CN109157578B - Florfenicol composite synergist and preparation method and application thereof - Google Patents
Florfenicol composite synergist and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a composite synergist, in particular to a florfenicol composite synergist and a preparation method and application thereof, wherein the composite synergist comprises the following components in parts by weight: 70-88 parts of purple sweet potato leaf extract and purification product, 5-12 parts of phellodendron fermentation liquor and purification product, 3-8 parts of eucommia fermentation liquor and purification product, 0.3-0.5 part of mildew preventive and 0.3-0.5 part of antioxidant. The composite synergist of the invention is used in combination with florfenicol, so that the antibacterial effect of the florfenicol can be enhanced, the treatment effect of the florfenicol is improved, and meanwhile, the composite synergist has no toxic or side effect on water body environment and fish bodies.
Description
Technical Field
The invention relates to a composite synergist, in particular to a florfenicol composite synergist and a preparation method and application thereof.
Background
Florfenicol (Florfenicol), also known as Florfenicol, is a new veterinary-dedicated amidol broad-spectrum antibacterial drug successfully developed in the late stage of the eighties of the twentieth century, generates a broad-spectrum antibacterial effect by inhibiting the activity of peptidyl transferase in an antibacterial mechanism, and has the advantage of wide antibacterial spectrum. Since its introduction to the market in 1990, it has been widely used in the treatment of bacterial diseases in most aquaculture species.
In recent years, the use of florfenicol in the treatment of bacterial diseases in aquaculture varieties has also exposed several significant problems. Firstly, due to the objective existence of the factors such as overproof residue, cross drug resistance and the like, the application of a plurality of drugs which are synergized with florfenicol at the aquaculture end is prohibited by policy, so that a safe and effective florfenicol synergist at the aquaculture end is blank. Secondly, the single dosage of the florfenicol used as a medicament for treatment is obviously increased gradually, so that the medicament cost is increased, and the culture benefit is reduced. Finally, the florfenicol is used as micro powder, the solubility of the florfenicol in water is very low under the conditions of normal temperature and normal pressure, and the lack of cosolvent can cause the reduction of the drug effect, which is also the core reason that the expected effect is sometimes difficult to achieve when the aquiculture end adopts water-soluble pharmaceutical baits.
Therefore, the safe and efficient synergist is screened out, the requirements of synergy with florfenicol, safety, no toxicity and solubilization-assisting synergy are met functionally, and great value is brought to the improvement of the application effect of the florfenicol in the aquaculture end.
Disclosure of Invention
Aiming at the defects, the invention provides a florfenicol composite synergist, a preparation method and application thereof, which aim to safely and efficiently solve the outstanding problems in the application process of florfenicol at the aquaculture end.
The invention achieves the above purposes through the following scheme:
in a first aspect, the florfenicol composite synergist comprises the following components in parts by weight: 70-88 parts of purple sweet potato leaf extract and purification product, 5-12 parts of phellodendron fermentation liquor and purification product, 3-8 parts of eucommia fermentation liquor and purification product, 0.3-0.5 part of mildew preventive and 0.3-0.5 part of antioxidant.
Preferably, the florfenicol composite synergist comprises the following components in parts by weight: 85 parts of purple sweet potato leaf extract purified product, 8 parts of phellodendron fermentation liquor purified product, 6 parts of eucommia fermentation liquor purified product, 0.5 part of mildew preventive and 0.5 part of antioxidant.
Preferably, the purple sweet potato leaf extract purified product is prepared by the following steps: extracting the purple sweet potato leaves by using a solvent to obtain an extract, and then carrying out vacuum freeze drying on the extract to prepare solid powder so as to obtain a purified purple sweet potato leaf extract.
Further preferably, the purple sweet potato leaf extract purified product is prepared by the following steps: weighing 100 parts of purple sweet potato leaves with the water content less than or equal to 20% according to parts by weight to prepare purple sweet potato leaf micro powder, wherein the volume ratio of the purple sweet potato leaves to a solvent (petroleum ether) is 1: (10-15) measuring petroleum ether which is 10-15 times of the volume of the purple sweet potato leaves, carrying out hot reflux extraction for 5-7 times, each time for 2-5 hours, combining extracting solutions, filtering, concentrating under reduced pressure, evaporating at constant temperature to obtain an extract, preferably carrying out low-temperature low-pressure extraction and steam treatment on the extract to form solid powder with the water content of 3-5%, and obtaining the purple sweet potato leaf extraction purification product.
In a preferred embodiment, the purple sweet potato leaf extract purified product is prepared by the following steps: weighing 100 parts of purple sweet potato leaves with the water content less than or equal to 20% by weight, sieving the purple sweet potato leaves with a 80-mesh sieve to prepare purple sweet potato leaf micro powder, wherein the volume ratio of the purple sweet potato leaves to the solvent is 1: (10-15) measuring petroleum ether (with a boiling range of 60-90 ℃) which is 10-15 times of the weight of the purple sweet potato leaves, carrying out hot reflux extraction for 5-7 times, each time for 2-5 hours, combining the extracting solutions, filtering, concentrating under reduced pressure, evaporating to dryness at a constant temperature of 55 ℃ to obtain an extract, carrying out low-temperature low-pressure extraction and steam treatment on the extract for 48 hours to form solid powder with the water content of 3-5%, and obtaining the purple sweet potato leaf extraction and purification product.
Preferably, the cortex phellodendri fermentation liquor purified product is prepared by the following steps: weighing cortex Phellodendri, micronizing, fermenting with lactobacillus to obtain fermentation liquid, ultrasonic crushing, dialyzing with running water, concentrating to obtain concentrate, and vacuum freeze drying to obtain solid powder.
Further preferably, the cortex phellodendri fermentation liquor purified product is prepared by the following steps: weighing 100 parts by weight of phellodendron amurense with coarse bark removed, preparing the phellodendron amurense into micro powder, adding the micro powder into a container filled with water with the mass volume ratio of 40-50 times, adding a lactobacillus liquid nutrient medium with the volume ratio of 3-5% of the added water, carrying out closed decoction for 30-45 minutes, cooling to normal temperature, adding more than or equal to 10 viable bacteria91000-1200 ml of cfu/ml lactobacillus liquid, 2mol of MgCl210-12 ml, sealing, heating to 37 ℃, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging the crushed residue liquid at 20000rpm for 10 minutes, removing residues, collecting supernatant, filling into a dialysis bag, dialyzing with running water for 36-48 hours, concentrating the high molecular weight polyethylene glycol to one fifth of the original volume, collecting concentrated solution, and performing low-temperature low-pressure steam extraction treatment on the concentrated solution for 36-48 hours to form solid powder with the water content of 3-5% to obtain the cortex phellodendri fermentation liquid purified product.
Further preferably, the cortex phellodendri fermentation liquor purified product is prepared by the following steps: weighing 100 parts by weight of phellodendron amurense with coarse bark removed, sieving the phellodendron amurense with a 80-mesh sieve to prepare micro powder, adding the micro powder into a container with distilled water with the mass volume ratio of 40-50 times, then adding the components of a common lactic acid bacteria liquid nutrient medium, carrying out closed decoction for 30-45 minutes at 100 ℃, cooling to normal temperature, adding more than or equal to 10 viable bacteria91000-1200 ml of cfu/ml lactobacillus liquid, 2mol of MgCl210 to 12ml, sealing, heating to 37 ℃, culturing at constant temperature until OD is 3.0 to 3.6, taking the culture for ultrasonic crushing treatment, repeating the treatment for three times, taking the crushed slag liquid, centrifuging at 20000rpm for 10 minutes, removing slag, collecting supernatant, filling into a dialysis bag with 0.45 nanometer aperture, dialyzing with running water for 36 to 48 hours,concentrating the high molecular weight polyethylene glycol to one fifth of the original volume, collecting the concentrated solution, and performing low-temperature low-pressure pumping steam treatment on the concentrated solution for 36-48 hours to form solid powder with the water content of 3-5% to obtain the cortex phellodendri fermentation liquor purified product.
Preferably, the purified eucommia ulmoides fermentation liquor is prepared by the following steps: weighing eucommia ulmoides produced in Sichuan province, carrying out micro powder treatment, fermenting by lactobacillus to generate fermentation liquor, carrying out ultrasonic crushing, running water dialysis and concentration on the fermentation liquor to obtain a concentrate, and carrying out vacuum freeze drying on the concentrate to prepare solid powder so as to obtain the purified eucommia ulmoides fermentation liquor.
Further preferably, the purified eucommia ulmoides fermentation liquor is prepared by the following steps: weighing 100 parts of eucommia bark, sieving the eucommia bark with a 80-mesh sieve to prepare micro powder, adding water with the mass volume ratio of 100-130 times, then adding a lactobacillus liquid nutrient medium, carrying out closed decoction for 30-45 minutes at 100 ℃, cooling to normal temperature, and adding the number of viable bacteria which is more than or equal to 109Adding ethanol into 1000-1200 ml of lactobacillus liquid of cfu/ml, fully mixing uniformly, sealing, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging to remove residues, collecting supernatant, filling into a dialysis bag, performing running water dialysis for 36-48 hours, concentrating high-molecular-weight polyethylene glycol to one fifth of the original volume, collecting concentrated solution, and performing low-temperature low-pressure steam extraction treatment on the concentrated solution for 36-48 hours to form solid powder with the water content of 3-5% to obtain the purified product of the eucommia fermentation liquid.
Further preferably, the purified eucommia ulmoides fermentation liquor is prepared by the following steps: weighing 100 parts of eucommia bark, sieving the eucommia bark with a 80-mesh sieve to prepare micro powder, adding water with the mass volume ratio of 100-130 times, then adding a lactobacillus liquid nutrient medium, carrying out closed decoction for 30-45 minutes at 100 ℃, cooling to normal temperature, and adding the number of viable bacteria which is more than or equal to 1091000-1200 ml of cfu/ml lactobacillus strain liquid is added with 5ml of ethanol, fully and uniformly mixed, sealed, heated to 37 ℃, cultured at constant temperature until OD is 3.0-3.6, the culture is taken for ultrasonic crushing treatment and repeated treatment for three times, the crushed residue liquid is taken for centrifugation at 20000rpm for 10 minutes to remove residue, the supernatant is collected and put into a dialysis bag with 0.45 nanometer aperture, running water dialysis is carried out for 36-48 hours,concentrating the high molecular weight polyethylene glycol to one fifth of the original volume, collecting the concentrated solution, and performing low-temperature low-pressure pumping steam treatment on the concentrated solution for 36-48 hours to form solid powder with the water content of 3-5% to obtain the purified product of the eucommia fermentation liquid.
Preferably, the lactic acid bacteria liquid nutrient medium: 11-15 parts of peptone, 3-5 parts of beef extract, 3-5 parts of sodium chloride and 1000 parts of distilled water by mass, and adjusting the pH to 7.0-7.4.
Wherein, the mildew preventive and the antioxidant can be common commercial feed-grade products, for example, the mildew preventive can adopt Kemeiba, dichlofluanid and the like; the antioxidant can be adaquine.
In a second aspect, a method for preparing the florfenicol composite synergist is provided, which comprises the following steps: uniformly mixing the purple sweet potato leaf extract purified product, the phellodendron fermentation liquor purified product, the eucommia fermentation liquor purified product, the mildew preventive and the antioxidant to obtain the composite synergist.
In a third aspect, a composite synergist prepared by the preparation method is provided.
In a fourth aspect, an application of the composite synergist is provided, and the application comprises diluting the composite synergist, and mixing the diluted composite synergist with the following components in a volume-to-mass ratio (1-3): 100 is used in combination with florfenicol.
Preferably, the application comprises diluting the composite synergist by 85-120 times, for example 100 times, with a neutral PBS buffer solution, and then mixing the diluted composite synergist and the neutral PBS buffer solution in a volume-to-mass ratio (1-3): 100 is used in combination with florfenicol.
Preferably, the neutral PBS buffer solution has a pH value of 7-7.4.
Further preferably, the application of the composite synergist comprises the following steps of diluting the composite synergist, and mixing the diluted composite synergist with the following components in a volume-to-mass ratio (1-3): 100 is used in combination with florfenicol.
Preferably, the application of the composite synergist is used for the synergism of florfenicol, and more preferably for the synergism of the florfenicol on G-escherichia coli.
The composite synergist of the invention is used in combination with florfenicol, so that the antibacterial effect of the florfenicol can be enhanced, the treatment effect of the florfenicol is improved, and meanwhile, the composite synergist has no toxic or side effect on water body environment and fish bodies.
Compared with the similar products in the market, the invention has the following beneficial effects:
1. the components of the invention all belong to natural plants, are convenient to obtain and low in price, and the active ingredients of the components have extremely high stability through advanced process extraction and compatibility, have no toxic or side effect on water body environment and fish bodies, and meet the requirement of pollution-free culture.
2. The components of the three purified products of the invention are scientifically proportioned, have synergistic antibacterial effect, and can achieve the antibacterial synergistic effect of the invention by using one component or two components in combination.
3. The florfenicol-containing antibacterial agent is used together with florfenicol aiming at aquatic cases infected by gram-negative bacteria, can enhance the antibacterial effect of the florfenicol and improve the treatment effect of the florfenicol.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1:
weighing 85 parts of purple sweet potato leaf extract purified product, 8 parts of phellodendron fermentation liquor purified product, 6 parts of eucommia fermentation liquor purified product, 0.5 part of mildew preventive Kemeiba and 0.5 part of antioxidant Fudaquine according to parts by weight, and uniformly mixing the components according to a predetermined proportion according to the requirement of aseptic operation to obtain the florfenicol composite synergist.
The purple sweet potato leaf extract purified product is prepared by the following steps: weighing 100 parts of purple sweet potato leaves with the water content less than or equal to 20% by weight, sieving the purple sweet potato leaves with a 80-mesh sieve to prepare purple sweet potato leaf micro powder, wherein the volume ratio of the purple sweet potato leaves to the solvent is 1: (10-15) measuring petroleum ether (with a boiling range of 60-90 ℃) which is 10-15 times of the weight of the purple sweet potato leaves, carrying out hot reflux extraction for 5-7 times, each time for 2-5 hours, combining the extracting solutions, filtering, concentrating under reduced pressure, evaporating to dryness at a constant temperature of 55 ℃ to obtain an extract, placing the extract in a vacuum drier, carrying out low-temperature low-pressure extraction and steam treatment on the extract for 48 hours to form solid powder with water content of 3-5%, and obtaining an extracted and purified purple sweet potato leaf, wherein the purified purple sweet potato leaf can be stored at 4 ℃ for later use.
The cortex phellodendri fermentation liquor purified product is prepared by the following steps: weighing 100 parts by weight of phellodendron amurense with coarse bark removed, sieving the phellodendron amurense with a 80-mesh sieve to prepare micro powder, adding the micro powder into a container which is pre-filled with sterile distilled water with the mass volume ratio of 40-50 times, then adding a common lactic acid bacteria liquid nutrient medium with the volume matched with the volume of the sterile distilled water, carrying out closed decoction for 30-45 minutes at 100 ℃, cooling to normal temperature, adding the number of viable bacteria which is more than or equal to 1091000-1200 ml of cfu/ml lactobacillus liquid, 2mol of MgCl210-12 ml, sealing, heating to 37 ℃, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging the crushed residue liquid at 20000rpm for 10 minutes, removing residues, collecting supernatant, filling into a dialysis bag with 0.45 nanometer aperture, dialyzing with running water for 36-48 hours, concentrating the high molecular weight polyethylene glycol to one fifth of the original volume, collecting concentrated solution, placing the concentrated solution in a box type vacuum dryer, performing low-temperature low-pressure steam pumping treatment for 36-48 hours to form solid powder with water content of 3-5%, and obtaining a purified substance of the phellodendron fermentation liquor, which can be stored at 4 ℃ for later use.
The purified eucommia ulmoides fermentation liquor is prepared by the following steps: weighing 100 parts of eucommia ulmoides by weight, sieving the eucommia ulmoides by a 80-mesh sieve to prepare micro powder, adding the micro powder into a container pre-filled with sterile distilled water with the mass volume ratio of 100-130 times, then adding common lactic acid bacteria liquid nutrient medium components matched with the volume of the sterile distilled water, carrying out closed decoction for 30-45 minutes at the temperature of 100 ℃, cooling to normal temperature, adding the number of viable bacteria more than or equal to 1091000-1200 ml of a cfu/ml lactobacillus liquid, adding 5ml of ethanol, fully mixing, sealing, heating to 37 ℃, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging the crushed residue liquid at 20000rpm for 10 minutes, removing residues, collecting supernatant, filling into a dialysis bag with a pore diameter of 0.45 nanometer, dialyzing for 36-48 hours with running water, concentrating high molecular weight polyethylene glycol to one fifth of the original volume, collecting concentrated solution, placing the concentrated solution in a box type vacuum drier, performing low-temperature low-pressure steam pumping treatment for 36-48 hours to form solid powder with water content of 3-5%, thus obtaining a purified eucommia fermentation liquid, which can be stored at 4 ℃ for later use.
Example 2
Weighing 82 parts of purple sweet potato leaf extract purified product, 12 parts of phellodendron fermentation liquor purified product, 5 parts of eucommia fermentation liquor purified product, 0.5 part of mildew preventive Kemeiba and 0.5 part of antioxidant Fudaquine according to parts by weight, and uniformly mixing the components according to a predetermined proportion according to the requirement of aseptic operation to obtain the florfenicol composite synergist. The purple sweet potato leaf extract purified product, the phellodendron fermentation liquor purified product and the eucommia ulmoides fermentation liquor purified product are prepared according to the method in the embodiment 1.
Test example 1
1. Test materials
1.1 test drugs
Florfenicol standard: purchased from China institute for veterinary medicine
Compound synergist: florfenicol composite synergist from preparation of example 1
1.2 test strains
Gram-positive bacteria: streptococcus agalactiae ATCC13813
Gram-negative bacteria: escherichia coli ATCC25922
1.3 Medium
TSA broth or LB broth
2. Test method
2.1 selection of drug concentration
The dilution of the drug combination assay was determined based on the MIC (minimum inhibitory concentration) of florfenicol and the compound potentiator. Generally, about 6 to 8 dilutions are selected. The highest concentration of each drug was 4 or 8 times its MIC, then serially diluted two-fold to 1/4 or 1/8 of its MIC.
2.2 determination of MIC (minimum inhibitory concentration) of Single drug
The concentration of the prepared florfenicol stock solution is 5120 mu g/ml, and the concentration of the composite synergist stock solution is 51200 mu g/ml
Respectively diluting the florfenicol stock solution concentration/the composite synergist stock solution concentration.
The concentration of the florfenicol stock solution is as follows:
512-256-128-64-32-16-8-4-2-1-0.5-0.25
concentration of the composite synergist stock solution:
5120-2560-1280-640-320-160-80-40-20-10-5-2.5-1.25-0.625-0.3125
the MICs of the individual drugs were determined according to the MIC method.
2.2.1 test tube numbering
Each group of drugs was done in 2 replicates. In each row, 2 tubes were labeled with "broth control tube" and "test bacteria growth control".
2.2.2 dilution of antibacterial drug
The concentration of the stock solution is diluted by 10 times to the concentration of the first test tube. Adding 1.8ml of MH broth into the 1 st tube, adding 0.2ml of antibacterial drug stock solution (such as 1280 mu g/ml) into the 1 st tube, uniformly mixing, sucking 1ml into the 2 nd tube, sucking 1ml into the 3 rd tube after uniformly mixing, diluting to the last tube in a multiple ratio in this way, sucking 1ml from the last tube, and discarding, wherein the last three tubes are growth control groups without drugs, and the last two tubes are test bacteria growth control groups. At the moment, the concentration of the drugs in each tube is the corresponding florfenicol series concentration or compound synergist series concentration in the step (2).
2.2.3 enrichment culture
The glycerol-preserved bacterial liquid is taken to room temperature and transferred to the broth, and the bacterial liquid after 3 generations of culture is reserved (the 3 rd generation bacterial liquid is mostly cultured for 4-6 h). The grown bacterial liquid is corrected to the 0.5 McLeod turbidity standard by using 3-5 ml of normal saline, and then diluted by using MH broth to obtain a mixture with the concentration of 1: 100 times of the total amount of the bacteria so that the bacteria content is 106CFU/ml。
2.2.4 inoculation
1m1 of test bacteria solution is added to each test tube (each micropore) in the first row from low concentration to high concentration in turn, and standard bacteria is added to each test tube in the second row. The final inoculation bacterial quantity is about 5X 105CFU/ml。
2.3 combination drug test
Florfenicol: in the row A, 50 mu L of 8MIC florfenicol is added into the front 7 holes, and 100 mu L of 4MIC florfenicol is added into the 8 th hole; in the row B, 50 mu L of 4MIC florfenicol is added into the front 7 holes, and 100 mu L of 2MIC florfenicol is added into the 8 th hole; c, adding 50 mu L of 2MIC florfenicol into the first 7 holes of the row, and adding 100 mu L of MIC florfenicol into the 8 th hole; and so on to row G.
Compound synergist: first 7 wells of column 1, 8MIC composite potentiators, 5 per well0 mu L, and 100 mu L of 4MIC compound synergist is added into the 8 th hole; 50 mu L of 4MIC compound synergist is added into the first 7 holes of the row 2, and 100 mu L of 2MIC compound synergist is added into the 8 th hole; 50 mu L of 2MIC compound synergist is added into the first 7 holes of the row 3, and 100 mu L of MIC compound synergist is added into the 8 th hole; and so on to column 8. Adding diluted bacterial liquid 10 into front 7 holes of A-G6CFU/ml 100. mu.L, final inoculum concentration 5 x 10 per well5CFU/ml. With H8 wells serving as growth controls.
2.4 determination of results of Combined drug susceptibility test
The results of the combined susceptibility test are of four types.
And (3) synergistic effect: the combined activity of the two antibacterial agents is obviously greater than the sum of the antibacterial effects of the single agents (1+1> 2).
Additive effect: the activity of the combination of the two antibacterial drugs is slightly increased compared with that of either single drug (1+ 1-2).
Unrelated effects: the activity of both antibacterial drugs is not affected by the other drug (1+1 ═ 1).
Antagonism: the activity of one antibacterial agent is attenuated by the other (1+1< 1).
And calculating partial bacteriostatic concentration in a laboratory as a judgment basis of a combined drug sensitivity test.
And (4) judging the standard:
3. results
3.1 Combined susceptibility test of two test substances against gram-Positive cocci
3.1.1 Single drug MIC
TABLE 1 MIC test results of two test substances for G + bacteria
3.1.2 Combined susceptibility testing
TABLE 2 Combined drug sensitivity test results of two tested substances to G + bacteria
Injecting: the fluorine single refers to a single florfenicol drug, and the composite single refers to a single composite synergist drug. The same applies below.
As shown in tables 1 and 2, the MIC of the single florfenicol drug is 4. mu.g/mL, the MIC of the single composite synergist drug is 640. mu.g/mL, and the MICs of the florfenicol and the composite synergist in combination are 4 and 1280. mu.g/mL, respectively. When the two medicines are used together, the MIC of the composite synergist is obviously higher than the MIC value of the single medicine, the FIC index of the combined medicine is obtained and judged:
FIC 8/8+1280/640 > 3, indicating that florfenicol and the compound synergist have antagonistic action.
3.2 Combined susceptibility test of two test substances to Escherichia coli ATCC25922
TABLE 3 results of MIC test of two test substances on Escherichia coli ATCC25922
3.2.1 Combined susceptibility testing
TABLE 4 Combined susceptibility test results of two test substances to G-E.coli
As shown in tables 3 and 4, the MIC of the single drug of florfenicol is 32 mug/ml, the MIC of the single drug of the composite synergist is more than 10240 mug/ml, and the MICs of the florfenicol and the composite synergist are 16 mug/ml and 320 mug/ml respectively when the florfenicol and the composite synergist are combined. When the two medicines are used together, the MIC of the composite synergist is obviously higher than the MIC value of the single medicine, the FIC index of the combined medicine is obtained and judged:
FIC＝16/32+320/10240＝0.5+1/32，FIC＝0.5～1
the florfenicol and the composite synergist have additive effect, and the MIC value of the florfenicol can be reduced by using the composite synergist.
4. Small knot
The combined drug sensitive result of the florfenicol and the composite synergist shows that the florfenicol and the composite synergist have antagonistic action when used together with G + coccus and the florfenicol. Therefore, florfenicol is prohibited from being used in combination with a composite synergist for cases due to gram-positive bacterial infection.
For G-bacillus escherichia coli ATCC25922, the composite synergist and the florfenicol have an additive effect, and the MIC value of the florfenicol can be reduced. Thus, in the case of G-bacillus E.coli infection, the combination of florfenicol and the composite synergist can reduce the dosage of florfenicol.
Test example 2
This example is intended to evaluate the safety of florfenicol for the internal administration of farmed fish when combined with a composite synergist.
1. Materials and methods
1.1 test Fish
Grass carp: purchased from a fine variety field of the Buddha mountain grass carp, and the weight specification is 100 plus or minus 5 g.
1.2 preparation of medicinal baits
The dosage of the florfenicol is 15mg per kilogram of fish body weight, and the dosages of the composite synergist are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight according to the mass-volume ratio.
Taking the dosage of the florfenicol and the composite synergist as a reference value, and taking the daily average feeding rate as 2.5 percent, three kinds of medicine baits of I-1#, I-2#, and I-3# are prepared in sequence, so that the assumed intake concentration of the florfenicol and the composite synergist in the baits reaches the reference value.
Two kinds of medicine baits I-4# and I-5# are prepared simultaneously, the florfenicol is added in zero, and the assumed intake concentration of the composite synergist in the baits respectively reaches 0.15ml/kg and 0.30 ml/kg.
The same-grade bait completely free of florfenicol and composite synergist is used as blank feed, which is called blank feed for short.
1.3 test grouping and management
The grass carp is randomly divided into A, B, C, D, E, F groups, each group is provided with 3 parallel groups, each group uses 100 fish tails, A, B, C, D, E five groups are respectively fed with five kinds of medicine baits of I-1#, I-2#, I-3#, I-4#, and I-5#, and the group F is fed with blank materials without any medicine and is set as a negative control group.
1.4 index determination
Evaluation of tissue toxicity: tissue samples were collected on days 7, 14, 21, and 28 of the test, respectively, to prepare sections, and histomorphometric analyses were performed on the liver, kidney, spleen, and intestinal tract of the test fish. If 0, 1, 2, 3 or 4 or more foci are observed in the tested tissue, they are marked as normal, plus, minus, plus, minus, plus, minus, plus, or minus, plus, is noted, or minus, is noted in the tested tissue.
Determination of transaminase activity: non-anticoagulated blood was collected from the caudal vein of each test group on test days 7, 14, 21, and 28, respectively, centrifuged at 5000rpm for 10 minutes, serum was collected, and the transaminase activity of the serum samples was measured on a blood biochemical analyzer according to the instructions.
2 results of the test
During the test period, tissue samples are collected at the stage continuous feeding time nodes respectively to prepare slices. The histomorphic analysis of the slices shows that when the slices are continuously fed for 7, 14 and 21 days, the 1-time mixed feeding of 15mg/kg of fish body weight does not have any obvious pathological damage to the liver, spleen, kidney and intestine of the grass carp. When the feed is continuously fed for 28 days, the florfenicol is singly used for detecting single focal lesions in the liver and intestinal tract of the grass carp, wherein the single lesion in the liver tissue is manifested by moderate edema and broadening of local tissue, no inflammation and no bleeding; while the single focus of intestinal tissue shows that a small amount of epithelial cells at the tail end of local intestinal villi are exfoliated, and the focus has no inflammation and bleeding. However, the liver tissue and the intestinal tissue of the grass carp compounded with the composite synergist on the basis of the florfenicol detect pathological morphological characteristics similar to those of the florfenicol single-use group.
Therefore, the compound synergist and the florfenicol have no new additive pathological changes to the grass carps within the compound dosage range with the mass volume ratio of 1-3%.
On the other hand, from the results of tissue section analysis of grass carps in the test group with the composite synergist used alone, the grass carps are free from any obvious lesions on the liver, kidney, spleen and intestine of the grass carps after being continuously fed for 7, 14, 21 and 28 days.
3 small knot
The test results are combined to conclude that the composite synergist and the florfenicol are used singly or combined with the florfenicol within the compound dosage range with the mass-volume ratio of 1-3%, and the composite synergist is used for 21 days continuously, so that the composite synergist has good internal safety for important tissues and organs of the grass carp. The time length is also obviously longer than the medication course length of the antibiotic preventive and therapeutic application, and meets the safety requirement of production and use.
Test example 3
This example is intended to evaluate the preventive effect of florfenicol on bacterial pathogenic infections when combined with a complex synergist.
1. Materials and methods
1.1 test Fish and test Strain
Grass carp: purchased from a fine variety field of the Buddha mountain grass carp, and the weight specification is 100 plus or minus 5 g.
Test strains: aeromonas hydrophila
1.2 preparation of medicinal baits
The dosage of the florfenicol is 15mg per kilogram of fish body weight, and the dosages of the composite synergist are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight according to the mass-volume ratio. And setting the concentration as a supposed Nie entry reference value.
Taking the dosage of the florfenicol and the composite synergist as a reference value, and taking the daily average feeding rate as 2.5 percent, three kinds of medicine baits of II-I #, II-2#, and II-3# are prepared in sequence, so that the assumed intake concentration of the florfenicol and the composite synergist in the baits reaches the reference value.
Bait completely containing no florfenicol and composite synergist is used as blank feed, which is called blank feed for short.
1.3 test grouping and management
The grass carp is randomly divided into A, B, C, D four groups, 3 parallel groups are arranged under each group, 100 tails of the grass carp are used in each parallel group, A, B, C four groups are respectively fed with three kinds of medicine baits of II-1#, II-2#, and II-3#, wherein the group A is a florfenicol positive control group, and the group D is fed with a blank material which does not contain any medicine and is set as a control group.
1.4 determination of survival to challenge
Three kinds of medicine baits II-1#, II-2#, and II-3# are continuously fed on the 1 st to 3 rd days of the test, while the control group is fed with common blank feedstuff. In the morning of 4 th day of experiment, before feeding, the test grass carp is artificially infected with aeromonas hydrophila by intraperitoneal injection, the infection dose is 0.2ml, and the concentration is 106cfu/ml, the feeding is resumed at noon, and the feeding is continued for 7 days at the feeding rate based on the satiation.
After the artificial infection operation, the observation is continued for 7 days, and the survival condition of the grass carp in each group is recorded. And the survival rate was calculated according to the following formula.
2 results of the test
According to the analysis of test results, the survival rate of the control group is only 22% during the observation period after the toxicity attack, and is obviously lower than that of the florfenicol single-use group and the florfenicol composite synergist combined group. Compared with the 63% survival rate of the florfenicol single-use group, the survival rates of the two dose groups of the florfenicol and the composite synergist are respectively and obviously higher by 14.3% and 19.0%.
3 small knot
The test result shows that the composite synergist has obvious defense synergistic effect on the fluorobenicol in resisting the infection of the aeromonas hydrophila within the test dosage range.
Test example 4
This example is intended to evaluate the preventive effect of florfenicol on bacterial pathogenic infections when used in combination with a composite synergist with the components replaced individually.
1. Materials and methods
1.1 test Fish and test Strain
Grass carp: purchased from a fine variety field of the Buddha mountain grass carp, and the weight specification is 100 plus or minus 5 g.
Test strains: aeromonas hydrophila
1.2 Replacing requirement and operation of individual Components of the composite synergist
Weighing 82 parts of purple sweet potato leaf extract purified product, 12 parts of phellodendron fermentation liquor purified product, 5 parts of eucommia fermentation liquor purified product, 0.5 part of mildew preventive Kemeiba and 0.5 part of antioxidant Fudaquine according to parts by weight, and uniformly mixing the components according to a predetermined proportion according to the requirement of aseptic operation to obtain the florfenicol composite synergist. The purple sweet potato leaf extract purified product, the phellodendron fermentation liquor purified product and the eucommia ulmoides fermentation liquor purified product are prepared according to the method in the embodiment 1.
The purple sweet potato leaf extract is weighed to replace the purple sweet potato leaf extract purified product in equal parts, and the purple sweet potato leaf extract purified product, the cortex phellodendri fermentation liquor purified product, the eucommia ulmoides fermentation liquor purified product, the mildew preventive Kemeiba and the antioxidant Fudaquine form an independent replacement group according to the requirements of the dosage parts, and the replacement is called as purple sweet potato leaf extract purified product replacement for short. The preparation method of the purple sweet potato leaf water extract comprises the following steps: purple sweet potato leaves with the water content of less than 12% are mixed according to the mass-volume ratio of 1: 3-5, adding distilled water, soaking for 30-45 min, decocting for 30-45 min with water for the first time, pouring out the water extract, and mixing the water extract with the water extract according to the mass-to-volume ratio of 1: 3-5, adding distilled water, decocting for 30-45 min, filtering, and concentrating the 2-time water extracts to obtain the purple sweet potato leaf water extract, wherein the quality control parameter is that 1ml of purple sweet potato leaf water extract is equivalent to 1g of purple sweet potato leaf.
Weighing equal parts of cortex phellodendri aqueous extract to replace the cortex phellodendri fermentation liquor purified product, and forming a replacement group with the purple sweet potato leaf extracted purified product, the cortex eucommiae fermentation liquor purified product, the mildewproof agent clomex and the antioxidant fordoquine according to the requirements of the parts by weight, wherein the replacement group is called as cortex phellodendri extracted purified product replacement for short. The preparation method of the phellodendron aqueous extract comprises the following steps: mixing cortex phellodendri according to a mass-volume ratio of 1: 3-5, adding distilled water, soaking for 30-45 min, decocting for 30-45 min with water for the first time, pouring out the water extract, and mixing the water extract with the water extract according to the mass-to-volume ratio of 1: 3 to 5, adding distilled water, decocting for 30 to 45min, filtering, completely concentrating the 2 water extracts, and preparing the phellodendron aqueous extract, wherein the quality control parameter is that 1ml of phellodendron aqueous extract is equivalent to 1g of phellodendron.
Weighing an equal amount of eucommia ulmoides aqueous extract to replace the eucommia ulmoides fermentation liquid purified product, and forming a replacement group with the purple sweet potato leaf extracted purified product, the phellodendron fermentation liquid purified product, the mildewproof agent kemuba and the antioxidant Fudaquine according to the requirement of the amount parts, wherein the replacement group is called phellodendron extracted purified product replacement for short. The preparation method of the eucommia ulmoides aqueous extract comprises the following steps: eucommia bark with the water content of less than 12 percent is mixed according to the mass volume ratio of 1: 3-5, adding distilled water, soaking for 30-45 min, decocting for 30-45 min with water for the first time, pouring out the water extract, and mixing the water extract with the water extract according to the mass-to-volume ratio of 1: 3-5, adding distilled water, decocting for 30-45 min, filtering, and concentrating the water extracts of 2 times to prepare the folium cortex eucommiae water extract, wherein the quality control parameter is that 1ml of folium cortex eucommiae water extract is equivalent to 1g of folium cortex eucommiae weight.
1.3 preparation of medicinal baits
The dosage of the florfenicol is 15mg per kilogram of fish body weight, and the dosages of the composite synergist are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight according to the mass-volume ratio. The dosages of the compound synergist replaced by the purified purple sweet potato leaf extract are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight. The dosages of the composite synergist replaced by the cortex Phellodendri extract purified product are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight. The dosages of the compound synergist replaced by the eucommia ulmoides extract purified product are respectively set to be 0, 0.15 and 0.30ml per kilogram of fish body weight.
Taking the dosage of the florfenicol and the composite synergist as a reference value, and taking the daily average feeding rate as 2.5 percent, three kinds of medicine baits of III-I #, III-2#, and III-3# are sequentially prepared, so that the assumed intake concentration of the florfenicol and the composite synergist in the baits reaches the reference value. And preparing IV-I #, IV-2#, IV-3# of the composite synergist group replaced by the purple sweet potato leaf extract and purification product, V-I #, V-2#, V-3# of the composite synergist group replaced by the cortex phellodendri extract and purification product, and VI-I #, VI-2#, and VI-3# of the composite synergist group replaced by the cortex eucommiae extract and purification product in sequence by the same method.
Bait completely containing no florfenicol and composite synergist is used as blank feed, which is called blank feed for short.
1.3 test grouping and management
Dividing grass carps into thirteen groups at random, setting 3 parallels under each group, feeding test materials corresponding to serial numbers of III-1#, III-2#, III-3#, IV-I #, IV-2#, IV-3#, V-I #, V-2#, V-3#, VI-I #, VI-2# and VI-3# by using 100 tails of the grass carps for each parallel, and the test materials are as follows:
1.4 determination of survival to challenge
On the 1 st to 3 rd days of the test, test materials corresponding to the numbers III-1#, III-2#, III-3#, IV-I #, IV-2#, IV-3#, V-I #, V-2#, V-3#, VI-I #, VI-2#, and VI-3# are continuously fed, and a control group is fed with common blank feed. In the morning of 4 th day of experiment, before feeding, the test grass carp is artificially infected with aeromonas hydrophila by intraperitoneal injection, the infection dose is 0.2ml, and the concentration is 106cfu/ml, the feeding is resumed at noon, and the feeding is continued for 7 days at the feeding rate based on the satiation.
After the artificial infection operation, the observation is continued for 7 days, and the survival condition of the grass carp in each group is recorded. And the survival rate was calculated according to the following formula.
2 results of the test
According to the analysis of test results, the survival rate of the control group is only 22% during the observation period after the toxicity attack, and is obviously lower than that of the florfenicol single-use group and the florfenicol composite synergist combined group. Compared with the 63% survival rate of the florfenicol single-use group, the survival rates of the two dose groups of the florfenicol and the composite synergist are respectively and obviously higher by 14.3% and 19.0%.
3 small knot
According to the test results, when the fermented and purified products of the three natural plants including the purple sweet potato leaves, the eucommia ulmoides and the golden cypress in the composite synergist are respectively replaced by the water extracts of the three natural plants, the defense synergistic effect on the fluorobenicol in the aspect of resisting the infection of the aeromonas hydrophila is obviously lower than that of the composite synergist prepared by the embodiment of the invention in the same added test dose gradient range. In other words, the fermented purified products of the purple sweet potato leaves, the eucommia ulmoides and the golden cypress have the synergistic effect on the florfenicol against the aeromonas hydrophila, but if the purified products are replaced by the water extracts of the purple sweet potato leaves, the eucommia ulmoides and the golden cypress, the combined synergistic effect of the composite synergist prepared by the embodiment of the invention on the florfenicol against the aeromonas hydrophila cannot be achieved even if the additive amount is equal.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.
Claims (4)
1. The florfenicol composite synergist is characterized by comprising, by weight, 70-88 parts of purple sweet potato leaf extract purified matter and phellodendron bark fermentation liquor purified matter5-12 parts of eucommia fermentation broth purified product, 3-8 parts of mildew preventive, 0.3-0.5 part of mildew preventive and 0.3-0.5 part of antioxidant; the purple sweet potato leaf extraction and purification product is prepared by the following steps: weighing 100 parts of purple sweet potato leaves with the water content less than or equal to 20% according to parts by weight to prepare purple sweet potato leaf micro powder, wherein the volume ratio of the purple sweet potato leaves to the solvent is 1: (10-15) measuring petroleum ether which is 10-15 times of the volume of the purple sweet potato leaves, carrying out hot reflux extraction for 5-7 times, each time for 2-5 hours, combining extracting solutions, filtering, concentrating under reduced pressure, evaporating at constant temperature to obtain an extract, and carrying out low-temperature low-pressure extraction and steam treatment on the extract to form solid powder with the water content of 3-5% so as to obtain an extracted and purified purple sweet potato leaf; the cortex phellodendri fermentation liquor purified product is prepared by the following steps: weighing 100 parts by weight of phellodendron amurense with coarse bark removed, preparing the phellodendron amurense into micro powder, adding the micro powder into a container filled with water with the mass volume ratio of 40-50 times, adding a lactobacillus liquid nutrient medium with the volume ratio of 3-5% of the added water, carrying out closed decoction for 30-45 minutes, cooling to normal temperature, adding more than or equal to 10 viable bacteria91000-1200 ml of cfu/ml lactobacillus liquid, 2mol of MgCl210-12 ml, sealing, heating to 37 ℃, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging crushed residue liquid at 20000rpm for 10 minutes, removing residues, collecting supernatant, filling into a dialysis bag, dialyzing with running water for 36-48 hours, concentrating high molecular weight polyethylene glycol to one fifth of the original volume, collecting concentrated solution, performing low-temperature low-pressure steam extraction treatment on the concentrated solution for 36-48 hours to form solid powder with water content of 3-5%, and obtaining a purified substance of phellodendron fermentation liquor; the purified eucommia ulmoides fermentation liquor is prepared by the following steps: weighing 100 parts of eucommia bark, sieving the eucommia bark with a 80-mesh sieve to prepare micro powder, adding 100-130 times of water by mass volume ratio, then adding a lactobacillus liquid nutrient medium, carrying out closed decoction for 30-45 minutes at 100 ℃, cooling to normal temperature, and adding more than or equal to 10 viable bacteria9Adding 1000-1200 ml of cfu/ml lactobacillus liquid, fully mixing, sealing, culturing at constant temperature until OD is 3.0-3.6, performing ultrasonic crushing treatment on the culture, repeating the treatment for three times, centrifuging to remove residues, collecting supernatant, filling into a dialysis bag, dialyzing with running water for 36-48 hours, and concentrating high molecular weight polyethylene glycol until the original material is obtainedAnd one fifth of the product is collected, the concentrated solution is subjected to low-temperature low-pressure steam extraction treatment for 36-48 hours to form solid powder with the water content of 3-5%, and the purified product of the eucommia fermentation liquor is obtained.
2. The florfenicol composite synergist according to claim 1, which is characterized by comprising 85 parts by weight of purple sweet potato leaf extract and purification product, 8 parts by weight of phellodendron fermentation liquor and purification product, 6 parts by weight of eucommia fermentation liquor and purification product, 0.5 part by weight of mildew preventive and 0.5 part by weight of antioxidant.
3. A method for preparing the florfenicol composite synergist of claim 1 or 2, characterized in that a purple sweet potato leaf extract purified product, a phellodendron fermentation liquor purified product, an eucommia fermentation liquor purified product, a mildew preventive and an antioxidant are uniformly mixed to obtain the composite synergist.
4. The application of the florfenicol composite synergist in claim 1 or 2, which is characterized in that the application is to dilute the florfenicol composite synergist and then to prepare the florfenicol composite synergist in a volume-to-mass ratio (1-3): 100 is used in combination with florfenicol for the synergism of the florfenicol on G-escherichia coli.
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