CN111521694B - Application of ligustilide in eliminating quinolone drug residues in cultured fishes - Google Patents
Application of ligustilide in eliminating quinolone drug residues in cultured fishes Download PDFInfo
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- CN111521694B CN111521694B CN202010282900.5A CN202010282900A CN111521694B CN 111521694 B CN111521694 B CN 111521694B CN 202010282900 A CN202010282900 A CN 202010282900A CN 111521694 B CN111521694 B CN 111521694B
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- ligustilide
- sarafloxacin
- quinolone
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- channel catfish
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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Abstract
The invention discloses application of ligustilide in eliminating residue of quinolone drugs (especially sarafloxacin) in cultured fishes, wherein the ligustilide can reduce the concentration of the residual quinolone drugs in the cultured fishes, and the ligustilide can obviously shorten the elimination half-life period and withdrawal period of the quinolone drugs in the cultured fishes. The ligustilide can be used for accelerating the acceleration of the elimination of the residue of the quinolone chemical drugs in the cultured fishes, and has important significance for ensuring the quality safety of aquatic products and reducing the risk of human bodies ingesting aquatic products containing the residue of the quinolone chemical drugs.
Description
Technical Field
The invention belongs to the technical field of aquatic animal pharmacology, and particularly relates to application of ligustilide in accelerating elimination of residue of quinolone drugs (especially sarafloxacin) in cultured fishes.
Background
Quinolone drugs are synthetic antibacterial drugs, and are widely used in the prevention and treatment of bacterial diseases in aquaculture animals due to their advantages of strong antibacterial ability, broad antibacterial spectrum, low price, etc. With the rapid development of the aquaculture industry in China, the yield of aquatic products in China is the top of the world, but along with the frequent occurrence of various bacterial diseases of aquatic animals, carbostyril drugs are used in large quantities, so that the bacterial diseases of the aquatic animals are treated, the problem of residual carbostyril drugs in the aquatic products is also caused, the problem of quality safety of the aquatic products is caused, and the physical health of consumers is influenced. The market supervision and management bureau of Heilongjiang province in 2019 publishes the condition of 20-stage food safety supervision spot check, 2 batches of aquatic products are notified due to the fact that enrofloxacin exceeds the standard, the food safety spot check information is issued by the food and drug administration of Hubei province in 2018 in 10 and 25 days, and as a result, 2 batches of enrofloxacin exceeds the standard, and 3 batches of aquatic products are issued by the food and drug administration of Shanxi province in 2016. Zhang cong et al (2018) reported that flumequine was detected in 4 kinds of aquaculture products in the Altai lake with a detection rate of 16.38%. Ietalurus punctatus (Ietalurus punetaus) was native to North America, belongs to the order Silurfornes, Ictaluraceae, was introduced by Hubei in 1984 and began to breed extensively within the country. The channel catfish is a large freshwater fish, has the advantages of fast growth, wide adaptability, strong disease resistance, superior meat quality, low price and the like, and is deeply loved by consumers. By 2015, since the annual output of the cultured channel catfish is higher than that of the channel catfish in the United states, China has become the first major country for culturing the channel catfish. However, with the expansion of the culture scale, the increasing of the intensive culture degree, the occurrence of the conditions such as variety degeneration, drug abuse and the like, the demand for the use of drugs is increasing. The problem of drug residue becomes one of the key problems which hinder the development of the channel catfish industry in China. Aiming at the problems, the invention adopts the natural product ligustilide monomer to reduce the residual concentration of quinolone drugs (especially sarafloxacin) in the channel catfish body, shortens the residual time and shortens the drug withdrawal period, thereby having important significance in ensuring the quality safety of aquatic products such as channel catfish and the like.
Disclosure of Invention
In order to rapidly eliminate the residual quinolone drugs in the cultured fishes, the invention provides the application of ligustilide in accelerating the elimination of the residual quinolone drugs (especially sarafloxacin) in the cultured fishes.
In order to achieve the purpose, the invention adopts the following technical measures:
in the embodiment of the invention, after the ictalurus punctatus is subjected to oral administration of sarafloxacin (the oral administration dosage is 20mg/kg of fish body weight) for 12h, distilled water and ligustilide (20mg/kg of fish body weight) are respectively subjected to oral administration, and the residual concentration of the sarafloxacin in the blood plasma and the belt skin muscle of the ictalurus punctatus is detected at different time points. The result shows that compared with a control group (oral lavage of sarafloxacin and distilled water), the elimination half-life period of the sarafloxacin in the blood plasma of the channel catfish of the test group (oral lavage of sarafloxacin and ligustilide) is shortened by 29.71h compared with that of the control group, and the half-life period of the sarafloxacin in skin and meat is shortened by 40.73 h; the drug withdrawal period of the sarafloxacin hydrate in the test group is shortened by 6.83 days compared with that in the control group.
The application of ligustilide in eliminating quinolone drug residues in cultured fishes comprises the following steps: irrigating the ligustilide orally for cultured fishes, or feeding a feed containing the ligustilide;
further, the quinolone medicine is sarafloxacin;
further, the effective amount of ligustilide is 20mg/kg fish body weight;
further, the cultured fishes include, but are not limited to, catfishes such as channel catfish, yellow catfish and the like, cyprinid fishes such as grass carp, black carp, silver carp, bighead carp, megalobrama amblycephala and the like, and perciformes fishes such as tilapia, snakehead, mandarin fish, weever and the like.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the ligustilide can shorten the drug withdrawal period of a quinolone drug-sarafloxacin in skin and meat (edible tissues) of channel catfish by 6.83 days, and the residual quantity of the sarafloxacin in the channel catfish body at the same time point is also obviously reduced to 1/5-4/5 (blood plasma) and 1/5-1/2 (skin and meat) of the original concentration.
(2) The invention can shorten the residual time and reduce the residual quantity of quinolone drugs (especially the sarafloxacin) in the channel catfish, and has important significance for ensuring the quality safety of aquatic products and reducing the risk of human body to ingest aquatic products containing the residual quinolone drugs.
Drawings
Fig. 1 is a curve of elimination of residual sarafloxacin in blood plasma of channel catfish of control group and test group.
Fig. 2 is a curve for eliminating sarafloxacin residue in the skin-carrying muscle of the channel catfish with natural proportion in the control group and the test group.
Detailed Description
And (3) testing the fish: ictalurus punctatus with average weight (180 +/-32) g purchased from Baishazhou large market in Wuhan, transported to a laboratory, placed in a cleaned aquarium (122cm multiplied by 80cm) sterilized by potassium permanganate and temporarily kept for 1 week, and the test water is fully aerated tap water. After the temporary rearing is finished, selecting individuals without diseases and injuries and with uniform specifications for testing, and controlling the water temperature to be 24 +/-1 ℃.
Sarafloxacin (purity is more than or equal to 98.0% and purchased from Shanghai-sourced leaf Biotech, Inc.), and ligustilide (purity is more than or equal to 98.0% and purchased from Shanghai-sourced leaf Biotech, Inc.).
The test is divided into two groups, one group is a control group of the oral cavity irrigation channel catfish sarafloxacin (the oral cavity irrigation dosage is 20mg/kg fish body weight), and the oral cavity irrigation of the control group is performed with the oral cavity irrigation of the sarafloxacin for 12h and then with the oral cavity irrigation of distilled water; and injecting the artemisinine (20mg/kg of fish weight) into the other group of channel catfish at the mouth for 12h, and then injecting the ligustilide (20mg/kg of fish weight) into the channel catfish at the mouth. The blood plasma and natural proportional belt skin muscle of the channel catfish are collected at 2h, 4h, 8h, 12h, 1d, 2d, 3d, 5d and 7d after the channel catfish is respectively irrigated with ligustilide and distilled water in the test group and the control group.
Pretreatment of a muscle sample:
after the frozen muscle tissue sample is naturally thawed at room temperature, 2.0g of muscle tissue is weighed into a 15mL centrifuge tube. 6mL of an extracting agent (acetonitrile containing 0.4% hydrochloric acid, volume fraction) is added into the sample, the mixture is placed on a mixer to be mixed for 30s by vortex oscillation, 2.0g of anhydrous sodium sulfate is added, and the mixture is placed on the mixer to be mixed for 30s by vortex oscillation. Centrifuge at 7000r/min for 5min and transfer the supernatant to another 15mL centrifuge tube. 6mL of an extractant (acetonitrile containing 0.4% hydrochloric acid, volume fraction) was added to the centrifuge tube containing the precipitate, and the mixture was vortexed and kneaded on a kneader for 30 seconds, followed by centrifugation at 7000r/min for 5 min. Mixing the supernatants into a 15mL centrifuge tube, placing the centrifuge tube on a 50 ℃ nitrogen blowing instrument for drying, adding 2mL of n-hexane solution, adding 2mL of constant volume solution (0.1% formic acid aqueous solution: acetonitrile 80:20, V: V), carrying out vortex oscillation and uniform mixing for 30s, centrifuging at 7000r/min for 5min, discarding the n-hexane layer on the upper layer, taking the liquid on the lower layer, filtering through a 0.22 mu m filter head, and carrying out UPLC analysis.
Treatment of plasma samples:
the cryopreserved plasma was thawed naturally at room temperature. After shaking up, 1.0mL of plasma was taken up in a 10mL centrifuge tube with a plug, 3mL of an extractant (acetonitrile containing 0.4% hydrochloric acid, volume fraction) was added, and the mixture was placed on a speed-adjustable mixer and vortexed and shaken for 30 s. After adding 1.0g of anhydrous sodium sulfate, the mixture is vortexed and mixed for 30 seconds. Centrifuge at 7000r/min for 5min and transfer the supernatant to another 10mL centrifuge tube. To the centrifuge tube with the precipitate was added 3mL of an extractant (acetonitrile containing 0.4% hydrochloric acid, volume fraction) and mixed by vortexing with a mixer for 30s and centrifuged with a centrifuge at 7000r/min for 5 min. The supernatant was combined into a 10mL centrifuge tube and dried on a 50 ℃ nitrogen blower. Adding 1mL of n-hexane solution, placing on a mixer, carrying out vortex oscillation for 30s, adding 1mL of constant volume solution (0.1% formic acid water solution: acetonitrile 80:20, V: V), and carrying out vortex oscillation for 30 s. Centrifuging at 12000r/min for 5min, discarding the upper n-hexane layer, collecting the lower layer, filtering with 0.22 μm filter head, and performing UPLC analysis.
Liquid chromatography conditions: an ultra-high performance liquid chromatography band fluorescence detector (Waters UPLC-FLR), wherein the phase A is 0.2% formic acid, the phase B is acetonitrile, isocratic elution is carried out, the volume ratio of the phase A to the phase B is 4:1, the flow rate is 0.3mL/min, the excitation wavelength is 295.7nm, the emission wavelength is 455.1nm, the column temperature is 30 ℃, and the sample injection amount is 5 mu L.
Verification of the analysis methodology:
and (3) standard recovery rate and precision: taking blank plasma and a natural proportional band skin muscle tissue, adding a proper amount of sarafloxacin standard solution to prepare plasma samples containing sarafloxacin concentrations of 10, 50, 100, 500, 1000 and 2000ng/mL and natural proportional band skin muscle tissue samples of 10, 50, 100, 500, 1000 and 2000 mug/kg, setting 3 concentrations in each concentration in a day in parallel, and repeating the operation for 5 times in a week. And carrying out UPLC detection. And calculating the recovery rate according to the ratio of the peak area of the medicament in the blood plasma and natural ratio dermal muscle tissue samples to the peak area of the standard solution with the same concentration, and calculating the precision within and between days.
Data processing: the sarafloxacin is eliminated from the channel catfish body according to the first-order kinetic process, namely obeying index elimination at the later stage of elimination: c ═ C0e-ktC represents the drug concentration, C0Is the intercept of the log curve of residual elimination (. mu.g/kg), k represents the elimination rate constant, the elimination half-life T1/20.693/k. The MRL of the sarafloxacin in the fish skin and the fish flesh is 30 mu g/kg according to the stipulation of China, and the withdrawal period is calculated by adopting WT1.4 software. The drug time curve graph is calculated and plotted using Microsoft Excel 2013.
Analysis of results
The method comprises the following steps of:
the recovery rate of the sarafloxacin labeling in the blood plasma of the channel catfish is 90.90 +/-5.35-97.63 +/-5.17%, the intra-day precision is 2.18-5.02%, and the inter-day precision is 3.18-6.86%; the recovery rate of sarafloxacin galanthamine in the skin muscle of channel catfish is 88.03 +/-6.63%, the precision in the day is 2.62-5.64%, and the precision in the day is 4.02-7.23% (see table 1). The LOD of the plasma detection limit of the channel catfish is 2.5 mug/mL, the LOQ of the quantitative limit is 5 mug/mL, the LOD of the sarafloxacin in the natural proportional belt muscle is 1 mug/kg, and the LOQ of the quantitative limit is 2.5 mug/kg.
TABLE 1 California punctata plasma and natural ratio sarafloxacin recovery and precision in the muscles with skin
The influence of ligustilide on the elimination of sarafloxacin in channel catfish bodies is as follows:
the concentration of the sarafloxacin in the blood plasma of the channel catfish of the test group (oral cavity sarafloxacin + ligustilide) is 1/5-4/5 of the concentration of the sarafloxacin at the same time point of the control group (oral cavity sarafloxacin + distilled water), and the concentration of the sarafloxacin in the skin and meat of the channel catfish of the test group is 1/5-1/2 of the control group. The elimination half-life periods of the sarafloxacin in the blood plasma of the channel catfish of the control group and the test group are 103.43h and 73.72h respectively, and the elimination half-life period of the sarafloxacin in the blood plasma of the channel catfish of the test group is shortened by 29.71h compared with that of the control group; the elimination half-life periods of the sarafloxacin in the skin and meat of the channel catfish of the control group and the test group are 119.48h and 78.75h respectively, and the elimination half-life period of the sarafloxacin of the test group is shortened by 40.73h compared with that of the sarafloxacin of the control group. The drug withdrawal period of the channel catfish skin and the sarafloxacin in the meat of the control group calculated by WT1.4 software is 20.66 days, the drug withdrawal period of the channel catfish skin and the sarafloxacin in the meat of the test group is 13.83 days, and the drug withdrawal period of the sarafloxacin of the test group is shortened by 6.83 days compared with that of the control group. Therefore, the ligustilide can promote the elimination of the residual sarafloxacin in the channel catfish body. In the embodiment, channel catfish is taken as a representative to verify the removing effect of ligustilide on quinolone drugs in cultured fishes, wherein the cultured fishes include, but are not limited to, catfishes of the order silurus, such as channel catfish, pseudobagrus fulvidraco and the like, carps of the family cyprinidae, such as grass carp, black carp, silver carp, bighead carp, megalobrama amblycephala and the like, and perciformes fishes, such as tilapia, snakehead, mandarin fish, weever and the like.
Claims (2)
1. Application of ligustilide in eliminating residue of quinolone drugs sarafloxacin in cultured fishes.
2. The use of claim 1, wherein the effective amount of ligustilide is 20mg/kg body weight of fish.
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