectin A to bamboo borer at 20mg/L was 77.52%, and the corrected mortality of the transformed product 4″-O-glucosyl tenvermectins A/B to bamboo borer at 20mg/L was 68.56%, 70.62%, respectively. Compared with tenvermectin A, the latter product 4″-O-glucosyl tenvermectins A/B had no significant difference in activity against bamboo borer.

Table 5: Activity of 4″-O-glucosyl tenvermectins A/B against bamboo borer

The preparation method of the 4"-O-glucosyl tenvermectins A/B of the present invention can prepare the 4"-O-glucosyl tenvermectins A/B with a high conversion rate. The 4″-O-glucosyl tenvermectins A/B has excellent effects in controlling agricultural and forestry pests or harmful mites.

The use of the 4″-O-glucosyl tenvermectins A/B of the present invention has been described through specific examples. Those skilled in the art can refer to the content of the present invention and appropriately change the raw materials, process conditions and other links to achieve other corresponding purposes. The changes do not depart from the content of the present invention, and all similar substitutions and modifications that are obvious to those skilled in the art are deemed to be included within the scope of the present invention.

The embodiments described above are only preferred solutions of the present invention, and do not limit the present invention in any form. There are other variations and modifications on the premise of not exceeding the technical solutions described in the claims.

Claims (10)

1.4 " preparation method of-O-glucosyl tenvermectins A/B, it is characterised in that comprise the steps:

1) red saccharopolyspora seed liquor is prepared: take the S. erythraea strain that preserving number is ATCC 11635 Saccharopolyspora erythraea, streak inoculation is on YMS solid medium, in the constant incubator of 20-28 DEG C Cultivate 5-7 days, by the strain transfer after activation in fluid medium, be placed in 20-28 DEG C, the horizontal shaker of 250rpm is cultivated 40 hours, obtain seed liquor；

2) 4 are prepared "-O-glucosyl tenvermectins A/B: seed liquor is transferred in converting in culture medium, cultivate 24 little The conversion of substrate sky dimension rhzomorph A/B that time after, addition is dissolved with organic solvent, at 20-28 DEG C, converts under conditions of 250rpm and cultivates After 150 hours, obtain fermentation liquid；4 are obtained through chromatographic isolation after using extractant extractive fermentation liquid and reclaiming extractant "-O- Glucosyl tenvermectins A/B sterling；Described 4 " chemical constitution of-O-glucosyl tenvermectins A/B It is characterized as:

The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that Described step 1) in the preparation method of fluid medium be: take glucose 10g, yeast extract 2g, beef extract 2g and egg White peptone 3g, after being settled to 1000ml with distilled water, regulates pH to 7.0 with the NaOH of 1mol/l, uses 250ml triangular flask every bottle point Dress 40ml, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.

The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that Described step 2) conversion culture medium include that M-1 culture medium that carbon-nitrogen ratio is different, M-2 culture medium, M-3 culture medium and M-4 cultivate Base, wherein,

The component of M-1 culture medium is: glucose 5g, brown sugar 10g, tryptone 5g, yeast extract 2.5g, ethylenediaminetetraacetic acid Sodium 0.036g, glycine betaine 1.2g and sodium propionate 0.11g；

The component of M-2 culture medium is: cerelose 44g, Carnis Bovis seu Bubali cream 5.0g, peptone 6.0g, yeast extract 4g, casein Fried soybean cake powder 11.0g, NaCl 1.5g and K of peptone 3.0g, heat₂HPO₄0.5g；

The component of M-3 culture medium is: glucose 50g, soybean cake powder 5g, yeast extract 5g, peptone 4g, NaCl 5g, K₂HPO₄2g and KH₂PO₄1g；

The component of M-4 culture medium is: cerelose 50g, NaCl 5g, (NH₄)₂SO₄ 2g、CaCO₃6g and Semen sojae atricolor powder 30g；

After above component distilled water is settled to 1000ml, regulate pH to 7.0-7.2,250ml triangular flask with the NaOH of 1mol/l Every bottle of subpackage 40ml, at 121 DEG C, sterilizing 20min under the conditions of 0.15MPa.

The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that Described step 2) organic solvent include one or more in methanol, ethanol, DMSO.

The most according to claim 14 " preparation method of-O-glucosyl tenvermectinsA/B, it is characterised in that Described step 2) extractant include ethyl acetate, n-butyl alcohol, isobutyl acetate, ether, petroleum ether, dichloromethane or three chloromethanes One or more in alkane.

The most according to claim 14 " preparation method of-O-glucosyl tenvermectins A/B, its feature exists In, described step 2) purification include that reversed-phase high-performance liquid chromatography chromatographs.

The most according to claim 64 " preparation method of-O-glucosyl tenvermectins A/B, its feature exists In, described step 2) purification also include silica gel column chromatography.

8. an according to claim 14 " purposes of-O-glucosyl tenvermectins A/B, its feature exists In, 4 "-O-glucosyl tenvermectins A/B is used for preparing preventing and treating agriculture and forestry injurious insect or evil demodicid mite medicine.

The most according to claim 84 " purposes of-O-glucosyl tenvermectins A/B, it is characterised in that institute State agriculture and forestry injurious insect and include that Lepidoptera diamond-back moth section, Lepidoptera Noctuidae, Lepidoptera Lasiocampidae, Lepidoptera snout moths larva section, coleoptera are kowtowed One or more in first section, Tylenchida Aphelenchoidea；Described harmful demodicid mite includes spider mite kind insect.

The most according to claim 84 " purposes of-O-glucosyl tenvermectins A/B, it is characterised in that institute State 4 "-O-glucosyl tenvermectins A/B includes single 4 "-O-glucosyl tenvermectinA, single 4 "-O-glucosyl tenvermectin B or both mixture.