CN103641873B

CN103641873B - A kind of preparation method of Avrmectin

Info

Publication number: CN103641873B
Application number: CN201310689642.2A
Authority: CN
Inventors: 杨德昌; 刘书琴; 高玉成; 张宾; 张丽强; 杨飞; 李学红; 王栋良
Original assignee: NORTH CHINA PHARMACEUTICAL GROUP AINO CO Ltd
Current assignee: NORTH CHINA PHARMACEUTICAL GROUP AINO CO Ltd
Priority date: 2013-12-17
Filing date: 2013-12-17
Publication date: 2015-10-28
Anticipated expiration: 2033-12-17
Also published as: CN103641873A

Abstract

The invention discloses a kind of preparation method <b> of high-purity Abamectin, </b> is by Avrmectin meal acetone solution, application of sample is to the chromatographic column that octadecylsilane chemically bonded silica reverse-phase chromatography filler is housed, the mixed solution of polar solvent and water is used to carry out gradient elution as eluent, Fraction collection, Liquid Detection, merge, concentrated, dry, obtain object product, AVERMECTIN B1 purity 98.5 ~ 99.5% in described object product, and 97% >=Avermectin B1a purity >=95%.Avermectin B1a in finished product of the present invention and the component ratio of avermectin B1b controlled, B1b≤5.0%, other impurity 1# ~ 9#≤0.1%.Concise in technology, stable yield, quality controllable, for production pharmaceutical grade raw material provides safer technical guarantee.

Description

A kind of preparation method of Avrmectin

Technical field

The invention belongs to industrial microbial technology field, be specifically related to a kind of method utilizing Avrmectin crude product to prepare the controlled high-purity Abamectin of component ratio.

Background technology

Avrmectin (Avermectins, AVM), also known as avermectin, it is the one group of macrolide antibiotics produced by Avrmectin streptomycete (Streptomyces avermitilis) fermentation, C5,8 kinds of homologues that on C22-C23 with C25 tri-positions, structure is different, i.e. 4 main ingredients (Ala, A2a, B1a and B2a) and 4 accessory constituents (A1b, A2b, B1b and B2b), concrete structure is as follows:

, wherein: .

Avrmectin is that a class has desinsection, kills mite, the macrolide antibiotics of the sixteen-ring structure of eelworm-killing activity, to body ecto-and endoparasites, there is extremely strong killing activity, nonreactive fungus and bacterium is active, can not the synthesis of arrestin matter chitin, but it has very strong expelling parasite and insecticidal activity, be widely used in treatment multiple carcass ecto-and endoparasites sick.Avrmectin mechanism of action is comparatively special, insect is not easily made to produce resistance, chloride channel mainly through acting on γ-aminobutyric acid (GABA) acceptor in insect peripheral nervous system and L-glutamic acid gate causes insect paralysis, death, the nerve mediated with γ-aminobutyric acid due to Mammals is positioned at central nervous system, Avrmectin is not easy to enter central nervous system by hemato encephalic barrier, so have highly selective and high security, under common dose, to people, animal safety, do not injure natural enemy, do not destroy ecology.Avrmectin because having novel structure, efficient, wide spectrum, low residue and to these advantages such as people and animals and environmental safety, and becomes a kind of important green ecotypical microbiotic.

21st century, life that is healthy, clean, green, environmental protection more and more became selection and the pursuit of people, and this brings unprecedented opportunity to the development of green ecotypical Avrmectin as an environmental and ecological century.Avrmectin both can be used as agricultural antibiotic insecticides simultaneously, can be used for animal repellent again, also can be used as domestic hygiene medication, also be the people's wormer driving away filaria simultaneously, human medical, agricultural chemicals, veterinary drug is can be used for as raw material, be called as the medicine of " trinity ", just bring good market outlook for Avrmectin and derived product because of its wide Application Areas.

Its prerequisite that is structurally-modified and commercial application is furtherd investigate in the separation and purification of Avrmectin further, in 8 compositions of Avrmectin, B1a component toxicity is minimum, biological activity is the strongest, B1a with B1b is not easily separated by batch production process, and the domestic and international bibliographical information about Avermectin B1a separation and purification is also less at present.English Patent GB2185480A provides a kind of preparation method of Avrmectin, it is centrifugal after the extraction with aqueous solution of mycelium ethanol, methyl alcohol or acetone is concentrated, precipitation non-aqueous solvent such as chloroform or methylene dichloride dissolve rear filtration, extract XAD macroporous resin, silicagel column are separated, and obtain Avrmectin; Chinese patent application (application number 93115294.1) fermented liquid is separated by methods such as chromatographic separation resin HP-20SS (Diaion), gel chromatographic columns Sephadex LH-20, HPLC are anti-phase and obtains Avrmectin.These methods all complex steps, batch processing amount is little, and cannot realize the ratio control to B1a and B1b.

The domestic research of the purification process to Avrmectin mainly concentrates in the optimization of crystallization purifying condition, crystallization purifications not only needs repeatedly to carry out recrystallization, complex operation step, and the many factors such as the temperature in crystallisation process, stirring, crystal seed all can impact the product purity finally obtained, in the product finally obtained, the purity of AVERMECTIN B1 can not ensure at a higher level (Xie Zhi etc., the research of Avermectin cooling and crystallizing process, China's microbiotic impurity, volume the 1st phase January the 30th in 2005, P58-61).Jiang Lin etc. were at (Jiang Lin in 2002, silica gel column chromatography is separated the effect preparing Avrmectin B_2, Shanghai Agricultural journal, 2002, 18 (1): 81-83) adopt chromatography to be separated and prepare Avrmectin, with the mixing solutions of V (sherwood oil): V (Virahol)=7:1 for elutriant, be that stationary phase has good separation performance to AVERMECTIN B1 with silicagel column, but this method cost is very high, be not suitable for suitability for industrialized production, and it is volatile also to there is solvent systems in this method, be unfavorable for the recovery of solvent and the shortcoming of recycle, thus cause cost up, and can to environment, be not suitable for modern environment-friendly production theory.

Summary of the invention

The object of this invention is to provide a kind of technique simple; stable yield; the preparation technology of the high-purity Abamectin B1 that the component ratio that is produced on a large scale is controlled, to solve complex steps existing for existing separation purification method, batch processing amount is little, and problem that is with high costs, contaminate environment.

The object of the invention is by following technical scheme realize:

A kind of preparation method of high-purity Abamectin, the method is by Avrmectin meal acetone solution, application of sample is to the chromatographic column that octadecylsilane chemically bonded silica reverse-phase chromatography filler is housed, the mixed solution of polar solvent and water is used to carry out gradient elution as eluent, Fraction collection, Liquid Detection, merge, concentrated, dry, obtain object product, AVERMECTIN B1 purity 98.5 ~ 99.5% in described object product, and 97% >=Avermectin B1a purity >=95%.

The preparation method of high-purity Abamectin of the present invention, 0 < foreign matter content≤0.1% in gained object product.

The preparation method of high-purity Abamectin of the present invention, described polar solvent is any one in methyl alcohol, ethanol and acetonitrile.

The preparation method of high-purity Abamectin of the present invention, described gradient elution, is first use the polar solvent of volume ratio 50 ~ 70% and the mixed solution wash-out of water to remove impurity, then uses the polar solvent of volume ratio 75 ~ 80% and the mixed solution of water to carry out wash-out.

The preparation method of high-purity Abamectin of the present invention, the flow velocity of the eluent of described gradient elution be 1 ~ 4 column volume/hour.

The preparation method of high-purity Abamectin of the present invention, described Avrmectin meal is the crude extract of abamectin fermented liquid.

Further, polar solvent used in the present invention is preferably methyl alcohol.

Further, during institute's gradient elution of the present invention, the flow velocity of eluent is preferably 2 ~ 3BV/h.

The present invention uses ODS reverse-phase chromatography filler to carry out gradient chromatography, effectively eliminate impurity and partial pigment in Avrmectin, increase substantially product purity, finally obtain AVERMECTIN B1 purity >=98.5%, and Avermectin B1a wherein and the component ratio of avermectin B1b controlled, wherein 97% >=B1a purity >=95.0%, other impurity 1# ~ 9#≤0.1%.

Method concise in technology of the present invention, stable yield, quality controllable, for production pharmaceutical grade raw material provides safer technical guarantee, and without the need to using solvent that the toxicity such as chloroform, toluene is large as moving phase in production process of the present invention, have better environment friendly, cost is also more cheap.

Accompanying drawing explanation

Fig. 1 is the Avrmectin crude product color atlas prepared by embodiment 1.

Fig. 2 is the high-purity Abamectin color atlas prepared by embodiment 3.

Specific embodiment

Following embodiment only realizes method of the present invention for setting forth, and should not be construed as limitation of the present invention, all modifications and variations of making based on thinking of the present invention are all attributed to protection scope of the present invention.

Octadecylsilane bonded-phase silica ODS reverse phase filler is that YMC company produces; The reagent such as acetone, ethanol, methyl alcohol, acetonitrile are commercially available.The high performance liquid chromatograph that the present invention uses is 996 type detectors, 515 pumps (Waters company).

Liquid Detection condition: chromatographic column: Shim – pack VP-ODS chromatographic column (5 μm, 4.6 × 250mm); Moving phase: 80% acetonitrile-20% aqueous solution (containing 0.05% trifluoroacetic acid); Flow velocity: 1mL/min; Determined wavelength: 254nm.

Embodiment 1: the preparation of Avrmectin crude product:

The present embodiment use slant medium, seed culture medium, fermention medium component as follows:

Slant medium: glucose 15g, extractum carnis 3g, asparagine 0.5g, KH2PO4 0.5g, agar 19g, add after tap water dissolves and be settled to 1000ml, pH value is adjusted to 7.2 ~ 7.5; 121 DEG C of sterilizing 30min.

Seed culture medium: W-Gum 30.0g, beans Hectometer powder 8.0g, groundnut meal 10g, yeast powder 5.0g, COCl2 2.0g, add tap water and dissolve, be settled to 1000 ml, pH value 6.8-6.9,121 DEG C of sterilizing 30 min.

Fermention medium: W-Gum 150.0g, beans Hectometer powder 30.0g, yeast powder 9.0g, (NH4) 2SO4 0.25g, COCl2 0.03g, Na2MoO4 0.025g, MnSO4 0.003g, CaCO3 1.0g, amylase 0.03g, add tap water to dissolve, be settled to 1000 ml, pH value 7.2-7.4,121 DEG C of sterilizing 30min.

(1) by Avid kyowamycin bacterial strain (streptomyces averditilis, numbering 192S9) be inoculated on slant medium, 28 DEG C, cultivate 10 days in the environment of relative humidity 55% after, taking inclined-plane lawn is seeded in seed culture medium, 28 DEG C, relative humidity 55%, 200rpm, rotation radius 50mm condition under cultivate 48h obtain seed liquor; Seed liquor is inoculated in the 50L fermentor tank that fermention medium is housed with the inoculum size of volume percent 5%, at tank temperature 28 DEG C, ambient relative humidity 55%, tank pressure 0.05 ± 0.01Mpa, air flow 20L/min, under 200rpm agitation condition, fermentation 240h obtains abamectin fermented liquid;

(2) get the abamectin fermented liquid of 10L (fermentation unit is 4845ug/ml) to filter, collect mycelium 2.5kg, then add 10L acetone, stirring and leaching was filtered after 1 hour, collected vat liquor; Vat liquor being concentrated into volume is 1L, then import Fraclite800 macroporous resin column and carry out adsorbing that (resin loading amount is 500mL, flow velocity is 2BV/h), resin after absorption is the aqueous ethanolic solution of 40%, the aqueous ethanolic solution wash-out of 80% by concentration successively, flow velocity is 1BV/h, Fractional Collections elutriant, by concentration be 80% ethanol eluate at 50 DEG C, be evaporated to solvent-free outflow, use ethanol periodic crystallisation, filter, drying obtains Avrmectin crude product (liquid chromatogram is shown in Fig. 1, and each component ratio is in table 1 and 2).

Embodiment 2: Avrmectin purifying crude process

Avrmectin crude product 5g prepared by Example 1, use 20mL acetone solution, application of sample is to the chromatography column (loading amount is 500ml) of ODS reverse-phase chromatography filler, first use the aqueous ethanolic solution (volumetric concentration 60%) of 10L as moving phase wash-out, then use the aqueous ethanolic solution of volumetric concentration 75% as moving phase wash-out (flow velocity is 2BV/h), all flow out to Avrmectin, Liquid Detection 75% ethanol eluate, elutriant is merged according to detected result, concentrated, vacuum-drying at 45 DEG C, obtain 1.5g AVERMECTIN B1 (B1a+B1b), purity is that each component ratio of 99.0%(is in table 1 and 2).

Embodiment 3: Avrmectin purifying crude process

Avrmectin crude product 10g prepared by Example 1, use 50mL acetone solution, application of sample is to the chromatography column (loading amount is 1000ml) of ODS reverse-phase chromatography filler, first use the methanol aqueous solution (volumetric concentration 70%) of 15L as moving phase wash-out, then the methanol aqueous solution wash-out of volumetric concentration 85% (flow velocity is 4BV/h) is used, all flow out to Avrmectin, Liquid Detection 85% meoh eluate, elutriant is merged according to detected result, concentrated, vacuum-drying at 45 DEG C, obtain 4.1g AVERMECTIN B1 (B1a+B1b), purity is that 99.4 %(liquid chromatograms are shown in Fig. 2, each component ratio is in table 1 and 2).

Embodiment 4: Avrmectin purifying crude process

Avrmectin crude product 10g prepared by Example 1, use 50mL acetone solution, application of sample is to the chromatography column (loading amount is 1000mL) of ODS reverse-phase chromatography filler, first use the acetonitrile solution (volumetric concentration 50%) of 10L as moving phase wash-out, then the acetonitrile solution wash-out of volumetric concentration 80% (flow velocity is 3BV/h) is used, all flow out to Avrmectin, Liquid Detection 80% acetonitrile liquid, elutriant is merged according to detected result, concentrated, vacuum-drying at 45 DEG C, obtains 3.5g, B1a+B1b is the Avrmectin of 98.9 %, and each component ratio is in table 1 and 2.

Table 1 embodiment 1 ~ 4 gained Avermectin B1a and B1b content (area normalization method):

In table 2 embodiment 1-4 gained Avrmectin except B1a and B1b, other components 1 ~ 9# content (area normalization method):

Embodiment	1#	2#	3#	4#	5#	6#	7#	8#	9#
										1	0.25	0.27	0.11	0.15	0.1	0.13	0.50	0.27	0.30
3	-	-	0.04	0.02	0.04	0.07	-	-	-
										4	-	-	0.02	0.07	0.03	0.06	-	-	-
5	-	-	0.05	0.08	0.03	0.05	-	-	-

As can be seen from the result of table 1, table 2 and Fig. 1, Fig. 2, the inventive method effectively can be separated the B1 component (purity is up to 94%) in Avrmectin crude product, especially B1a component (purity is up to 96.8%), and the content of impurity reduces greatly in product, 1#, 2#, 7# ~ 9# does not detect, and the content of 3# ~ 6# is all below 0.1%.

Comparative example:

Utilize common chromatograph packing material (silica gel and Fraclite800 resin) according to a conventional method and select different moving phase, carry out purifying to the Avrmectin crude product that embodiment 1 obtains, and coordinate crystallization purifying, the purity detecting of final products obtained therefrom the results are shown in Table 3.

Table 3: use silica gel and Fraclite800 resin to carry out the Liquid Detection result of purifying products obtained therefrom for filler:

As can be seen from above detected result, utilize Fraclite800 chromatographic resin to carry out purifying, B1a+B1b >=98.5% in products obtained therefrom, but B1a≤95.0%, and other components do not reach requirement yet; Utilize silica gel to carry out purifying, can obtain B1a+B1b content and the satisfactory sample of ratio, but need to use chloroform, toluene equal solvent, toxicity is comparatively large, and the index of other component 1#-9#≤0.1% still fails to realize.

Claims

1. the preparation method of an Avrmectin, it is characterized in that the method is, by Avrmectin meal acetone solution, application of sample, to the chromatographic column that octadecylsilane chemically bonded silica reverse-phase chromatography filler is housed, uses the mixed solution of polar solvent and water to carry out gradient elution as eluent, Fraction collection, Liquid Detection, merges, concentrated, dry, obtains object product, AVERMECTIN B1 purity 98.5 ~ 99.5% in described object product, and 97% >=Avermectin B1a purity >=95%; Described gradient elution, is first use the polar solvent of volume ratio 50 ~ 70% and the mixed solution wash-out of water to remove impurity, then uses the polar solvent of volume ratio 75 ~ 80% and the mixed solution of water to carry out wash-out; The flow velocity of the eluent of described gradient elution be 1 ~ 4 column volume/hour; Described polar solvent is any one in methyl alcohol, ethanol and acetonitrile.

2. the preparation method of Avrmectin according to claim 1, is characterized in that, foreign matter content≤0.1% in gained object product.

3. the preparation method of Avrmectin according to claim 1, is characterized in that, described Avrmectin meal is the crude extract of abamectin fermented liquid.