阅读说明：本技术 迷迭香提取物在防控多子小瓜虫病药物制备中的应用 (Application of rosemary extract in preparation of medicine for preventing and controlling ichthyophthiriasis ) 是由 顾泽茂 胡静波 杨浩 李丹 于 2021-10-09 设计创作，主要内容包括：本发明公开了迷迭香(Rosemarinus officinalis)提取物在防控多子小瓜虫病药物制备中的应用,属于渔业病害防控技术领域。本发明首次将迷迭香提取物应用于防控多子小瓜虫病,在有效剂量范围能实现100％杀灭多子小瓜虫。迷迭香提取物对金鱼的半致死浓度分别为其杀灭小瓜虫掠食体和滋养体的半数有效浓度的28.6和9.4倍,在杀虫的有效范围内对鱼安全。迷迭香资源丰富,其提取物的制备工艺简单,具有开发成新型防控小瓜虫病的绿色替代药物的潜力。(The invention discloses application of rosemary (Rosemarinus officinalis) extract in preparation of a drug for preventing and controlling ichthyophthiriasis, belonging to the technical field of fishery disease prevention and control. The rosemary extract is applied to preventing and controlling the ichthyophthiriasis for the first time, and 100 percent of ichthyophthiriasis can be killed within the effective dose range. The semi-lethal concentration of the rosemary extract on goldfish is 28.6 times and 9.4 times of the half effective concentration of the rosemary extract on killing ichthyophthirius multifiliis and trophozoite respectively, and the rosemary extract is safe to the goldfish within the insecticidal effective range. The rosemary has rich resources, the preparation process of the extract is simple, and the rosemary has the potential of being developed into a novel green substitute medicament for preventing and controlling ichthyophthiriasis.)

1. Application of herba Rosmarini officinalis extract in preparing medicine for preventing and controlling ichthyophthiriasis is provided.

2. Use according to claim 1, characterized in that: the rosemary is the leaf of rosemary.

3. Use according to claim 1, characterized in that: the rosemary extract is an ethanol extract of rosemary.

4. A method for killing the Polychachis bicolor is characterized in that: the method comprises the step of applying rosemary extract to the insect solution of the chaulmoogra.

5. The method for killing the Polychachis bicolor according to claim 4, wherein: the effective insecticidal concentration of the rosemary extract is 4-32mg/L, wherein the effective concentration to a predator is more than or equal to 4mg/L, the effective concentration to a trophosome is more than or equal to 8mg/L, and the effective concentration to cyst is more than or equal to 16 mg/L.

Technical Field

The invention belongs to the technical field of fishery disease prevention and control, and particularly relates to application of a rosemary extract in preparation of a drug for preventing and controlling ichthyophthiriasis.

Background

The ichthyophthiriasis is a parasitic disease caused by the fact that Ichthyophthirius multifiliis Fouquet (called ichthyophthiriasis for short) parasitizes on the body surfaces and gills of freshwater fishes, is wide in distribution, quick in morbidity and high in mortality rate, and can cause a large amount of death of the fishes in serious conditions to cause economic loss. At present, the production of prevention and control of the ichthyophthiriasis mainly depends on chemical drugs such as formalin, copper sulfate and the like, but the use of the drugs faces the problems of high residue, high toxicity, environmental unfriendliness and the like. Therefore, there is an urgent need to find safe and effective green substitute drugs.

The Chinese herbal medicine as a natural product has the advantages of small toxic and side effects, low residue, environmental friendliness and the like, and is an important resource for developing green medicines. Wherein Rosmarinus officinalis (Rosemarinus officinalis Linn) is a medicinal plant of Rosmarinus of Labiatae, has antioxidant, antibacterial, antitumor, antiinflammatory, and cardiovascular diseases resisting effects, and can be used for treating asthma, cough, dyspepsia, rheumatic toothache, and heart disease. At present, there is no report that rosemary is used for preventing and controlling ichthyophthiriasis.

Disclosure of Invention

The invention aims at providing the application of rosemary extract in the preparation of a medicament for preventing and controlling ichthyophthiriasis, and also aims at providing a method for killing ichthyophthiriasis.

The above object is achieved by the following scheme:

the in vitro insecticidal test result shows that: the rosemary extract has killing effect on predators, trophoblasts and cysts of the ichthyophthirius multifiliis and has dose-effect relationship, and the ichthyophthiriasis can be prevented and controlled by killing the ichthyophthiriasis.

The medicinal part of the rosemary is the leaf of the rosemary.

The herba Rosmarini officinalis extract is obtained by extracting herba Rosmarini officinalis with water or ethanol, preferably ethanol.

A method for killing the water melon worms with fruit worms comprises the step of applying rosemary extract to the worm liquid of the water melon worms with fruit worms.

Preferably, the effective insecticidal concentration of the rosemary extract is 4-32mg/L, wherein the effective concentration for a predator is more than or equal to 4mg/L, the effective concentration for a trophosome is more than or equal to 8mg/L, and the effective concentration for an cyst is more than or equal to 16 mg/L.

Compared with the prior art, the invention has the following advantages:

(1) the rosemary extract is applied to the preparation of the drug for preventing and controlling the ichthyophthiriasis of the majority for the first time, and the ichthyophthiriasis can be killed by 100 percent within the effective dose range.

(2) The plant raw materials are rich in resources in China, easy to obtain, simple in preparation process and wide in application prospect.

(3) semi-Lethal Concentration (LC) of rosemary extract to goldfish of the invention₅₀) 48.7mg/L, which is the half Effective Concentration (EC) of the extract on the predator and the trophozoite respectively₅₀) 28.6 and 9.4 times of the total weight of the composition, and is safe to fish within the insecticidal effective range.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The materials used in the following examples are: the host model is goldfish with a total length of 7.1-10.0(8.2 + -0.7) cm, a body length of 4.6-5.8(5.1 + -0.3) cm and a body weight of 3.1-7.0(4.7 + -1.0) g, and is from a certain breeding farm in Shanghai. The parasite model is a Pectinatus ichthyophthirius, and is derived from the aquiculture base of the university of agriculture in Huazhong. The rosemary extract is extracted in a laboratory of the inventor.

Before the test begins, the applicant selects more than 70 Chinese herbal medicines such as dandelion, isatis root, common andrographis herb, desmodium, heartleaf houttuynia herb, rosemary and the like, and observes the killing effect on the predators of the ichthyophthirius multifiliis in vitro after water or ethanol extraction, and preliminarily screens out a plurality of Chinese herbal medicines with strong activity, wherein the rosemary has strong insecticidal activity on the ichthyophthirius multifiliis, and some Chinese herbal medicines have no activity or very low activity. Then, the insecticidal activity of the rosemary on other common freshwater fish parasites (dactylogyrus and gyrodactylous) is investigated, and the result shows that the rosemary can not effectively kill the dactylogyrus and the gyrodactylous, which indicates that the rosemary has certain specificity on the pesticide effect of the ichthyophthirius multifiliis, and then the following experiments are carried out.

The extraction process of the rosemary extract comprises the following steps:

the rosemary leaves are dried in an oven at the temperature of 55 ℃ to constant weight, and then are crushed by a crusher and filtered by a 60-mesh screen to obtain medicine powder. Taking 1kg of powder, adding 10L of anhydrous ethanol according to a ratio of 1:10, leaching for 2 times in a 45 ℃ water bath kettle, extracting for 2h each time, combining the extracting solutions, and filtering residual medicine powder to obtain a filtrate. Concentrating the filtrate at 55 deg.C under reduced pressure, and drying the concentrated product in 55 deg.C oven to remove residual ethanol to constant weight to obtain rosemary ethanol extract.

Example 1: rosemary extract killing effect on melon worm infected larva

(1) Preparing the medicine: accurately weighing 0.1g of rosemary extract, using 1mL of dimethyl sulfoxide to promote dissolution, preparing 100g/L of liquid medicine, and diluting to the concentration required by the test. Meanwhile, a control treatment solution containing 0.02% of dimethyl sulfoxide and no compound was prepared.

(2) Set up 6 test groups, 1 dmso control group, and 1 blank control group, each set up 3 replicates, and the assay was completed in a 96-well plate. 160 μ L of the predator-containing solution was added to each well of the 96-well plate, and 6 drops of 10 μ L of the worm solution were pipetted into each well and counted on a glass slide to estimate the number of predators contained in 100 μ L of that well. Each well was filled with 100. mu.L of each drug solution at different concentrations to give final drug concentrations of 8, 4, 2, 1, 0.5, and 0.25mg/L in this order, and a group containing only distilled water and only 0.02% dimethyl sulfoxide was used as a negative control. The test temperature is 24 ℃, the 100 percent lethal time of the predators of different treatment groups is recorded, the number of the predators in each hole is counted after 4 hours, and the mortality rate and the half Effective Concentration (EC) of the predators are calculated₅₀) The calculation formula is as follows:

mortality (%). percent (1-test group number of surviving predators/number of predators not dosed) x 100%

The results show that: the killing effect of the rosemary extract on the ichthyophthirius multifiliis dose-dependent, and the higher the concentration is, the shorter the killing time is. When the concentration is 8.0mg/L, 100% of predators can be killed within 30 min; when the concentration is 4.0mg/L, 100% of predators can be killed within 150 min. Calculating EC of rosemary extract on ichthyophthirius multifiliis through SPSS₅₀It was 1.7 mg/L.

TABLE 1 Effect of Rosemary extracts on Pest grazing of Pachyrhizus

Concentration (mg/L)	Death time (min)	Mortality (%)
			8	28.7±2.5	100.0±0.0
4	149.5±6.5	100.0±0.0
			2	﹣	68.7±5.5
1	﹣	14.9±12.0
			0.5	﹣	0.0±0.0
0.25	﹣	0.0±0.0
			0(H2O)	﹣	0.0±0.0
0(0.02％DMSO)	﹣	0.0±0.0

Example 2: rosemary extract with effect of killing adult (trophozoite) of ichthyophthirius multifiliis

(1) The drug formulation was the same as in example 1.

(2) Set 5 test groups, 1 dmso control group, and 1 blank control group, each set 3 replicates, and the assay was completed in 12-well plates. 1mL of insect solution containing about 30 mature trophozoites was added to each well of a 12-well plate, the number of trophozoites in each well was accurately counted, 1mL of drug solutions with different concentrations were added, respectively, so that the final drug concentrations were 32, 16, 8, 4, and 2mg/L in this order, and a group to which only distilled water and only 0.05% dimethylsulfoxide were added was set as a negative control. After incubation in an incubator at 24 ℃ for 6h, the number of surviving worms was recorded for each treatment group and the mortality was calculated. Standing a 12-hole plate in a constant-temperature incubator at 24 ℃, counting the number of the predators in each hole after 12 hours, and calculating the number of the predators hatched from each cyst in each treatment group according to the following calculation formula:

percent mortality (%) < 100% (number of dead trophozoites/number of non-medicated trophozoites in test group).)

The average number of the predator hatched from each cyst is the total number of the predator per each pore/the number of cysts per pore

The result shows that when the concentration of the rosemary extract is more than 8.0mg/L, 100 percent of trophozoite can be killed within 6 hours. Calculating EC of the water melon worm trophosome through SPSS₅₀It was 5.2 mg/L.

TABLE 2 effect of rosemary extract on the trophozoite of the Polychachis

Concentration (mg/L)	Mortality rate	Number of hatchings
			32	100.0±0.0	0.0±0.0
16	100.0±0.0	0.0±0.0
			8	100.0±0.0	0.0±0.0
4	10.0±8.8	551.5±84.4
			2	3.3±3.3	650.5±63.7
0(H2O)	0.0±0.0	713.6±16.9
			0(0.05％DMSO)	0.0±0.0	696.5±73.7

Example 3: rosemary extract killing effect on chaulmoogra cyst

(1) The drug formulation was the same as in example 1.

(2) Set 5 test groups, 1 dmso control group, and 1 blank control group, each set 3 replicates, and the assay was completed in 12-well plates. Adding 1mL of insect solution containing about 30 mature trophozoites into each hole of a 12-hole plate, accurately counting the number of trophozoites in each hole, putting the 12-hole plate in a constant-temperature incubator at 24 ℃ for incubation for 4h, accurately counting the number of cysts in each hole after the trophozoites develop into cysts, then respectively adding 1mL of liquid medicines with different concentrations to ensure that the final medicine concentration is 32, 16, 8, 4 and 2mg/L in sequence, and taking the group added with only distilled water and only 0.05 percent of dimethyl sulfoxide as negative control. Placing a 12-hole plate in a constant-temperature incubator at 24 ℃ for incubation for 12h, recording the death rate of cysts of each treatment group, counting the number of predators in each hole, and calculating the number of the predators incubated by each cyst of each treatment group according to the following calculation formula:

percent mortality (%) - (number of dead capsules in test group/number of capsules formed in test group) × 100%

The average number of the predator hatched from each cyst is the total number of the predator per each pore/the number of cysts per pore

The results show that when the concentration of the rosemary extract is 32.0mg/L, the lethality to the capsule in 12h is 100%; when the concentration is 16.0mg/L, the grazing body form hatched by the cysts is abnormal. Calculating the EC of the seed coat on the ichthyophthirius multifiliis through SPSS₅₀It was 10.753 mg/L.

TABLE 3 Effect of rosemary extract on Pectinatus fasciatus eradication

Concentration (mg/L)	Mortality rate	Number of hatchings
			32	100.0±0.0	0.0±0.0
16	100.0±0.0	0.0±0.0
			8	5.6±6.9	360.7±113.9
4	4.4±3.8	518.6±89.9
			2	0.0±0.0	680.9±85.7
0(H2O)	0.0±0.0	715.6±71.3
			0(0.05％DMSO)	0.0±0.0	695.6±36.7

Example 4: acute toxicity test of rosemary extract on goldfish

(1) Preparing a liquid medicine: the procedure is as in example 1.

(2) Pre-experiment: the rosemary extract was measured to give a total death concentration of 100mg/L in 24 hours and 20mg/L in 96 hours for the test goldfish.

(3) 3L of aerated tap water and 10-tailed goldfish were put into 21 aquaria, respectively, and the test temperature was maintained at 23 + -0.5 deg.C, and oxygen was supplied by an oxygen pump. Setting 100, 80, 60, 40 and 20mg on the basis of pre-testFive concentration groups were set, and a group to which only distilled water and only 0.1% dimethyl sulfoxide were added was set as a negative control, and 3 replicates were set for each concentration. And (3) observing the poisoning condition of the test fish under various concentrations at any time during the test period, and if death of poisoning of the goldfish is found, timely fishing out the goldfish to avoid damaging water quality and influencing the test result. Counting the test result for 96h, and calculating the semilethal concentration (LC)₅₀) And 95% confidence intervals (calculated using the SPSS software probit program).

The results of acute toxicity tests of the rosemary extract on goldfish show that the total lethal concentration of the rosemary extract on goldfish is 100.0mg/L and the total lethal concentration of the rosemary extract on goldfish LC (liquid chromatography) is 100.0mg/L within 96h₅₀48.7mg/L, which is the EC for the predator and the trophozoite respectively₅₀28.6 and 9.4 times of the amount of the extract, it can be seen that the rosemary extract is safe for fish in the range of insecticidal effectiveness.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.