Application of Baohuoside I in the prevention and treatment of Cryptocaryon irritans disease

Abstract

The present invention discloses the application of Baohuoside I in preventing and treating Cryptocaryon irritans disease, and proves that Baohuoside I extracted from the traditional Chinese herbal medicine Epimedium can effectively kill Cryptocaryon irritans, and low concentrations of Baohuoside I can quickly inhibit the growth and reproduction of Cryptocaryon irritans, reduce its infringement on farmed animals, and reduce the infection rate and mortality rate of aquaculture animals. From an environmental protection perspective, compared with some chemically synthesized drugs, Baohuoside I is derived from natural plants and does not leave harmful substances that are difficult to degrade in water bodies and farmed organisms during use, does not pollute the breeding environment, and is conducive to maintaining ecological balance. In addition, Epimedium is widely distributed in my country and is rich in resources, which makes the source of Baohuoside I very extensive, provides a solid foundation for large-scale production and application, and can also reduce breeding costs to a certain extent and improve economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of application technology of traditional Chinese medicine extracts and drugs for preventing and treating Cryptocaryon irritans disease, and in particular to the application of baohuoside I in preventing and treating Cryptocaryon irritans disease. Background Art

[0002] White spot disease in marine fish is a globally prevalent parasitic disease caused by Cryptocaryon irritans. It typically occurs from May to October, with a peak in summer and autumn. The outbreak of white spot disease in marine fish is primarily dependent on water temperature. Cryptocaryon irritans can infect fish in water temperatures between 22 and 32°C, with widespread infestation occurring at temperatures between 25 and 27°C. The larvae of Cryptocaryon irritans swim continuously in the water in search of a host. Upon infestation, they develop into trophozoites, which ingest host cells and tissue fluids. The trophozoites stimulate the epidermal tissues of the fish, causing them to proliferate and then envelop the larvae, resulting in visible "white spots" on the fish's surface. The trophozoites shed from the host and develop into swimming cyst precursors, which then settle on the bottom and develop into cysts. The cysts proliferate, releasing larvae to begin a new life cycle. Because the fish's epidermis encases the trophozoites, immersion medications cannot directly penetrate the epidermal tissues and reach them, making prevention and control more difficult. Moreover, with the increasing degree of aquaculture intensification in recent years, the breeding density has increased, the breeding environment has deteriorated, and the "white spot disease" of marine fish has frequently broken out, causing huge economic losses and seriously restricting the development of the marine aquaculture industry.

[0003] Currently, the prevention and treatment of Cryptocaryon irritans disease are primarily physical and chemical. Physical methods include freshwater immersion, heat treatment, conversion culture, ultraviolet irradiation, drying, and ozone treatment. These methods are complex to operate and their effectiveness in preventing and treating Cryptocaryon irritans is less than ideal. Chemical methods, on the other hand, primarily involve soaking diseased fish or parasites in drugs. Commonly used drugs include potassium permanganate, copper sulfate, and formalin. Additionally, biological dyes, formalin, acriflavine, methylene blue, sodium hypochlorite, sulfathiazole chloride, penicillin, and benzalkonium chloride have also been shown to have inhibitory effects on Cryptocaryon irritans. While chemical methods are effective, they are not suitable for long-term use and can lead to drug resistance in parasites. Furthermore, some drugs can cause irreversible environmental damage. Therefore, it is necessary to develop effective and environmentally friendly prevention and control measures to curb the spread of the pathogen, reduce economic losses to the aquaculture industry, and promote the development of marine aquaculture. Summary of the Invention

[0004] In view of this, the object of the present invention is to propose a reliable, environmentally friendly and flexible use of Baohuoside I in preventing and treating Cryptocaryon irritans disease.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The use of Baohuoside I in the preparation of drugs for preventing and treating Cryptocaryon irritans disease, wherein the chemical structure of Baohuoside I is shown in Formula 1:

[0007]

[0008] Based on the above, this solution also proposes a method for preparing Baohuoside I, which comprises the following steps:

[0009] S01, extracting and separating the epimedium powder N times by an ethanol reflux method, and then collecting the extracts and concentrating them under reduced pressure to obtain an extract, i.e., a preliminarily separated epimedium ethanol extract;

[0010] S02, diluting the extract with water, then extracting it with petroleum ether and ethyl acetate in sequence, concentrating it under reduced pressure using a rotary evaporator, and finally taking out the crude extract to obtain a petroleum ether extract, an ethyl acetate extract, and a water extract, respectively;

[0011] S03. Mixing methanol with the ethyl acetate extract to dissolve the ethyl acetate extract to obtain a solute, then filtering the solute using a Büchner funnel and filtration flask. Using a macroporous adsorption resin column, gradient elution with methanol-water according to a preset ratio is performed, with each gradient being 1.5 L, and each flask receiving 500 mL. Then, each gradient eluate is collected to obtain different fractions; wherein, the fractions eluted from the macroporous adsorption resin column are monitored using a thin layer chromatography plate, using 5%-10% concentrated ethanolic sulfuric acid as a color developer, and heated until color is developed. Fractions showing the same point on the silica gel plate are combined to obtain 8 fractions, which are respectively designated as Fr.1-Fr.8;

[0012] S04. Fr.1 was separated by MCI column, gradient elution was performed with methanol-water, each gradient eluate was collected, and the fractions of the same part were combined together after monitoring with silica gel plate to obtain 4 parts, named Fr.A-Fr.D; Fr.D was separated by MCI column, gradient elution was performed with methanol-water, each gradient eluate was collected, and the fractions of the same part were combined together after monitoring with silica gel plate to obtain 4 parts Fr.d1-Fr.d4. Fr.d2 was purified by MCI column, eluted with methanol-water in proportion, and the precipitate was filtered at normal pressure to obtain the compound, which is Baohuoside I.

[0013] As a preferred implementation option, preferably, in this scheme S03, when eluting with methanol-water according to a preset ratio gradient, the methanol ratio is gradually increased, and the ratio gradient is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

[0014] As a preferred implementation option, preferably, in this solution S04, when Fr.1 is separated on the MCI column, methanol-water is used for multiple elution at a gradient of 10%-100%.

[0015] As a preferred implementation option, preferably, in this solution S04, when Fr.D is separated using an MCI column, it is eluted multiple times with methanol-water at a gradient of 20%-70%.

[0016] Based on the above, this solution also proposes a drug for preventing and treating Cryptocaryon irritans disease, which includes Baohuoside I prepared by the above-mentioned preparation method.

[0017] Based on the above, this solution also proposes a medicine for aquaculture animals, which includes Baohuoside I prepared by the above-mentioned preparation method.

[0018] Traditional Chinese herbal medicines are widely used in aquaculture. They are highly valued by the aquaculture industry for their good efficacy, low residue, no drug resistance and environmental protection. They are a potential fishery medicine library for the prevention and treatment of "white spot disease" of marine fish. Epimedium brevicornu Maxim is a traditional Chinese herbal medicine. Its main active ingredient is flavonoids. Among them, epimedium A, epimedium B, epimedium C, icariin and icariin are the most representative and have a high content. Epimedium has the effects of tonifying kidney yang, strengthening muscles and bones, and removing rheumatism. It plays a good role in the treatment of osteoporosis, breast diseases and reproductive system diseases. Based on the application of existing Chinese herbal medicines in aquaculture, the present invention extracts the flavonoid compound brevicornuside I from epimedium. This scheme finds that brevicornuside I has the effect of resisting Cryptocaryon irritans through in vitro anti-insect experiments, providing a new, efficient and environmentally friendly way for the prevention and treatment of "white spot disease" of marine fish.

[0019] Adopting the above-mentioned technical scheme, the present invention has the beneficial effect compared with the prior art: this scheme discloses a Chinese medicine compound for preventing and treating Cryptocaryon irritans disease in marine fish, and proves that the baohuoside I extracted from the traditional Chinese herbal medicine Epimedium can effectively kill Cryptocaryon irritans, and low concentration of baohuoside I can quickly inhibit the growth and reproduction of Cryptocaryon irritans, reduce its infringement on farmed animals, and reduce the infection rate and mortality rate of aquaculture animals. From the perspective of environmental protection, compared with some chemical synthetic drugs, baohuoside I is derived from natural plants, and during use, no harmful substances that are difficult to degrade will remain in water bodies and farmed organisms, and no pollution will be caused to the breeding environment, which is conducive to maintaining ecological balance. In addition, Epimedium is widely distributed in my country and rich in resources, which makes the source of baohuoside I very extensive, provides a solid foundation for large-scale production and application, and can also reduce breeding costs to a certain extent and improve economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the preparation process of Baohuoside I in the embodiment of this scheme;

[0022] Figure 2 1 is a schematic diagram comparing the effects of Baohuoside I on Cryptocaryon irritans larvae in the embodiments of this solution;

[0023] Figure 3 Schematic diagram of the embodiment of this scheme, wherein the morphology of Cryptocaryon irritans larvae is observed under a stereomicroscope, Figure 3 a shows the normal larval morphology of Cryptocaryon irritans, with a complete body, a clearly visible large nucleus inside, and a body surface covered with cilia. The cell mouth can also be seen, as indicated by the arrow; Figure 3 b shows that after 10 minutes of treatment with 20 μg / mL Baohuoside I solution, the cilia of the insect body began to fall off and vacuolization occurred. Figure 3 c: After 30 min of treatment with 40 μg / mL Baohuoside I solution, the worm body became round, the cell membrane ruptured, the contents flowed out, and the cells began to lyse;

[0024] Figure 4 In the embodiment of this scheme, the half inhibitory concentration (IC 50 ) representation diagram;

[0025] Figure 5 This is a comparative diagram of the inhibitory effect of Baohuoside I on Cryptocaryon irritans cysts in the embodiments of this solution;

[0026] Figure 6 Schematic diagram of the morphology of Cryptocaryon irritans cysts observed under a stereomicroscope in the embodiment of this scheme; wherein, Figure 6 a. Figure 6 b. Figure 6 c is the hatching process of Cryptocaryon cysts under normal stimulation. Figure 6 a A complete cyst can be seen, and the material inside the cyst is uniform. Figure 6 b is the cyst hatching process, with beaded masses visible inside (as indicated by the arrows), and the cyst wall thickening. Figure 6 c is the state of cyst hatching, the larvae break out from the direction indicated by the arrow, and the remaining cyst is an empty shell; d, e, and f are the states of cyst hatching after 6 hours of treatment with 40 μg / mL Baohuoside I solution, the cyst development is blocked, the internal color becomes lighter, and it appears brown ( Figure 6 f), Figure 6 d shows the phenomenon of cyst wall separation, which also inhibits the cysts in the hatching process, causing the larvae to die and unable to break out of the wall ( Figure 6 e);

[0027] Figure 7 In the embodiment of this scheme, the half inhibitory concentration (IC50) of Baohuoside I for inhibiting the cyst hatching of Cryptocaryon spp. at 6 h of action is 50 ). DETAILED DESCRIPTION

[0028] The present invention will be described in further detail below with reference to the accompanying drawings and examples. It is particularly noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Similarly, the following examples are only some embodiments of the present invention and are not intended to be exhaustive. All other embodiments obtained by those of ordinary skill in the art without creative effort are intended to fall within the scope of protection of the present invention.

[0029] Example 1

[0030] Extraction, Preparation and Identification of Baohuoside I

[0031] 1.1 Extraction of flavonoids from Epimedium

[0032] 1.1.1 Preparation of Epimedium ethyl acetate extract

[0033] Combine Figure 1 As shown, the herb is dried in the sun and crushed in a grinder to obtain 1.5 kg of powder. It is then soaked in a 95% ethanol solution for three days, extracted once a day, and initially separated by ethanol reflux. The solid portion is retained and the ethanol is recovered. The three extracts are mixed and concentrated under reduced pressure to obtain a total of 366.5 g of extract. The initially separated herb ethanol extract is then diluted with an appropriate amount of water. The petroleum ether and ethyl acetate extracts are sequentially extracted and concentrated under reduced pressure using a rotary evaporator to obtain a crude ethyl acetate extract. The petroleum ether extract (34.4 g), the ethyl acetate extract (78 g), and the water extract (254.1 g) are obtained respectively.

[0034] 1.1.2 Separation of Epimedium ethyl acetate extract

[0035] Combine Figure 1As shown, a certain amount of methanol is added to the ethyl acetate portion of the ethanol extract of Epimedium to dissolve it, and filtered with a Buchner funnel and suction flask, and then applied to a macroporous adsorption resin column. The mixture is eluted with methanol-water in a certain proportion gradient (the proportion of methanol is gradually increased, which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), each gradient is washed with 1.5L, each bottle is filled with 500mL, and each gradient eluate is collected to obtain multiple fractions.

[0036] Fractions eluted from the macroporous adsorption resin column were monitored by thin-layer chromatography (TLC) using 5%-10% concentrated ethanolic sulfuric acid as a color developer. The fractions were heated until color developed. Fractions showing identical spots on the silica gel plate were then pooled together, yielding eight pools, designated Fr.1 through Fr.8. Based on the spot color development, the Fr.1 pool, containing the most prominent spots, was selected as the basis for further development of the protocol.

[0037] 1.1.3 Isolation and extraction of baohuoside I

[0038] The combined fractions of Fr.1 were separated on an MCI column and gradient eluted with methanol-water (10%-100% gradient, increasing by 10%). Each gradient eluate was collected and then monitored on a silica gel plate. Fractions showing identical spots on the plate were combined to obtain four fractions, designated Fr.A-Fr.D. Fr.D contained the most prominent color spot, so the combined fraction of Fr.D served as the basis for further development of the protocol.

[0039] The combined Fr.D fractions were separated using an MCI column and gradient eluted with methanol-water (20%-70% in 10% increments). Each gradient eluate was collected and monitored on a silica gel plate. Fractions showing identical spots on the plate were combined to yield four fractions, designated Fr.d1-Fr.d4. Fr.d2 contained the most prominent color spot, so the combined Fr.d2 fraction served as the basis for further development of the protocol.

[0040] The combined fractions of Fr.d2 were purified on an MCI column and gradient eluted with methanol-water (gradient ratio of 10%-100%, increasing by 10%). The gradient eluates were collected and monitored on a silica gel plate. The fractions showing the same prominent color spots on the silica gel plate were combined together and the precipitate was filtered at normal pressure to obtain compound 1 (23.6 mg).

[0041] 1.2 Identification of compound 1

[0042] 1.2.1 Structural determination and identification of flavonoids

[0043] Compound 1 (yellow powder) was isolated from the extract, and its chemical composition was studied and identified using column chromatography and modern spectroscopic techniques.

[0044] After component analysis, the molecular formula of compound 1 was obtained: C 27 H 30 O 10 The identification data is: 1 H-NMR (MeOD, 600MHz) δppm: 12.39 (1H, s, HO-5), 7.73 (2H, d, J=8.8.Hz, H-2' / 6'), 6.91 (2H, d, J=8.8.Hz, H-3' / 5'), 6.17 (1H, s, H-6), 5.08 (1H, t, J=6.6.Hz, H-12), 3.62 (1H, m, Ha-11), 3.36 (1H, m, Hb-11), 3.75 (3H, s, MeO-4'), 1.69 (3H, s, H-14), 1.54 (3H, s, H- 15), 5.26 (1H, dd, J = 1.2Hz, H-1”), 3.10-3.68 (H-2”-5”), 0.79 (3H, d, J = 5.9Hz, H-6”). 13 C-NMR (MeOD, 150MHz) δppm: 154.0 (C-2), 129.6 (C-3), 177.6 (C-4), 161.2 (C-5), 102.9 (C-6), 161.1(C-7),107.5(C-8),157.0(C-9),105.7(C-10),24.1(C-11),122.7(C-12),131.6(C-13) ,27.9(C-14),20.3(C-15),123.1(C-1'),130.7(C-2' / 6'),158.6(C-4'),114.4(C-3' / 5'),5 7.0(MeOD-4’), 99.6(C-1”), 72.0(C-2”), 72.1(C-3”), 72.5(C-4”), 72.0(C-5”), 19.5(C-6”).

[0045] Based on the above data, which were consistent with the data reported in the literature, compound 1 was determined to be baohuoside I (reference: Zhao YD, Chen SF, Wang YD, et al. Effect of drying processes on prenylflavonoid content and antioxidant activity of Epimedium koreanum Nakai [J]. Journal of Food and Drug Analysis, 2017, 1-11.). The specific structure is shown in Formula 1 below:

[0046]

[0047] 1.3 Anti-Cryptocercariae larvae test

[0048] Incubation of Cryptocaryon irritans: Place morphologically normal Cryptocaryon irritans cysts in seawater and incubate in a 26°C water bath. When a large number of larvae hatch in the water, take 100 μL and count them under a microscope. Concentrate to 1000-1500 larvae / mL for later use.

[0049] Sample preparation: Compound 1 (hereinafter referred to as Baohuoside I) was dissolved in methanol solution, and the drug solution concentrations were prepared in sequence to 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL, and stored in a refrigerator at 4°C. The drug pH was 6-7.

[0050] Take 1 μL of sample solution and 99 μL of the above-mentioned Cryptocaryon irritans suspension and incubate them in a 96-well plate. Methanol solution and seawater are used as controls. Three parallels are set for each concentration gradient. The cells are counted under a microscope every 30 minutes and the number of dead Cryptocaryon irritans in each well is recorded. The results are shown in the table. Figure 2 shown.

[0051] Figure 2 Figure 2 shows the incubation of Cryptocaryon irritans larvae with different concentrations of Baohuoside I for 0, 30, 60, 90, and 120 minutes, followed by observation of larval mortality using a stereomicroscope. (n.s.) indicates no significant difference in larval mortality compared to the seawater control group; *p < 0.05, **p < 0.01, ***p < 0.001, with seawater as the control.

[0052] Depend on Figure 2It can be seen that compared with the seawater control, 1.25μg / mL, 2.5μg / mL, and 5μg / mL of Baohuoside I did not cause significant death in Cryptocaryon larvae within 120 minutes of stimulation, and only the movement speed of some larvae decreased; 20μg / mL and 40μg / mL of Baohuoside I caused significant death in Cryptocaryon larvae after 30 minutes of stimulation. Under the action of the drug, the movement of the larvae slowed down, became round, and finally the cell membrane ruptured and the contents flowed out (results see Figure 3 ), the larval mortality rate reached 100% within 60 minutes, indicating that Baohuoside I has the effect of killing Cryptocaryon irritans larvae at a concentration of 10 μg / mL and above, and the insecticidal effect is dose- and time-dependent. Its IC 50 was 7.361 μg / mL (see Figure 4 ).

[0053] Figure 3 middle, Figure 3 a shows the normal larval morphology of Cryptocaryon irritans, with a complete body, a clearly visible large nucleus inside, and a body surface covered with cilia. The cell mouth can also be seen, as indicated by the arrow; Figure 3 b shows that after 10 minutes of exposure to 20 μg / mL Baohuoside I solution, the worm began to lose its cilia and vacuolize. Figure 3 c: After 30 min of treatment with 40 μg / mL Baohuoside I solution, the worm body became round, the cell membrane ruptured, the contents flowed out, and the cells began to lyse.

[0054] 1.4 Anti-Cryptocercariae cyst test

[0055] Sample preparation: Baohuoside I was diluted with methanol solution, and the concentration of the solution was set to 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, and 16 mg / mL. The solution was stored in a refrigerator at 4°C, and the pH of the drug was 6-7.

[0056] 990 μL of fresh seawater containing about 20 cysts was added to each well of a 24-well microplate, and then 10 μL of Baohuoside I solution was added to each well to make the final concentrations of the compound reach 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL, respectively. Methanol solution and seawater with the same highest concentration of the compound were used as negative controls. After 6 hours of action, the original solution in each well was replaced with fresh, filtered, and sterilized seawater. The plates were then placed in a 28°C incubator for 7 days, with 1 mL of fresh, filtered, and sterilized seawater replaced every morning and evening. The cyst hatching status was recorded, and the results are shown in the table. Figure 5 .

[0057] Figure 5Cryptocaryon irritans cysts were incubated with different concentrations of Baohuoside I for 6 hours, and cyst hatching was observed using a stereomicroscope. ns indicates no significant difference in cyst hatching rate compared with the seawater control group; *p < 0.05, **p < 0.01, ***p < 0.001, with seawater as the control.

[0058] Depend on Figure 5 It can be seen that after 6 hours of treatment with different concentrations of Baohuoside I, the cyst hatching rate of Cryptocaryon cysts was stimulated. Among them, under the action of 10μg / mL Baohuoside I, there was no significant difference in the cyst hatching rate compared with the seawater group, indicating that this concentration did not inhibit the hatching of Cryptocaryon cysts; under the action of 20μg / mL Baohuoside I, the cyst hatching rate was significantly lower than that of the seawater group; under the action of 40, 80, and 160μg / mL Baohuoside I, the hatching rate was extremely significantly lower than that of the seawater group. Cyst wall separation occurred in the late stage of cyst incubation, and the cysts in the hatching process were inhibited by the drug, and the larvae in the cysts died and could not break through the wall ( Figure 6 ), indicating that 20 μg / mL and above Baohuoside I can significantly inhibit the hatching of Cryptocaryon cysts for 6 h, and its IC 50 was 24.98 μg / mL (see Figure 7 ).

[0059] Figure 6 middle, Figure 6 a. Figure 6 b. Figure 6 c is the hatching process of Cryptocaryon cysts under normal stimulation. Figure 6 a shows a complete cyst with uniform internal material. b shows the cyst hatching process with beaded masses (as indicated by arrows) visible inside and thickened cyst walls. Figure 6 c is the state where the cyst is hatched, the larvae break out from the direction indicated by the arrow, and the remaining cyst is an empty shell; Figure 6 d. Figure 6 e. Figure 6 Figure f is the hatching state of the cysts after 6 hours of treatment with 40 μg / mL Baohuoside I solution. The cyst development is blocked and the internal color becomes lighter and brown (Figure f). Figure 6 d shows the phenomenon of cyst wall separation, which also inhibits the cysts in the process of hatching, causing the larvae to die and unable to break out of the wall (see Figure 6 e).

[0060] The above descriptions are only some embodiments of the present invention and do not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. The use of Baohuoside I in the preparation of a drug for preventing and treating Cryptocaryon irritans disease, characterized in that: The chemical structural formula of the Baohuoside I is shown in Formula 1:

Images