Application of baohuoside I in prevention and treatment of cryptocaryon irritans disease

Abstract

The invention discloses an application of baohuoside I in prevention and treatment of cryptocaryon irritans, which proves that baohuoside I extracted from traditional Chinese herbal medicine herba epimedii can effectively kill cryptocaryon irritans, and low-concentration baohuoside I can quickly inhibit the growth and reproduction of cryptocaryon irritans and reduce the infringement of cryptocaryon irritans to bred animals. The infection rate and the death rate of aquaculture animals are reduced. From the perspective of environmental protection, compared with some chemical synthetic drugs, baohuoside I is derived from a natural plant, harmful substances which are difficult to degrade are not left in a water body and a cultured organism in the use process, the culture environment is not polluted, and the ecological balance is favorably maintained. Besides, herba epimedii is widely distributed in China and is rich in resource, so that the baohuoside I is very wide in source, a solid foundation is provided for large-scale production and application, the breeding cost can be reduced to a certain extent, and the economic benefit can be improved to a certain extent.

Description

Technical Field

[0001] The present invention relates to the application technology of Chinese medicine extracts and the field of 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] Marine fish white spot disease is a parasitic disease caused by Cryptocaryon irritans that is prevalent worldwide. The disease often occurs from May to October, concentrated in summer and autumn. The outbreak of marine fish white spot disease mainly depends on the water temperature. Cryptocaryon irritans can infect fish in water bodies of 22-32℃, especially in large areas of fish at a water temperature of 25-27℃. The larvae of Cryptocaryon irritans can swim continuously in the water to find a host. After infecting the host, they develop into trophozoites, ingest host cells and tissue fluid. The epidermal tissue of the fish proliferates due to the stimulation of the trophozoites, and then wraps the worm body, forming "white spots" visible on the surface of the fish. The trophozoites fall off from the host and develop into swimming cyst precursors. When they attach to the bottom, they develop into cysts. The cysts proliferate and release the larvae to enter a new life cycle. Because the fish epidermis wraps the trophozoites in the tissue, the drugs cannot directly penetrate the epidermal tissue to act on the trophozoites by immersion, which increases the difficulty of prevention and control. 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] At present, the prevention and treatment of Cryptocaryon irritans disease is mainly divided into physical methods and chemical methods. The physical methods mainly include freshwater immersion, heat treatment, conversion breeding, ultraviolet irradiation, drying and ozone treatment. These methods are complicated to operate and the prevention and treatment effects of Cryptocaryon irritans are not ideal. The chemical method mainly uses drugs to soak sick fish or worms to achieve the therapeutic effect. Commonly used drugs include potassium permanganate, copper sulfate, formalin, etc. In addition, biological dyes, formalin, acriflavine, methylene blue, sodium hypochlorite, sulfathiazole chloride, penicillin, benzalkonium chloride, etc. have also been proven to have inhibitory effects on Cryptocaryon irritans. Although the chemical method is effective, it is not suitable for long-term use, which will cause the worm to develop drug resistance, and some drugs will cause irreversible harm to the environment. Therefore, it is necessary to develop an efficient and environmentally friendly prevention and control measure to curb the spread of pathogenic worms, reduce economic losses in the aquaculture industry, and promote the development of the marine aquaculture industry. 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 the prevention and treatment of 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 structural formula of the Baohuoside I is shown in Formula 1:

[0007]

[0008] Based on the above, this scheme 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 for reduced pressure concentration 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 turn, 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 partial extract to dissolve the ethyl acetate partial extract to obtain a solute, and then filtering the solute with a Buchner funnel suction bottle, using a macroporous adsorption resin column, using methanol-water gradient elution according to a preset ratio, each gradient flushing 1.5L, each bottle receiving 500mL, and then collecting each gradient eluate to obtain different fractions; wherein, the fraction eluted from the macroporous adsorption resin column is monitored by a thin layer chromatography plate, using 5%-10% concentrated sulfuric acid ethanol as a color developer, heating until color is developed, and the fractions showing the same point on the silica gel plate are combined together to obtain 8 parts, which are named Fr.1-Fr.8 respectively;

[0012] S04. Separate Fr.1 by MCI column, perform gradient elution with methanol-water, collect each gradient eluate, monitor through silica gel plate, combine the fractions of the same part together to obtain 4 parts, named Fr.A-Fr.D; separate Fr.D by MCI column, perform gradient elution with methanol-water, collect each gradient eluate, monitor through silica gel plate, combine the fractions of the same part together to obtain 4 parts Fr.d1-Fr.d4, purify Fr.d2 by MCI column, elute with methanol-water in proportion, filter and precipitate 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 gradient ratio, the methanol ratio is gradually increased, and the gradient ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

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

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

[0016] Based on the above, the present scheme 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, the present scheme 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 potential fishery medicines for the prevention and treatment of "white spot disease" in marine fish. Epimedium brevicornu Maxim is a traditional Chinese herbal medicine. Its main active ingredients are flavonoid components, among which 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, and plays a good role in the treatment of osteoporosis, breast diseases and reproductive system diseases. The present invention is based on the application of existing Chinese herbal medicines in aquaculture, and extracts the flavonoid compound baohuoside I from epimedium. This scheme uses in vitro anti-insect experiments to find that baohuoside I has the effect of resisting irritant cryptonucleus, which provides a new way of efficient and environmentally friendly prevention and treatment of "white spot disease" in marine fish.

[0019] By adopting the above-mentioned technical scheme, the present invention has the beneficial effect compared with the prior art: the present scheme discloses a Chinese medicine compound for preventing and treating Cryptocaryon irritans of 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 no harmful substances that are difficult to degrade will remain in the water body and farmed organisms during use, 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, providing 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

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

[0022] Figure 2 This is a schematic diagram of the comparison of the effects of Baohuoside I on Cryptocaryon irritans larvae in the embodiments of this scheme;

[0023] Figure 3 Schematic diagram of observing the morphology of Cryptocaryon irritans larvae under a stereomicroscope in the embodiment of the present scheme, wherein: Figure 3 a is the normal larval morphology of Cryptocaryon irritans, with a complete body, a large nucleus clearly visible inside, cilia on the body surface, and a cell mouth visible in it, as indicated by the arrow; Figure 3 b shows that after 10 min of exposure to 20 μg / mL Baohuoside I solution, the cilia of the insect body began to fall off and cavitation occurred. Figure 3 c is that after 30 min of exposure to 40 μg / mL Baohuoside I solution, the insect 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 ) characterization diagram;

[0025] Figure 5 This is a comparison chart of the inhibitory effect of Baohuoside I on Cryptocaryon irritans cysts in the embodiment of this scheme;

[0026] Figure 6 This is a 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 process of cyst hatching, 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 hatching states of cysts 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 cyst wall separation phenomenon, which will also inhibit the cysts in the hatching process, causing the larvae to die and unable to break through the wall ( Figure 6 e);

[0027] Figure 7 is the half inhibitory concentration (IC) of Baohuoside I in the embodiment of this scheme for inhibiting the hatching of Cryptocaryon cysts at 6h. 50 ). DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are 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] Combination Figure 1 As shown, the epimedium is dried and crushed with a pulverizer to obtain 1.5kg of powder, which is soaked in 95% ethanol solution for three days, extracted once a day, and initially separated by ethanol reflux method, and the solid part is retained, and ethanol is recovered; the extracts obtained three times are mixed and concentrated under reduced pressure to obtain a total of 366.5g of extract. After that, the preliminarily separated epimedium ethanol extract is diluted with an appropriate amount of water. The petroleum ether and ethyl acetate extracts are extracted in turn, concentrated under reduced pressure with a rotary evaporator, and the ethyl acetate extract crude product is taken out. The petroleum ether partial extract (34.4g) and the ethyl acetate partial extract (78g) and the water partial extract (254.1g) are obtained respectively.

[0034] 1.1.2 Separation of Epimedium ethyl acetate extract

[0035] Combination Figure 1As shown, a certain amount of methanol is added to the ethyl acetate part of the ethanol extract of Epimedium to dissolve it, and filtered with a Buchner funnel and suction bottle, and then placed on a macroporous adsorption resin column, and 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] The fractions eluted from the macroporous adsorption resin column were monitored by thin layer chromatography (TLC), using 5%-10% concentrated sulfuric acid ethanol as a color developer, heated until color was developed, and then the fractions showing the same spots on the silica gel plate were combined together to finally obtain 8 combined parts, named Fr.1-Fr.8. Based on the spot color development, the Fr.1 combined fraction containing the most prominent color spots was selected as the basis for further development of the plan in the subsequent steps.

[0037] 1.1.3 Isolation and extraction of baohuoside I

[0038] The combined fraction of Fr.1 was separated on an MCI column and gradient eluted with methanol-water (gradient ratio of 10%-100%, increasing by 10%), and each gradient eluate was collected and monitored on a silica gel plate. The fractions showing the same spots on the silica gel plate were combined together to obtain 4 parts, named Fr.A-Fr.D. Among them, Fr.D contained the most prominent color spots, so the combined fraction of Fr.D was used as the basis for further development of the plan in the subsequent steps.

[0039] The combined fraction of Fr.D was separated by MCI column, and gradient eluted with methanol-water (gradient ratio of 20%-70%, increasing by 10%), and each gradient eluate was collected and monitored by silica gel plate. The fractions showing the same spots on the silica gel plate were combined together to obtain 4 parts, named Fr.d1-Fr.d4. Among them, Fr.d2 contained the most prominent color spot, so the combined fraction of Fr.d2 was used as the basis for the subsequent steps to further develop the plan.

[0040] The combined fractions of Fr.d2 were purified on an MCI column with gradient elution using methanol-water (gradient ratio of 10%-100%, with a gradient increase of 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 spectroscopy techniques.

[0044] After component analysis, the molecular formula of compound 1 was obtained: C 27 H 30 O 10 . The identification data are: 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 being 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.), and the specific structure is shown in Formula 1 below:

[0046]

[0047] 1.3 Experiment on anti-Cryptocercariasis larvae

[0048] Incubation of Cryptocaryon irritans: Place normal morphological Cryptocaryon irritans cysts in seawater and incubate them in a 26°C water bath. When a large number of larvae hatch out of 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 concentrations of the solutions were prepared in sequence to be 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 In the figure, different concentrations of Baohuoside I were co-incubated with Cryptocaryon irritans larvae for 0, 30, 60, 90, and 120 min, and then the larval mortality was observed under a stereomicroscope. Among them, ns means there is no significant difference in larval mortality compared with 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 the larvae of Cryptocaryon larvae within 120 minutes of stimulation, and only the movement speed of some larvae decreased; 20μg / mL and 40μg / mL of Baohuoside I stimulated Cryptocaryon larvae for 30 minutes and caused significant death of the larvae. Under the action of the drug, the larvae moved slower and became round, and finally the cell membrane ruptured and the contents flowed out (results in Figure 3 ), the larval mortality rate reached 100% within 60 min, 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 7.361 μg / mL (see Figure 4 ).

[0053] Figure 3 middle, Figure 3 a is the normal larval morphology of Cryptocaryon irritans, with a complete body, a large nucleus clearly visible inside, cilia on the body surface, and a cell mouth visible in it, as indicated by the arrow; Figure 3 b: After 10 minutes of exposure to 20 μg / mL Baohuoside I solution, the cilia of the insect body began to fall off and vacuolization occurred. Figure 3 c: After being treated with 40 μg / mL Baohuoside I solution for 30 min, the insect body became round, the cell membrane ruptured, the contents flowed out, and the cells began to lyse.

[0054] 1.4 Anti-Cryptocercariasis 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. It was stored in a refrigerator at 4°C, and the drug pH 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, fresh filtered and sterilized seawater was used to replace the original solution in each well. Then, the plates were placed in a 28°C incubator and cultured for 7 days. 1 mL of fresh filtered and sterilized seawater was replaced every morning and evening. The cyst hatching was recorded. The results are shown in the table. Figure 5 .

[0057] Figure 5Different concentrations of Baohuoside I were co-incubated with Cryptocaryon irritans cysts for 6 h, and the cyst hatching was observed under 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 the hatching of Cryptocaryon cysts was stimulated by different concentrations of Baohuoside I for 6 hours. Under the action of 10μg / mL Baohuoside I, the cyst hatching rate was not significantly different from that of the seawater group, indicating that this concentration would not inhibit the hatching of Cryptocaryon cysts; while under the action of 20μg / mL Baohuoside I, the cyst hatching rate was significantly lower than that of the seawater group, and under the action of 40, 80, and 160μg / mL Baohuoside I, the hatching rate was extremely significantly lower than that of the seawater group. The cyst wall separation phenomenon 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 out of the wall ( Figure 6 ), indicating that 20 μg / mL and above of Baohuoside I can significantly inhibit the hatching of Cryptocaryon irritans cysts for 6 h, and its IC 50 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 the arrow) and thickened cyst wall. 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 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 cyst wall separation phenomenon, which will also inhibit 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 are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, 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:

2. A method for preparing Baohuoside I, characterized in that: It includes the following steps: S01, extracting and separating the epimedium powder N times by an ethanol reflux method, and then collecting the extracts for reduced pressure concentration to obtain an extract, i.e., a preliminarily separated epimedium ethanol extract; S02, diluting the extract with water, then extracting it with petroleum ether and ethyl acetate in turn, 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; S03, mixing methanol with the ethyl acetate partial extract to dissolve the ethyl acetate partial extract to obtain a solute, and then filtering the solute with a Buchner funnel suction bottle, using a macroporous adsorption resin column, using methanol-water gradient elution according to a preset ratio, each gradient flushing 1.5L, each bottle receiving 500mL, and then collecting each gradient eluate to obtain different fractions; wherein, the fraction eluted from the macroporous adsorption resin column is monitored by a thin layer chromatography plate, using 5%-10% concentrated sulfuric acid ethanol as a color developer, heating until color is developed, and the fractions showing the same point on the silica gel plate are combined together to obtain 8 parts, which are named Fr.1-Fr.8 respectively; S04. Separate Fr.1 by MCI column, perform gradient elution with methanol-water, collect each gradient eluate, monitor through silica gel plate, combine the fractions of the same part together to obtain 4 parts, named Fr.A-Fr.D; separate Fr.D by MCI column, perform gradient elution with methanol-water, collect each gradient eluate, monitor through silica gel plate, combine the fractions of the same part together to obtain 4 parts Fr.d1-Fr.d4, purify Fr.d2 by MCI column, elute with methanol-water in proportion, filter and precipitate at normal pressure to obtain the compound, which is Baohuoside I.

3. The method for preparing Baohuoside I according to claim 2, characterized in that: In S03, when eluting with methanol-water according to a preset gradient ratio, the methanol ratio is gradually increased, and the gradient ratio is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

4. The method for preparing Baohuoside I according to claim 2, characterized in that: In S04, when Fr.1 was separated on an MCI column, it was eluted multiple times with methanol-water at a gradient of 10%-100%.

5. The method for preparing Baohuoside I according to claim 2, characterized in that: In S04, when Fr.D was separated using an MCI column, it was eluted multiple times with methanol-water at a gradient of 20%-70%.

6. A drug for preventing and treating Cryptocaryon irritans disease, characterized in that: It comprises the baohuoside I prepared by the preparation method according to any one of claims 2 to 5.

7. A drug for aquaculture animals, characterized in that: It comprises the baohuoside I prepared by the preparation method according to any one of claims 2 to 5.

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