AU2020102410A4 - Method for screening chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using tetrahymena thermophila - Google Patents
Method for screening chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using tetrahymena thermophila Download PDFInfo
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Abstract
The present invention discloses a method for screening Chinese herbal
medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena
thermophila. The method includes: Si, culturing the Tetrahymena thermophila; S2,
determining a growth curve of the Tetrahymena thermophila; S3, preparing
Chinese herbal medicine decoction or a traditional Chinese medicine monomer test
solution; S4, culturing and exposing the Tetrahymena thermophila by drugs; S5,
performing cell counting on a Tetrahymena thermophila test solution exposed by
drugs, and analyzing a median inhibitory concentration of the drugs inhibiting
growth of the Tetrahymena thermophila; S6, determining cell viability change
along with a drug concentration by a CCK-8 method, and calculating CC50 by
SPSS software; S7, evaluating effects of Chinese herbal medicines for inhibiting or
killing the Tetrahymena thermophila; S8, designing the drug concentration, adding
the drugs into feed, and evaluating an action effect of the fishes on the parasites.
Parasite-resistant drugs are subjected to high-throughput screening by replacing the
ciliated parasites of fishes with the Tetrahymena thermophila that may be
artificially cultured. Tested materials are readily available, are not limited by time,
temperature and season, and have excellent repeatability, economy, applicability
and rapidness.
Drawings of Description
Tetrahymena Cell counting
thermophila
-P - CbZ
Culture and - &
incubate
OD value determined by microplate
Chinese herbal medicine or reader and cell viability calculated by
purified product CCK-8 method
FIG. 1
A B
Rheum officinale
decoction 140 Rheu officinaledecoction
SSweet
wormwood herb 2 -- Sweet wornwood herb decoction
decoction
10-20
40 0
0- U,
4j 440
Drug concentration (g/L) Drug concentration (g/L)
FIG. 2
1
Description
Drawings of Description
Tetrahymena Cell counting thermophila -P - CbZ
Culture and -
& incubate
OD value determined by microplate Chinese herbal medicine or reader and cell viability calculated by purified product CCK-8 method
FIG. 1
A B Rheum officinale decoction 140 Rheu officinaledecoction SSweet wormwood herb 2 -- Sweet wornwood herb decoction decoction
40 0 10-20 0- U,
4j 440
Drug concentration (g/L) Drug concentration (g/L)
FIG. 2
Description
Technical Field
The present invention relates to the technical field of aquaculture, and particularly relates to a method for screening Chinese herbal medicine ingredients resistant to ciliated parasites by using Tetrahymena thermophila.
Background
Ciliated parasites of freshwater fishes include chthyophthirius multifiliis, Trichodina sp., Chilodonella sp., Trichodinella sp. and the like. The Ichthyophthirius multifiliis belongs to ciliated oligohymenophorea, Hymenostomatida, Ophryoglenidae, chthyophthirius. The Trichodina sp. and Trichodinella sp. belong to ciliated oligohymenophorea, Peritrichia, Peritrichida, Urceolariidae, Trichodina and Trichodinella; the Chilodonella sp. belongs to Kinetofragminophorea, Hypostomatia, Cyrtophorida, Chilodonellidae Chilodonella. The chthyophthirius multifiis has the most serious damage. When anti-parasitic drugs were screened in the past, fishes infected with parasitic were directly tested. It is understood from production practice that, due to individual differences, test material fishes of the same batch may have different sensitivities to the same parasites, and parasitic infection abundance of the fish individuals often has great differences. Effects of the anti-parasitic drugs are difficultly tested among the fishes having lower infection abundance. However, because of damage of the parasites, the fishes having higher infection abundance may easily suffer from acute death before drug actions. Therefore, since the action objects, i.e., the parasites, are difficultly stably obtained in batches and the fishes have non-uniform infection levels, the anti-parasitic drugs are difficultly screened.
Description
Predecessors made a lot of attempts for screening the anti-parasitic drugs such as the chthyophthiriusmultifiliis and the Trichodina sp. However, for existing drugs used in production, some parasites have produced drug resistance, and some drug residues may threaten safety of the environment and aquatic products. Thus, the drugs urgently need to be replaced. Since natural drugs derived from plants, animals and minerals have diversified sources and complex ingredients, if each kind of the drugs is tested by fishes infected with the parasites, the workload is heavy, the fishes infected with the parasites are insecure, and a great limitation may be caused to test screening. Tetrahymena thermophilahas wide development and application prospects in an aspect of resisting ciliated parasites of fishes. Thus, the present invention provides a method for screening Chinese herbal medicine ingredients resistant to ciliated parasites by using Tetrahymena thermophila.
Summary
A purpose of the present invention is to provide a method for screening Chinese herbal medicine ingredients resistant to ciliated parasites by using Tetrahymena thermophila, so as to solve problems proposed in the background. To achieve the above purpose, technical solutions of the present invention are as follows: the method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophila includes the following steps: S I, culturing the Tetrahymena thermophila transferring 1.0 mL of Tetrahymena thermophila liquid preserved in a culture medium at a room temperature into a black-cap bottle filled with 9.0 mL of a Tetrahymena thermophila culture medium; culturing the Tetrahymena thermophila liquid to a logarithmic phase under conditions of 28-32°C and 100 rpm; and transferring the resuscitated Tetrahymena thermophila into a conical flask containing 20 mL of the Tetrahymena thermophila culture medium so as to be cultured to an initial logarithmic phase;
Description
S2, determining a growth curve of the Tetrahymena thermophila inoculating the Tetrahymena thermophila according to initial density of 3000-5000 cells/mL; setting 3 parallels; culturing the Tetrahymena thermophila in a shaker at 28°C and 100 rpm; sampling once every 6 h for counting within 0-72 h; performing sampling every 12 h for counting within 72-144 h; 144 h later, performing counting every 24 h; counting the cells by a blood counting chamber or a cell counter; and drawing a growth curve of the Tetrahymena thermophila along with a concentration change by taking time as a horizontal coordinate and taking the quantity of the Tetrahymena thermophila as a vertical coordinate; S3, preparing Chinese herbal medicine decoction or a traditional Chinese medicine monomer test solution (1) preparation of the Chinese herbal medicine decoction weighing 30 g of original Chinese herbal medicines; adding 300 mL of distilled water to soak the medicines for 30-60 min; heating the solution to a boiling state; heating the solution by slow fire for 30-45 min; filtering the solution by sterile gauze; filtering out liquid medicine; decocting the medicines twice; merging the filtrate twice; concentrating the filtrate to be 30 mL so as to obtain original medicine liquid having a concentration of 1 g/nL; filtering the liquid by a 0.22um filter membrane; sterilizing the liquid; and preserving the liquid in a refrigerator at 4°C for later use; (2) preparation of the traditional Chinese medicine monomer test solution weighing 100 mg of traditional Chinese medicine monomers; adding 10 mL of a cosolvent such as DMSO; immediately adding 90 mL of sterile water; preparing a mother solution containing 1.0 mg/mL (1.0 g/L) of liquid medicine; enabling content of the DMSO to be 0.1 mL/mL (10% DMSO, about 0.1 mg/mL); and preserving the mother solution in the refrigerator at 4°C for later use; S4, drug exposure
Description
inoculating the Tetrahymena thermophila preserved at a room temperature into a 100 mL of sterilized Tetrahymena thermophila culture medium at an inoculation volume fraction of 5%; culturing the Tetrahymena thermophila to a logarithmic phase (24 h-48 h) in a shaker at 28-30°C and 100-150 rpm, and counting cells of the Tetrahymena thermophila; diluting the Tetrahymena thermophila culture medium to 10 mL of Tetrahymena thermophila cell suspension having a concentration of 1.0x10 4 -6.0x104 cells/mL; adding the
prepared tested drug in S3 that has a death rate of 0-100% and is provided with 5-6 concentrations according to an equal logarithmic interval; setting 3 parallel samples in each concentration group; setting blank control (culture medium only) and negative control (with the culture medium and Tetrahymena thermophila and without any drug); and culturing and exposing the Tetrahymena thermophila in the shaker at 28-30°C and 100-150 rpm for 24 h; S5, determining inhibitory growth concentrations of drugs taking the Tetrahymena thermophila test solution exposed by the drug of each concentration for 24 h in the step S4; counting living cells; counting the cells of each sample for 3 times; drawing a curve by taking the drug concentration as a horizontal coordinate and taking the number of living Tetrahymena thermophila as a vertical coordinate; analyzing a median inhibitory concentration (IC50) with 95% confidence limit of the drugs inhibiting growth of the Tetrahymena thermophila by SPSS software; S6, determining cell viability by CCK-8 method determining the cell viability by utilizing a reagent containing WST-8 by a CCK-8 method; determining OD value of the cells at a wavelength 450 nm by a microplate reader so as to determine cell viability change along the drug concentration; and calculating the CC50 with 95% confidence limit by the SPSS software (a corresponding drug concentration while producing cytotoxicity to 50% of the Tetrahymena thermophila);
Description
S7, result determination determining the median inhibitory concentration (IC50 in the S5) of the drug inhibiting the growth of the Tetrahymena thermophila and the concentration of the CCK-8 cell viability (CC50 in the S6); and evaluating an inhibitory killing effect of the Chinese herbal medicine or active ingredients thereof on the Tetrahymena thermophila; S8, effect of the drug for preventing the fishes from being infected with parasites based on the median inhibitory concentration for cell proliferation of the Tetrahymena thermophila, with reference to determination of the cell viability by CCK-8, designing the drug concentration; adding the drugs into feed, and evaluating an action effect of the fishes on the parasites. In one preferred embodiment, in the step S1, the Tetrahymena thermophila culture medium includes the following raw materials: 1.7-2.5% of polypolypeptone, 1.7-2.5% of yeast extract, 1.7-5.0% of D-glucose and 1 mL of 0.9 g/dL ferric trichloride storage solution. Distilled water is added until the culture medium is 1000 mL; and then the culture medium is sterilized at a high temperature of 121°C for 30 min and preserved at a room temperature after cooled. In one preferred embodiment, in the (1) of the S3, the original Chinese herbal medicine is Rheum officinale powder or sweet wormwood herb powder; and in the (2) of the S3, the traditional Chinese medicine monomers include rhein (content of 98.2%, powder), emodin (content of 99.93%, powder) and aloe-emodin (content of 99.54%, powder). 100 mg of each traditional Chinese medicine monomer is weighed; 10 mL of a cosolvent DMSO is added; 90 mL of sterile water is immediately added; and a mother solution containing 1.0 mg/mL (1.0 g/L) of liquid medicine is prepared, wherein the content of the DMSO is 0.1 mL/mL (10%, 0.1 mg/mL).
Description
In one preferred embodiment, in the steps S4 and S5, the cell counting method is as follows: the cells are counted by a Count star automatic cell counter, a Beckman Counter cell counter or a blood counting chamber; cells of the Tetrahymena thermophila are counted after fixed with ethanol or an iodine staining solution; and the data is stored in a computer. In one preferred embodiment, in the step S5, specific operations of the drugs for inhibiting growth of the Tetrahymena thermophila are as follows: 1 mL (equivalent to 1 g) of the Chinese herbal medicine decoction is sucked and then diluted with a Tetrahymena thermophila culture solution; test solutions having final concentrations such as 2 g/L (that equals to, 2 mg/mL), 1.25 g/L, 0.8 g/L, 0.5 g/L and 0.32 g/L are set according to equal logarithmic intervals; active ingredients of the traditional Chinese medicine monomers are also set according to equal logarithmic intervals by virtue of preparatory experiments; the solutions are respectively diluted into 5-6 appropriate concentrations such as 50 mg/L, 32 mg/L, mg/L, 12.5 mg/L, 8.0 mg/L and 5.0 mg/L; corresponding DMSO cosolvents with corresponding concentrations such as 5.0 mg/L, 3.2 mg/L, 2.0 mg/L, 1.25 mg/L, 0.8 mg/L and 0.5 mg/L are set as control; Tetrahymena thermophila strainCu428.2 cells growing actively are inoculated, wherein an initial concentration of the Tetrahymena thermophila is about 1.0x104 cells/mL; the Tetrahymena thermophila cells are cultured in a shaker at 28°C and 100 rpm for 24 hours; cells of the Tetrahymena thermophila are counted by an IC1000 Countstar automatic cell counter; and the median inhibitory concentration IC50 of Chinese herbal medicine water extract to the Tetrahymena thermophila is calculated by utilizing the SPSS software. In one preferred embodiment, in the step S6, the cell viability is determined by utilizing a reagent containing WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazol e monosodium salt] by the CCK-8 method. The reagent is reduced into an
Description
orange-yellow formazan dye that has high water solubility and is soluble in a tissue culture-medium by dehydrogenase in living cell mitochondrion in presence of an electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). OD value of the cells is determined at a wavelength 450 nm by a microplate reader. A specific operation method is as follows: 100 uL of cell suspension of different concentrations exposed by the traditional Chinese medicine decoction and the traditional Chinese medicine monomer agent for 24 h is added into a 96-well plate respectively (except wells in the outermost circle of the 96-well plate); 3-6 complex wells are set in each concentration group; 10 uL of CCK-8 solution is added into each well in the dark; normal cell control (including cells, drug dissolution media of the same concentration, a culture solution and CCK-8) and blank control (without cells and the culture medium and CCK-8 are added) are simultaneously set in the experiment; a culture plate is placed in an incubator at 28°C for standing and incubating in the dark for 4 h; and absorbance at 450 nm is determined by the microplate reader. The cell viability is calculated by utilizing the following formula: Cell viability %= (OD sample-OD blank)/(OD control-OD blank) x100, and the CC50 is calculated by the SPSS software. In one preferred embodiment, in the step S8, a preventive effect of goldfish on chthyophthirius multifiliis is evaluated; and the method is as follows: a concentration experiment group is set according to the effect of the drug on the Tetrahymena thermophila; the drug is added into the feed to prepare a drug bait; the goldfish is fed with the bait in an amount of 2% of the fish body weight; and the effect of the drug for preventing the goldfish from being infected with the Ichthyophthirius multifiliis is evaluated. Compared with the prior art, the present invention has beneficial effects as follows: 1. The parasite-resistant drugs are subjected to high-throughput screening by replacing the ciliated parasites of fishes with the Tetrahymena thermophila that
Description
may be artificially cultured. Tested materials are readily available, are not limited by time, temperature and season, and have the advantages of excellent repeatability, economy, applicability and rapidness. 2. The median inhibitory concentration of the traditional Chinese medicine for Tetrahymena thermophila proliferation can be rapidly obtained by complete experiment design and calculation methods of the median inhibitory concentration of the Chinese herbal medicine for Tetrahymena thermophila proliferation; and experimental results are clear and reliable. 3. The cell viability of the traditional Chinese medicine to the Tetrahymena thermophila is detected by the CCK-8 method; the orange-yellow formazan dye produced from the CCK-8 reagent by oxidation-reduction of the dehydrogenase in the cells is soluble in the tissue culture-medium; the cell viability may be directly determined; an experimental error is small; and the test is simple and rapid. 4. The effect of the drug to the Tetrahymena thermophila is evaluated in combination with determination of the living cell density and determination of the cell viability by the CCK-8 method. 5. With respect to the median inhibitory concentration design of the drug to the density and CCK-8 viability of the Tetrahymena thermophila, on the basis of the preliminary experiment safe to the fishes, the method in the present invention is validated by an experiment of adding the drugs into the fish feed for preventing the goldfish from being infected with the chthyophthirius multifiiis. Therefore, the effect of the Chinese herbal medicine preventing the fishes from being infected with the parasites screened in the present invention is effectively confirmed. The method is high in applicability and has popularization values.
Description of Drawings Fig. 1 is a schematic diagram of a technical route for screening a Chinese herbal medicine preventing fishes from being infected with ciliated parasites by utilizing Tetrahymena thermophila in the present invention;
Description
Fig. 2 shows living cell density and cell viability of Tetrahymena thermophila Cu 428.2 in two kinds of Chinese herbal medicine decoction for 24 h in the present invention; and Fig. 3 shows living cell density and cell viability of Tetrahymena thermophila Cu 428.2 in 3 rheum officinale monomers in the present invention. In the figures: A is a measurement result of a cell counting method; and B is a measurement result of a CCK-8 method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and fully described below in combination with the drawings in the embodiments of the present invention. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. Referring to Figs. 1-3, the present invention provides a technical solution as follows: a method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophila includes the following steps: S I, culturing the Tetrahymena thermophila 1.0 mL of Tetrahymena thermophila liquid preserved in a culture medium at a room temperature was transferred into a black-cap bottle filled with 9.0 mL of a Tetrahymena thermophila culture medium; the Tetrahymena thermophila liquid culture was cultured to a logarithmic phase under conditions of 28-32°C and 100 rpm; and the resuscitated Tetrahymena thermophila cells were transferred into a conical flask containing 20 mL of the Tetrahymena thermophila culture medium so as to be cultured to an initial logarithmic phase;
Description
S2, determining a growth curve of the Tetrahymena thermophila the Tetrahymena thermophila was inoculated according to initial density of 3000-5000 cells/mL; 3 parallels were set; the Tetrahymena thermophila was cultured in a shaker at 28°C and 100 rpm; sampling was performed once every 6 h for counting within 0-72 h; then sampling was performed every 12 h for counting within72-144 h; 144 h later, the Tetrahymena thermophila was counted every 24 h; the cells were counted by a blood counting chamber or a cell counter; and a growth curve of the Tetrahymena thermophila was drawn along with a concentration change by taking time as a horizontal coordinate and taking the quantity of the Tetrahymena thermophila as a vertical coordinate; S3, preparing Chinese herbal medicine decoction or a traditional Chinese medicine monomer test solution (1) preparation of the Chinese herbal medicine decoction 30 g of Rheum officinale powder or sweet wormwood herb powder was weighed; 300 mL of distilled water was added to soak the powder for 30-60 min; the solution was heated to a boiling state; then the solution was heated by slow fire for 30-45 min; the solution was filtered by gauze; the liquid medicine was filtered out; the medicine was decocted twice; the filtrate was merged twice; the filtrate was concentrated to be 30 mL so as to obtain original medicine liquid having a concentration of 1 g/nL; the liquid was filtered by a 0.22um filter membrane; and the liquid was sterilized, and preserved in a refrigerator at 4°C for later use; (2) preparation of the traditional Chinese medicine monomer test solution Rhein (content of 98.2%, powder), emodin (content of 99.93%, powder) and aloe-emodin (content of 99.54%, powder) were weighed; 100 mg of each traditional Chinese medicine monomer was weighed; 10 mL of a cosolvent DMSO was added; 90 mL of sterile water was immediately added; and a mother solution containing 1.0 mg/mL (1.0 g/L) of liquid medicine was prepared, wherein the content of the DMSO was 0.1 mE/mL (about 0.1 mg/mL);
Description
S4, drug exposure the Tetrahymena thermophilapreserved at a room temperature was inoculated into a 100 mL of sterilized Tetrahymena thermophila culture medium at an inoculation volume fraction of 5%; the Tetrahymena thermophila was cultured to a logarithmic phase (24 h-48 h) in a shaker at 28-30°C and 100-150 rpm, and cells of the Tetrahymena thermophila were counted; the Tetrahymena thermophila culture medium was diluted to 10 mL of Tetrahymena thermophila cell suspension having a concentration of 1.0x104 -6.0x104 cells/mL; the prepared tested drug in S3 that had a death rate of 0-100% and was provided with 5-6 concentrations was added according to an equal logarithmic interval; 3 parallel samples were set in each concentration group; blank control (culture medium only) and negative control (with the culture medium and Tetrahymena thermophila and without any drug) were set; and the Tetrahymena thermophila was cultured and exposed in the shaker at 28-32°C and 100-150 rpm for 24 h; S5, determining inhibitory growth concentrations of drugs the Tetrahymena thermophila test solution exposed by the drug of each concentration for 24 h in the step S4 was taken; living cells were counted; the cells of each sample were counted for 3 times; a curve was drawn by taking the drug concentration as a horizontal coordinate and taking the number of living Tetrahymena thermophila cells as a vertical coordinate; and a median inhibitory concentration (IC50) with 95% confidence limit of the drugs inhibiting growth of the Tetrahymena thermophila was analyzed by SPSS software; S6, determining cell viability by CCK-8 method the cell viability was determined by utilizing a reagent containing WST-8 by a CCK-8 method; OD value of the cells was determined at a wavelength 450 nm by a microplate reader so as to determine cell viability change along the drug concentration; and the CC50 with 95% confidence limit was calculated by the
Description
SPSS software (a corresponding drug concentration while producing cytotoxicity to 50% of the Tetrahymena thermophila); S7, result determination the median inhibitory concentration (IC50 in the S5) of the drug inhibiting the growth of the Tetrahymena thermophila and the concentration of the CCK-8 method (CC50 in the S6) were determined; and an inhibitory/killing effect of the traditional Chinese medicine to the Tetrahymena thermophila was evaluated; and S8, effects on parasites based on the median inhibitory concentration for cell proliferation of the Tetrahymena thermophila, with reference to determination of the cell viability by CCK8, a drug concentration was designed; the drugs were added into feed on premise of ensuring that a preliminary experiment is safe to fishes; and action effects of the fishes to the parasites were evaluated. In the step S, the Tetrahymena thermophila culture medium includes the following raw materials: 1.7-2.5% of polypeptone, 1.7-2.5% of yeast extract, 1.7-5.0% of D-glucose and 1 mL of 0.9 g/dL ferric trichloride storage solution. Distilled water was added until the culture medium is 1000 mL; and then the culture medium was sterilized at a high temperature of 121C for 30 min and preserved at a room temperature after cooled. In the steps S4 and S5, the cell counting method is as follows: the cells were counted by a Count star automatic cell counter, a Beckman Counter cell counter or a blood counting chamber; cells of the Tetrahymena thermophila were counted after fixed with ethanol or an iodine staining solution; and the data was stored in a computer. Specific operations of the drugs for inhibiting growth of the Tetrahymena thermophila are as follows: ImL (equivalent to 1 g) of the Chinese herbal medicine decoction was sucked and then diluted with a Tetrahymena thermophila culture solution; test solutions having final concentrations such as 2 g/L (that is, 2
Description
mg/mL), 1.25 g/L, 0.8 g/L, 0.5 g/L and 0.32 g/L were set according to equal logarithmic intervals; active ingredients of the traditional Chinese medicine monomers were also set according to equal logarithmic intervals by virtue of preparatory experiments; the solutions were respectively diluted into 5-6 appropriate concentrations such as 50 mg/L, 32 mg/L, 20 mg/L, 12.5 mg/L, 8.0 mg/L and 5.0 mg/L; corresponding DMSO cosolvents with corresponding concentrations such as 5.0 mg/L, 3.2 mg/L, 2.0 mg/L, 1.25 mg/L, 0.8 mg/L and 0.5 mg/L were set as control; Cu 428.2 Tetrahymena thermophila cells that grow actively were inoculated, wherein an initial concentration of the Tetrahymena thermophila was about 1.0x104 cells/mL; the Tetrahymena thermophila was cultured in a shaker at 28°C and 100 rpm for 24 hours; cells of the Tetrahymena thermophila were counted by an IC1000 Countstar automatic cell counter; and the median inhibitory concentration IC50 of Chinese herbal medicine water extract to the Tetrahymena thermophila was calculated by utilizing the SPSS software. In the step S6, the cell viability was determined by utilizing a reagent containing WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazol e monosodium salt] by the CCK-8 method. The reagent was reduced into an orange-yellow formazan dye that has high water solubility and is soluble in a tissue culture-medium by dehydrogenase in living cell mitochondrion in presence of an electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). OD value of the cells was determined at a wavelength 450 nm by a microplate reader. A specific operation method was as follows: 100 uL of cell suspension of different concentrations exposed by the traditional Chinese medicine decoction and the traditional Chinese medicine monomer agent for 24 h was added into a 96-well plate respectively (except wells in the outermost circle of the 96-well plate); 3-6 complex wells were set in each concentration group; 10 uL of CCK-8 solution was added into each well in the dark; normal cell control (with cells, drug dissolution
Description
media of the same concentration, a culture solution and CCK-8) and blank control (without cells and the culture medium and CCK-8 are added) were simultaneously set in the experiment; a culture plate was placed in an incubator at 28°C for standing and incubating in the dark for 4 h; and absorbance at 450 nm was determined by the microplate reader. The cell viability was calculated by utilizing the following formula: Cell viability %=(OD sample-OD blank)/(OD control-OD blank) x100, and the CC50 was calculated by the SPSS software. The median effective concentrations of 5 drugs to the Cu 428.2 Tetrahymena thermophila determined by the living cell counting method and the CCK-8 method respectively are as shown in the following table: Table 1 Median inhibitory concentrations of 5 drugs to the Cu 428.2 Tetrahymena thermophila determined by the living cell counting method and the CCK-8 method respectively
Drug Living cell counting method (mg/L) CCK-8 method (mg/L)
IC50 95% confidence limit CC50 95% confidence limit
Chinese Rheum officinale decoction 518.2 377.7-658.7 1046.5 849.9-1414.7
herbal Sweet wormwood herb decoction 1180.2 1046.0-1349.7 1683.6 1452.6-2051.1
medicine
Traditional Rhein 19.66 10.74-38.82 46.01 32.40-85.89
Chinese Emodin 3.77 1.908-5.543 6.07 4.39-7.65
Medicine Aloe-emodin 1.70 0.18-3.82 5.92 3.97-7.87
monomers
It can be seen from Figs. 2 and 3 that, a corresponding relation exists between the living cell density determined by the cell counting method and the living cell density determined by CCK-8 method. With the increase of concentrations of the
Description
drugs (that is, Rheum officinale decoction, sweet wormwood herb decoction, rhein, emodin and aloe-emodin), the living cell density of the Tetrahymena thermophila determined by the counting method and the cell viability determined by the CCK-8 method are decreased. With respect to the 2 kinds of Chinese herbal medicine decoction: with the adoption of the cell counting method, the Rheum officinale has a more excellent inhibitory effect than the sweet wormwood herb; and with the adoption of the CCK-8 method, the Rheum officinale also has a more excellent inhibitory effect than the sweet wormwood herb. With respect to the 3 kinds of traditional Chinese medicine monomers: with the adoption of the cell counting method, intensity of the determined inhibitory effects is as follows in sequence: aloe-emodin> emodin> rhein; and intensity of the cell viability determined by the CCK8 is as follows: aloe-emodin, emodin> rhein ( aloe-emodin and emodin have no significant difference ) . Results show that, the results determined by the two methods are basically consistent but have slight differences. In combination with the two methods, the action results of the drugs to the Tetrahymena thermophila are accurately evaluated. Further, a common methyl thiazolyl tetrazolium (MTT) method is used for detecting the cell viability of the Tetrahymena thermophila. However, compared with the CCK-8 method, since a product formazan produced by reduction of the MTT is insoluble in water and can be detected only after dissolved, the workload is increased, and the accuracy of experimental results is affected. The orange-yellow formazan dye produced from the CCK-8 reagent by oxidation-reduction of the dehydrogenase in the cells is soluble in the tissue culture-medium and can be directly determined, and the experimental error is small. Therefore, the solution in the present invention has significant superiority. In the step S8, a preventive effect of goldfish onchthyophthirius multifiliis was evaluated; and the method was as follows: a concentration experiment group
Description
was set according to the effect of the drug on the Tetrahymena thermophila; the drug was added into the feed to prepare a toxic bait; the goldfish was fed with the bait in an amount of 2% of the body weight daily for 7 days; and the effect of the drug for preventing the goldfish from being infected with theIchthyophthirius multifiliis was as shown in Table 2. The drugs were added into the feed for feeding the fishes according to a ratio that is 10-20 times that of the IC50. Based on the weight, the fishes were fed with 196.6 mg/Kg of rhein, 37.7 mg/Kg of emodin and 33.9 mg/Kg of aloe-emodin and then infected with theIchthyophthirius multifiliis within 7 days. Excellent preventive effects were achieved. Table 2 Effects of the rhein, emodin and aloe-emodin for preventing goldfishes from being infected withIchthyophthirius multifiliis Drug Dosage Number of dead Death rate of Relative protection
(mg/Kg fishes/number of fishes infected ratio=(death rate of the control
based on experimental with group-death rate of the
weight of fishes Ichthyophthirius experimental group)/the death
the fishes) multflliis rate of the control group
Control 0 53/60 88.3% /
Rhein 196.6 21/60 35.0% 60.3%
Emodin 37.7 15/60 25.0% 71.7%
Aloe-emodin 33.9 19/60 31.7% 64.2%
In conclusion, in combination with the prior art, the fishes infected with the parasites are directly tested. Due to the characteristic that the parasites may reproduce by virtue of fish bodies and cannot be cultured in vitro, the test objects cannot be readily available. Moreover, due to different infection degrees of the fishes and great individual differences of the drugs to the fishes, anti-parasite drugs are difficultly screened.
Description
According to the "method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophila" in the patent of the present invention, the parasite-resistant drugs are subjected to high-throughput screening by replacing the ciliated parasites of fishes with the Tetrahymena thermophila that may be artificially cultured. Tested materials are readily available, are not limited by time, temperature and season, and have the advantages of excellent repeatability, economy, applicability and rapidness. Although the embodiments of the present invention have been illustrated and described, various changes, modifications, replacement and transformations may be made to these embodiments by those ordinary skilled in the art without departing from the principle and spirit of the present invention. The scope of the present invention is defined by appended claims and equivalents thereof.
Claims (7)
1. A method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophila, comprising the following steps: S1, culturing the Tetrahymena thermophila transferring 1.0 mL of Tetrahymena thermophila liquid preserved in a culture medium at a room temperature into a black-cap bottle filled with 9.0 mL of a Tetrahymena thermophila culture medium; culturing the Tetrahymena thermophila liquid to a logarithmic phase under conditions of 28-32°C and 100 rpm; and transferring the resuscitated Tetrahymena thermophila into a conical flask containing 20 mL of the Tetrahymena thermophila culture medium so as to be cultured to an initial logarithmic phase; S2, determining a growth curve of the Tetrahymena thermophila inoculating the Tetrahymena thermophila according to initial density of 3000-5000 cells/mL; setting 3 parallels; culturing the Tetrahymena thermophila in a shaker at 28°C and 100 rpm; sampling once every 6 h for counting within 0-72 h; performing sampling every 12 h for counting within 72-144 h; 144 h later, performing counting every 24 h; counting the cells by a blood counting chamber or a cell counter; and drawing a growth curve of the Tetrahymena thermophila along with a concentration change by taking time as a horizontal coordinate and taking the quantity of the Tetrahymena thermophila as a vertical coordinate; S3, preparing Chinese herbal medicine decoction or a traditional Chinese medicine monomer test solution (1) preparation of the Chinese herbal medicine decoction weighing 30 g of original Chinese herbal medicines; adding 300 mL of distilled water to soak the medicines for 30-60 min; heating the solution to a boiling state; heating the solution by slow fire for 30-45 min; filtering the solution by gauze; filtering out liquid medicine; decocting the medicines twice; merging the filtrate twice; concentrating the filtrate to be 30 mL so as to obtain original medicine liquid having a concentration of 1 g/mL; filtering the liquid by a 0.22um
Claims
filter membrane; sterilizing the liquid; and preserving the liquid in a refrigerator at 4°C for later use; (2) preparation of the traditional Chinese medicine monomer test solution weighing 100 mg of traditional Chinese medicine monomers; adding 10 mL of a cosolvent (such as DMSO); immediately adding 90 mL of sterile water; preparing a mother solution containing 1.0 mg/mL (1.0 g/L) of liquid medicine; and enabling content of the DMSO to be 0.1 mL/mL (10%DMSO, about 0.1 mg/mL); S4, drug exposure inoculating the Tetrahymena thermophila preserved at a room temperature into a 100 mL of sterilized Tetrahymena thermophila culture medium at an inoculation volume fraction of 5%; culturing the Tetrahymena thermophila to a logarithmic phase (24 h-48 h) in a shaker at 28-30°C and 100-150 rpm, and counting cells of the Tetrahymena thermophila; diluting the Tetrahymena thermophila culture medium to 10 mL of Tetrahymena thermophila cell suspension having a concentration of 1.x10 4 -6.Ox104 cells/mL; adding the
prepared tested drug in S3 that has a death rate of 0-100% and is provided with 5-6 concentrations according to an equal logarithmic interval; setting 3 parallel samples in each concentration group; setting blank control (culture medium only) and negative control (with the culture medium and Tetrahymena thermophila and without any drug); and culturing and exposing the Tetrahymena thermophila in the shaker at 28-30°C and 100-150 rpm for 24 h; S5, determining inhibitory growth concentrations of drugs taking the Tetrahymena thermophila test solution exposed by the drug of each concentration for 24 h in the step S4; counting living cells; counting the cells of each sample for 3 times; drawing a curve by taking the drug concentration as a horizontal coordinate and taking the number of living Tetrahymena thermophila as a vertical coordinate; analyzing a median inhibitory concentration (IC50) with 95%
Claims
confidence limit of the drugs inhibiting growth of the Tetrahymena thermophila by SPSS software,; S6, determining cell viability by CCK-8 method determining the cell viability by utilizing a reagent containing WST-8 by a CCK-8 method; determining OD value of the cells at a wavelength 450 nm by a microplate reader so as to determine cell viability change along with the drug concentration; and calculating the CC50 with 95% confidence limit by the SPSS software (a corresponding drug concentration while producing cytotoxicity to 50% of the Tetrahymena thermophila); S7, result determination determining the median inhibitory concentration (IC50 in the S5) of the drug inhibiting the growth of the Tetrahymena thermophila and the concentration of the CCK-8 cell viability (CC50 in the S6); and evaluating an inhibitory killing effect of the Chinese herbal medicine on the Tetrahymena thermophila; S8, effect on parasites based on the median inhibitory concentration for cell proliferation of the Tetrahymena thermophila, with reference to determination of the cell viability by CCK-8, designing the drug concentration; adding the drugs into feed, and evaluating an action effect of the fishes on the parasites.
2. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophile according to claim 1, wherein in the step S, the Tetrahymena thermophila culture medium comprises the following raw materials: 1.7-2.5% of polypeptone, 1.7-2.5% of yeast extract, 1.7-5.0% of D-glucose and 1 mL of 0.9 g/dL ferric trichloride storage solution; distilled water is added until the culture medium is 1000 mL; and then the culture medium is sterilized at a high temperature of 121C for 30 min and preserved at a room temperature after cooled.
Claims
3. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophile according to claim 1, wherein in the (1) of the S3, the original Chinese herbal medicine is decoction piece or powder processed by Chinese herbal medicine; the Chinese herbal medicine is such as one or more of rhubarb decoction piece, Artemisia annua decoction piece, Rheum officinale powder and sweet wormwood herb powder; and in the (2) of the S3, the traditional Chinese medicine monomers are one or more of rhein (content of 98.2%, powder), emodin (content of 99.93%, powder) and aloe-emodin (content of 99.54%, powder).
4. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophile according to claim 1, wherein in the steps S4 and S5, the cell counting method is as follows: the cells are counted by a Count star automatic cell counter, a Beckman Counter cell counter or a blood counting chamber; cells of the Tetrahymena thermophila are counted after fixed with ethanol or an iodine staining solution; and the data is stored in a computer. 5. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophile according to claim 1, wherein in the step S5, specific operations of the drugs for inhibiting growth of the Tetrahymena thermophila are as follows: 1 mL (equivalent to 1 g) of the Chinese herbal medicine decoction is sucked and then diluted with a Tetrahymena thermophila culture solution; test solutions having final concentrations such as 2 g/L (that is, 2 mg/mL), 1.25 g/L, 0.8 g/L, 0.5 g/L and 0.32 g/L are set according to equal logarithmic intervals; active ingredients of the traditional Chinese medicine monomers are also set according to equal logarithmic intervals by virtue of preparatory experiments; the solutions are respectively diluted into 5-6 appropriate concentrations such as 50 mg/L, 32 mg/L, 20 mg/L, 12.5 mg/L, 8.0 mg/L and 5.0 mg/L; corresponding DMSO cosolvents with corresponding concentrations such as
Claims
5.0 mg/L, 3.2 mg/L, 2.0 mg/L, 1.25 mg/L, 0.8 mg/L and 0.5 mg/L are set as control; Tetrahymena thermophila cells growing actively are inoculated, wherein an initial concentration of the Tetrahymena thermophila is about 1.0xI04 cells/mL; the cells are cultured in a shaker at 28°C and 100 rpm for 24 hours; cells of the Tetrahymena thermophila are counted by an IC1000 Countstar automatic cell counter; and the median inhibitory concentration (IC50) of Chinese herbal medicine water extract to the Tetrahymena thermophila is calculated by utilizing the SPSS software.
6. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophile according to claim 1, wherein in the step S6, the cell viability is determined by utilizing a reagent containing WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazol e monosodium salt] by the CCK-8 method; the reagent is reduced into an orange-yellow formazan dye that has high water solubility and is soluble in a tissue culture-medium by dehydrogenase in living cell mitochondrion in presence of an electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS); OD value of the cells is determined at a wavelength 450 nm by a microplate reader; a specific operation method is as follows: 100 uL of cell suspension of different concentrations exposed by the traditional Chinese medicine decoction and the traditional Chinese medicine monomer agent for 24 h is added into a 96-well plate respectively (except wells in the outermost circle of the 96-well plate); 3-6 complex wells are set in each concentration group; 10 uL of CCK-8 solution is added into each well in the dark; normal cell control (with cells, drug dissolution media of the same concentration, a culture solution and CCK-8) and blank control (without cells and the culture medium and CCK-8 are added) are simultaneously set in the experiment; a culture plate is placed in an incubator at 28°C for standing and incubating in the dark for 4 h; and absorbance at 450 nm is determined by the
Claims
microplate reader; the cell viability is calculated by utilizing the following formula: Cell viability %=(OD sample-OD blank)/(OD control-OD blank) x100, and the CC50 is calculated by the SPSS software.
7. The method for screening Chinese herbal medicine ingredients resistant to ciliated parasites of fishes by using Tetrahymena thermophila according to claim 1, wherein in the step S8, a preventive effect of goldfish (Carassius auratus) on Ichthyophthirius multifiliis is evaluated; and the method is as follows: a concentration experiment group is set according to the effect of the drug on the Tetrahymena thermophila; the drug is added into the feed to prepare a drug bait; the goldfish is fed with the bait in an amount of 2% of the body weight daily; and the effect of the drug for preventing the goldfish from being infected with the Ichthyophthirius multifiliis is evaluated.
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| WO2008034121A2 (en) * | 2006-09-16 | 2008-03-20 | Board Of Regents, The University Of Texas System | Parasite vaccine |
| CN102286530A (en) * | 2011-06-16 | 2011-12-21 | 中国科学院水生生物研究所 | Tetrahymena expression vectors capable of introducing exogenous genes by one-step method, and construction method application thereof |
| CN107157968B (en) * | 2017-05-18 | 2019-07-19 | 暨南大学 | Rheum emodin kills the application in fish epizoite infusorian drug in preparation |
| CN108653261A (en) * | 2018-05-04 | 2018-10-16 | 浙江省淡水水产研究所 | Application of the 6- n-pentyloxies Rhein in prevention saprolegniasis and ich |
| CN108619384A (en) * | 2018-07-25 | 2018-10-09 | 贵州贵丰农业发展有限公司 | A method of prevention soft-shelled turtle parasitic disease |
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