CN101371831B - Use of Fructus arctii aglycone in medicament for killing ectoparasite of aquatic animal - Google Patents

Abstract

The invention discloses a new usage of arctigenin in insecticide for killing ectoparasites in the exterior body of an aquatic animal, in particular relates to a dactylogyrus. The invention also discloses a control agent containing the compound for killing ectoparasites in the exterior body of an aquatic animal and a preparation method thereof. As a novel green pollution-free fishery drug, the arctigenin has low toxicity to fish, is not liable to cause drug resistance, and can kill dactylogyrus thoroughly at one time, with noticeable result, low manufacturing cost, simple usage and convenient popularization.

Description

The application of arctigenin in killing aquatic animal vermin medicine

Technical field

The present invention relates to arctigenin (C ₂₁H ₂₄O ₆) purposes, relate in particular to the purposes in killing aquatic animal vermin medicine.

Technical background

Arctigenin is the seed from medicinal plants Fructus Arctii (Arctium lappa L. is a Compositae Compositae Arctium Arctium plant)---a kind of chemical compound of extraction separation the Fructus Arctii, and its structural formula is:

The molecular formula of this chemical compound is C ₂₁H ₂₄O ₆, molecular weight is 534, colourless bulk crystals, and easily molten in organic solvents such as chloroform, methanol, ethanol, indissoluble in the petroleum ether, fusing point are 111 ℃～112 ℃.This chemical compound is known can be as the active component of multiple medicines such as antibiotic, antitumor, blood sugar lowering, treatment acute and chronic nephritis, blood pressure lowering and anti HIV-1 virus.

Summary of the invention

The object of the present invention is to provide the new purposes of arctigenin, promptly kill application in the aquatic animal vermin medicine in preparation.

Say that further main purpose of the present invention is to provide arctigenin to kill application in aquatic animal vermin-dactylogyrider medicine in preparation.

A further object of the invention has provided a kind of verminal control agent of aquatic animal of killing, it is made up of following raw materials by weight percent: arctigenin 1%～20%, surfactant 5%～10%, transdermal agent 1%～4%, all the other are solvent, and the summation of above-mentioned raw materials is 100%;

Described surfactant is a tween 20;

Described transdermal agent is water solublity azone (XW-02), and name is called 1-positive dodecyl aza cyclohepta-2-ketone, molecular formula C ₁₈H ₃₅NO;

Described solvent is a dehydrated alcohol.

The preferred solvent of the present invention is a dehydrated alcohol, can also select other solvents well known to those skilled in the art for use, for example methanol.

Last purpose of the present invention provides the above-mentioned preparation method of killing external parasite preventing and treating agent for aquatic product animals, carries out according to the following steps:

1) take by weighing arctigenin, surfactant, transdermal agent, each raw material of solvent, standby;

2) arctigenin and solvent are stirred to dissolving fully;

3) surfactant, transdermal agent are added step 2) stir in the mixed solution of gained, promptly get and kill external parasite preventing and treating agent for aquatic product animals.

The using method of killing external parasite preventing and treating agent for aquatic product animals of the present invention is simple, directly presses 12mL～20mL/m ³Dosage splash in the pond can effectively preventing or kill Fish vermin, particularly dactylogyrider.

The present invention compared with prior art has the following advantages:

1) the present invention has excavated new purposes to the known compound arctigenin, has opened up new application.

2) arctigenin is the novel fisheries drug of a kind of nuisanceless green, low to toxicity in fish, the difficult drug resistance that produces, and the disposable dactylogyrider of thoroughly killing of energy, its effect is remarkable.

3) using method is simple.

The specific embodiment

Essence for a better understanding of the present invention below will be with arctigenin to fish acute toxicity experiment, pharmacological testing, and the pharmacological testing and the result of control agent illustrate its new purposes in killing the external worm medicine of Fish aquatic animal.

Test example 1 arctigenin is to the Brachydanio rerio acute toxicity test

1 materials and methods

1.1 material

1.1.1 for test agent

Testing used arctigenin purity is 99.50%, is provided by Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's animal technical college aquatic science prepared in laboratory.Accurately take by weighing 1.0g arctigenin monomer, with fully dissolving of dehydrated alcohol (available from Xi'an chemical reagent factory), standardize solution is in the 100mL volumetric flask, and being made into concentration is 10mgmL ^-1, 4 ℃ of refrigerators are preserved standby.

1.1.2 experimental animal

Test used Brachydanio rerio and purchase in Shaanxi Province's Aquatic product institute and view and admire the fish farm, require the body constitution stalwartness, specification homogeneous, average weight 0.47g.Buy the back back and support about 7d temporarily in the laboratory aquarium, throw something and feed 1-2 every day, and the feedstuff of input (laboratory self-control feedstuff) is eaten up in 10min and exceeded.Carry out toxicity test again after waiting to adapt to laboratory environment condition.

1.1.3 test apparatus

One of ALC-1100.2 percentage electronic balance, Beijing Sai Duolisi instrument system company limited; KQ-500E type ultrasonic cleaner; 5～10 μ L, 20～200 μ L pipettor: VoluMate.Made inGermany; Temperature automatically controlled heating rod, Zhoushan Zhejiang fishing gear factory produces; Plastic tub (volume is 8L) etc.

1.2 test method

1.2.1 experimental condition

Adopt hydrostatic formula test method(s), every basin adds the tap water 5L of fully sudden and violent gas, pH7.2, and control water temperature is 25 ± 1 ℃, adds test earlier behind the abundant mixing of adding test medicinal liquid again and uses fish, every basin is put 10 tails in a suitable place to breed.After putting fish in a suitable place to breed, require to keep dissolved oxygen in water at 5mg.L ^-1More than.In order to keep constant drug level, change water dosing again every 24h.

1.2.2 the setting of trial drug concentration

At first, carry out trial test repeatedly, determine the approximate range of formal test liquor strength, promptly test the liquor strength (12.0mgL that fish all is poisoned to death in 24h ^-1) and 96h in dead liquor strength (5.5mgL does not take place ^-1).Then on the trial test basis, at liquor strength 5.5～12.0mgL ^-1Press concentration logarithm equal difference (tolerance d=0.0376) insertion 5.9984,6.5418,7.1335,7.7796,8.4840,9.2512,10.0897,11.0027mgL in the scope ^-1Eight concentration test group are carried out formal test, observe the situation of being poisoned to death of test fish under each concentration.

In the death condition of duration of test observed and recorded test at any time fish, as finding ichthyism death, should pull out immediately, in order to avoid influence water quality, influence result of the test.The method of judging fish death is that (gill cover moves and stops) touching the caudal peduncle portion of fish with Glass rod or tweezers when fish ceases breathing after, if the fish body does not produce any stress within 3min, can judge death.

The duration of test statistics is tested the death condition of fish under each drug level in 24h, 48h, 72h and 96h.Duration of test should be kept quite, and avoids any interference to the test fish as far as possible.When the mortality rate of matched group less than 5%, the mortality rate of test fish can be proofreaied and correct in each time period; If the mortality rate of matched group then should be proofreaied and correct greater than 5%, updating formula adopts the Abbott formula; If the matched group mortality rate reaches more than 20%, then after searching reason, carry out toxicity test again.

Abbott formula: p=p '-C/1-C

P: corrected mortality, promptly sheerly drug-induced mortality rate

P ': test group mortality rate, i.e. natural cause and drug-induced mortality rate

C: matched group mortality rate, the i.e. mortality rate that causes of natural cause

1.2.3 arctigenin minimum lethal concentration scope, median lethal concentration (LC ₅₀)

Observed and recorded is for the death condition of examination fish in 24h, 48h, 72h and 96h, and test each time period of fish begins to occur dead least concentration and next concentration is the minimum lethal concentration scope of this time period, and calculates fatality rate.Utilize return law of the straight line, with linear regression equation y=a+bx (the concentration logarithm of x-drug level; The probability unit that the y-mortality rate converts to; A, b are respectively collinear intercept and slope.), obtain a and b, the computing formula of a and b is:

$a=\frac{\mathrm{\Σ}{x}^2\mathrm{\Σy}-\mathrm{\Σx\Σ}\left(\mathrm{xy}\right)}{\mathrm{k\Σ}{x}^2-{\left(\mathrm{\Σx}\right)}^2}$

$b=\frac{\mathrm{k\Σ}\left(\mathrm{xy}\right)-\mathrm{\Σx\Σy}}{\mathrm{k\Σ}{x}^2-{\left(\mathrm{\Σx}\right)}^2}$

K: the group number of drug level (mortality rate is that 0 and 100% concentration does not count)

After obtaining a, b, just can determine regression equation, and then bring the probability unit y=5.0 of 50% mortality rate into equation, obtain x, get its antilogarithm, just draw the LC in this time ₅₀, and calculate its fiducial limit of 95%, formula is:

In the formula: $s=\frac{1}{b}$

N: for the animal sum (mortality rate is that 0 and 100% concentration does not count) of examination

X:LC ₅₀The logarithm of value

1.2.4 the calculating of arctigenin safe concentration

According to Turbell formula computationally secure concentration:

2 results and analysis

2.1 arctigenin median lethal concentration (LC ₅₀)

2.1.1 each experimental concentration of arctigenin in different time to Brachydanio rerio intoxicating situation

The Brachydanio rerio death condition of each trial drug concentration in 24h, 48h, 96h sees Table 6.At drug level is 15.0mgL ^-1The high concentration test group, test ichthyism reaction is very obvious, occurs unusual performance after the medication about 2h, Brachydanio rerio emerges head, is slow in action then, the side trip occurs, begins to occur paralysis in the near future, disequilibrium, abdominal part up and dead.In concentration is 0.5mgL ^-1The low concentration test group in, Brachydanio rerio performance is quiet, is quick on the draw to external world, and is movable normal.

Table 6 arctigenin is to the result that is poisoned to death of Brachydanio rerio

2.1.2 minimum lethal concentration scope, median lethal concentration (LC ₅₀)

The death condition of statistics variable concentrations test medicinal liquid in 24h, 48h and 96h the results are shown in Table 7.

Each experimental concentration is got its logarithm, get its concentration logarithm, and with the percentage rate of each test ichthyism death time period table look-up its probit, probit substitution formula with the drug level logarithm of each time period and corresponding mortality rate thereof, obtain regression equation, can calculate the median lethal concentration (LC of each time period ₅₀) and its LC ₅₀95% fiducial limit.

Table 7 arctigenin is to the acute toxicity result of Brachydanio rerio

The LC of each time period of table 8 ₅₀And 95% fiducial limit

As shown in Table 8, the regression equation of gained is in the 12h: y=-15.68549+16.21641x, LC ₅₀=9.27385mgL ^-1, 95% the credible 8.78319～9.81108mgL that is limited to ^-1The regression equation of gained is in the 24h: y=-14.01489+14.78145x, LC ₅₀=8.87442mgL ^-1, 95% the credible 8.38627～9.40652mgL that is limited to ^-1The regression equation of gained is in the 48h: y=-16.53071+17.86879x, LC ₅₀=8.41621mgL ^-1, 95% the credible 7.99022～8.86380mgL that is limited to ^-1The regression equation of gained is y=-21.01241+23.18235x in the 96h, LC ₅₀=8.06114mgL ^-1, 95% the credible 7.69333～8.44165mgL that is limited to ^-1

2.1.3 safe concentration result of calculation

Calculate the safe concentration of arctigenin according to the Turbell formula to Brachydanio rerio.

$=2.2709\mathrm{mg}\·L^{-1}$

Show that from the result arctigenin is not very high to the safe concentration of Brachydanio rerio, still, in the reality, concentration is at 6.5418mgL ^-1Situation under, the generation of fish death does not appear in 48h, is 2.50mgL in conjunction with the effective dose of killing dactylogyrider ^-1Take all factors into consideration, arctigenin still has excellent popularization as pesticide for fish and is worth.

Test example 2 arctigenins are to the dactylogyrider killing effect

1 materials and methods

1.1 experimental animal

Host's model is Carassius auratus (Carassius auratus), and body weight derives from Xianyang, Shanxi province city Changxing Carassius auratus plant all less than 5.0g; The parasite model is medium-sized dactylogyrider (D.intermedius), is Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's aquatic science laboratory live population.

1.2 trial drug

Testing used arctigenin purity is 90.0%, is that Fructus Arctii separates acquisition through extraction, multistage silica gel column chromatography.Provide by Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's animal technical college aquatic science prepared in laboratory.

1.3 test test method

Use the enamel basin of volume, contain abundant aeration subsoil water 5L, use 25 ± 1 ℃ of temperature automatically controlled heating rod control water temperature, pH7.0～7.5 as 8L.During on-test, at first the Carassius auratus material that has infected medium-sized dactylogyrider is inspected by random samples 10 tails immediately, when parasite mean parasitized quantity and infection rate reach test requirements document, in each basin, throw in respectively and infect Carassius auratus 10 tails.Add each flow point in aforementioned each separating step respectively in water body according to the concentration set up of test then, stir.The film-making of depletion fish holobranch, microscopy during 48h are observed various samples killing effect to dactylogyrider under variable concentrations, the fish mortality rate when statistics optimum effective concentration, the highest killing rate, ichthyism concentration and poisoning concentration.Test repeats 3 times, and establishes matched group.

Get for the film-making of examination fish holobranch, microscopy observation and statistics dactylogyrider from gill portion dropping situations in the setting parasite killing time, if do not come off, whether deadly observe, mouthpart does not shrink the person at 1～2min around the polypide cephalic gland, be shown death, if gill portion do not have worm or worm death determine that all killing rate is 100%; Come off as fruit part, add up every tail fish gill portion survival parasite quantity, calculating mean value, and compare with matched group, calculate killing rate, estimate insecticidal effect with this.

2.1 kill the dactylogyrider effect

2.1.5 arctigenin insecticidal activity assay

Purity is that 90.0% arctigenin is measured it and killed the dactylogyrider activity and see Table 5.

Table 5 arctigenin 48h is to the activity of killing of medium-sized dactylogyrider

As shown in Table 5, when the concentration of arctigenin from 2.5mgL ^-1Beginning reaches 100% to the killing rate of dactylogyrider; Reach 8.0mgL and work as concentration ^-1The time, beginning fish is shown certain toxicity, the mortality rate of this macrura reevesii is 20%.

Real 3 the present invention of test kill the pharmacodynamics test of external parasite preventing and treating agent for aquatic product animals

1 materials and methods

1.1 material

1.1.1 experimental animal

Host's model is Carassius auratus (Carassius auratus), and the parasite model is medium-sized dactylogyrider, is Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's aquatic science laboratory live population; Infect the Carassius auratus body weight all less than 5g, derive from Aquatic product institute culture of ornamental fish field, Shaanxi Province.

1.1.2 for the reagent product

Contain 15.0% arctigenin external parasite preventing and treating agent for aquatic product animals, provide by Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's animal technical college aquatic science prepared in laboratory.

1.1.3 instrument and equipment

XH-B vortex mixer: the healthy medical apparatus company limited in Jiangyan City; BX41 optical microscope: OLYMPUS.Made in Japan; Satorius ten thousand/electronic balance; Aquarium and temperature regulating device.

1.2 test method

The insecticidal test method is specially as previously mentioned: uses volume to be 50cm * 40cm * 30cm8 glass aquarium, contains abundant aeration subsoil water, and pH7.0～7.5,25 ± 1 ℃ of control water temperature are thrown in respectively and are infected Carassius auratus 100 tails that medium-sized dactylogyrider is arranged.Set each extract concentrations at first on a large scale and carry out trial test, concentration according to parasite killing and Carassius auratus poisoning resets according to the gradient increasing or decreasing again, add each concentration medicine respectively and stir, the film-making of depletion fish holobranch, microscopy during 48h, observe various samples killing effect to dactylogyrider under variable concentrations, statistics optimum effective concentration, the highest killing rate.Test repeats 3 times, and establishes matched group.

2 results

2.1 matched group

The matched group fish that test is set up in carrying out is generally 100 tails, and 0h, the 48h in test inspects 30 tails immediately by random samples respectively, dissects holobranch microscopy statistics and infects medium-sized dactylogyrider par.At the 0h microscopy of test, medium-sized dactylogyrider par is 20.2 a/tail, and 48h is carried out in test, and medium-sized dactylogyrider par is 23.6 a/tail.Result with this and drug monitoring calculates parasitic killing rate.

2.2 the external parasite preventing and treating agent for aquatic product animals of killing of the present invention is killed the result to dactylogyrider

15.0% arctigenin is killed the Fish external parasite preventing and treating agent, the results are shown in Table 9 what 48h killed medium-sized dactylogyrider.As can be seen from Table 9, when concentration be 1.0mL/m ³The time, insecticidal power a little less than, only be 9.7%; When concentration is 12.0mL/m ³The time, its killing rate can reach 91.9%, and concentration is 15.0mL/m ³The time killing rate be 100%; When concentration is 21.0mL/m ³The time, intoxicating phenomenon does not still appear in the test fish, shows that said preparation is the good Biocidal fisheries drug of a kind of application prospect.

Table 9 15.0% arctigenin at 48h to medium-sized dactylogyrider killing effect

Essence for a better understanding of the present invention.The specific embodiment that provides below in conjunction with the inventor describes arctigenin in detail and kills the external parasite preventing and treating agent for aquatic product animals preparation method, and these only are the present invention embodiment preferably, but are not limited to these embodiment.

Embodiment 1

Take by weighing after arctigenin 1.0g and solvent 86.0g mix to fully dissolving, add tween 20 10.0g and transdermal agent water solublity azone 3.0g, fully mix homogeneously promptly gets arctigenin and kills external parasite preventing and treating agent for aquatic product animals.

Embodiment 2

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 4.0g, surfactant 8.0g, transdermal agent 2.0g, solvent 86.0g.

Embodiment 3:

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 8.0g, surfactant 10.0g, transdermal agent 2.0g, solvent 80.0g.

Embodiment 4:

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 12.0g, surfactant 7.0g, transdermal agent 2.0g, solvent 79.0g.

Embodiment 5:

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 16.0g, surfactant 5.0g, transdermal agent 1.0g, solvent 78.0g.

Embodiment 6:

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 20.0g, surfactant 5.0g, transdermal agent 4.0g, solvent 71.0g.

Embodiment 7:

Kill external parasite preventing and treating agent for aquatic product animals, mass percent is: arctigenin 16.0g, surfactant 7.0g, transdermal agent 2.0g, solvent 75.0g.

Embodiment 8:

Kill fish and water and produce agent for preventing and controlling ectoparasite of animals, mass percent is: arctigenin 1.0g, surfactant 10.0g, transdermal agent 4.0g, solvent 85.0g.

Claims (3)

1. arctigenin is killed application in aquatic animal vermin-dactylogyrider medicine in preparation.

2. kill aquatic animal vermin-dactylogyrider control agent for one kind, it is characterized in that: it is made up of following raw materials by weight percent: arctigenin 1%～20%, surfactant 5%～10%, transdermal agent 1%～4%, all the other are solvent, and the summation of above-mentioned raw materials is 100%;

Described surfactant is a tween 20;

Described transdermal agent is the water solublity azone;

Described solvent is a dehydrated alcohol.

3. the described preparation method of killing aquatic animal vermin-dactylogyrider control agent of claim 2 is characterized in that, carries out according to the following steps:

1) take by weighing arctigenin, surfactant, transdermal agent, each raw material of solvent, standby;

2) arctigenin and solvent are stirred to dissolving fully;

3) surfactant, transdermal agent are added step 2) stir in the mixed solution of gained, promptly get and kill external parasite preventing and treating agent for aquatic product animals.