Discovery and application of anti-ciliate active flavone molecule in fructus psoraleae

Abstract

The invention relates to discovery and application of anti-ciliate active flavone molecules in a natural product, in particular to discovery and application of three anti-ciliate flavone compounds in a natural product fructus psoraleae, and the anti-ciliate flavone compounds are neopsoralen isoflavone, psoralen isoflavone A and psoralen B. The invention further relates to a preparation method of the anti-ciliate active flavone molecules. In-vitro insecticidal experiments show that the compound provided by the invention has an insecticidal effect on a model organism tetrahymena piriforme. The current research shows that ciliates appear in the breeding process. When the fish body is injured, the water environment is deteriorated and the like, the cultured fish is infected to a certain degree. When the ciliates are seriously infected, a large amount of ciliates can die, and huge economic loss is caused. Therefore, the anti-ciliate flavonoid compound provided by the invention provides a candidate compound for the development of ciliate drugs.

Description

Technical Field

[0001] The present invention relates to the application of anti-ciliate active molecules in natural products, specifically the application of five anti-ciliate flavonoid compounds in the natural product psoralea corylifolia, wherein the anti-ciliate flavonoid compounds are neopsoralen isoflavone, psoralen isoflavone A and psoralen B. The application mainly relates to aquaculture ciliate disease. Background Art

[0002] Psoralea corylifolia is the dried mature fruit of the genus Psoralea in the leguminous family. It is recorded in ancient books such as "Yao Xing Lun", "Rihuazi Bencao", "Kaibao Bencao", "Compendium of Materia Medica", and "Yuzhu Yaojie". It is pungent, bitter, and warm in nature. It enters the kidney and spleen meridians, and has the effects of warming the kidney and assisting yang, nourishing the kidney and strengthening bones. It is clinically used to treat osteoporosis, fractures, osteoarthritis and other diseases (Dilihuma Dilimulati et al. Research progress on the safe application of Psoralea corylifolia based on the evolution of toxicity / effect records [J]. Chinese Pharmacovigilance: 2023, 1-16). Modern studies have shown that Psoralea corylifolia has anti-tumor, antioxidant, antibacterial and anti-inflammatory pharmacological activities, which are all related to its chemical components (Lu, Y., Zhang, X., Wang, J., et al. Research Progress on Chemical Constituents and Pharmacological Actions of Psoralea Fructus. Chinese Journal of Experimental Traditional Medical Formulae. 2019, 25, 180-189.). The chemical components reported in Psoralea corylifolia mainly include coumarins, benzofurans, flavonoids and monoterpene phenols (Tan Wei. Research on Chemical Composition and Pharmacological Actions of Psoralea corylifolia [D]. Donghua University, 2017.). Although Psoralea corylifolia has good pharmacological activity, its anti-ciliate components and the mechanism of action of the active components have not been reported.

[0003] Ciliates are a type of pathogenic parasite that is harmful to farmed fish. They are facultative parasites. For example, the more harmful Tetrahymena can live freely in freshwater and seawater, but in some special circumstances, such as excessive stocking density, damage to the body surface of farmed fish, eutrophication of farmed water, etc., they can cause a certain degree of infection to farmed animals (Li Wenyu. Identification of two parasitic Tetrahymena species in ornamental fish and research on their infection mechanism [D]. Harbin Normal University). After the ciliates infect and invade the fish body, they will multiply in large numbers and stimulate the host fish's epidermis to produce a large amount of mucus. Fish infected with ciliates swim slowly and eat less. The symptoms on the body surface are usually manifested as darkening of the body surface and then swelling and redness, necrotic ulcers on the skin and subcutaneous muscles, and a large number of ciliates can be seen by microscopic examination of the mucus scraped from the surface lesions; in severe cases, it can cause systemic infection of ciliates, and the presence of ciliates can be observed in blood and internal organs by microscopic examination. Ciliates usually like to gather in areas rich in bacterial flora, so sick fish often show symptoms of bacterial infection (Ma Lidan, Wang Dianfu, Yu Xiaojie, etc. Marine cercariasis cases in farmed pufferfish [J]. Chinese Journal of Veterinary Medicine, 2012, 48 (3): 69-70.). At present, the commonly used therapeutic drugs for various types of aquatic ciliates are zinc sulfate, copper sulfate and its compound drugs, potassium permanganate, formalin, glutaraldehyde, surfactants, etc. (Rintamaki-Kinnunen, P., Rahkonen, et al. Treatment of ichthyophthiriasis after malachite green. I. Concrete tanks at salmonidfarms. Diseases of Aquatic Organisms. 2005, 64, 69-76.). However, the frequent use of these drugs brings threats such as anthelmintic resistance, residual risk, environmental pollution, and toxicity to the host.

[0004] Some Chinese herbal extracts have good killing effects on fish protozoan parasites, and some reports have been made on their application in the prevention and treatment of aquatic ciliates (Carvalho, A., Domingues, I., Carvalho, C., Silva, AMS, Soares, AMVM, Marques, CR, 2023. In vitro antiprotozoal activity of hibiscus sabdariffa extract against a ciliate causing high mortalities in turbotaquaculture. Biology-Basel. 12, 912.). Based on the traditional efficacy of the Chinese medicine psoralea corylifolia, this patent will search for new anti-ciliate active molecules from it, laying the foundation for the development of safe and efficient anti-ciliate fishery drugs.

[0005] We prepared the EtOAC extract of Psoralea corylifolia by liquid chromatography and combined it with antiparasitic screening to obtain 6 active fractions, which were then analyzed by HPLC-tandem high-resolution mass spectrometry to deduce insecticidal active molecules. Finally, 4 flavonoids with inhibitory activity against Tetrahymena pearformis were obtained. Currently, the anti-ciliate active molecule activity of this type of component has not been reported. Summary of the invention

[0006] The present invention relates to the application of anti-ciliate active molecules in natural products. One of the purposes is to provide compounds with anti-ciliate activity in natural products, including psoralen, neopsoralen isoflavone, psoralen isoflavone A and psoralen dihydroflavone methyl ether; and also includes pharmaceutically acceptable carriers or excipients. The second purpose is to provide the application of such compounds in the field of aquaculture, involving the treatment of aquatic ciliate diseases.

[0007] In vitro insecticidal experiments show that the natural compounds in the present invention have a killing effect on ciliates. Current studies have shown that ciliates appear in the breeding process. When the fish body is injured or the water environment deteriorates, the farmed fish will be infected to a certain extent. Severe infection will cause a large number of deaths, resulting in huge economic losses. Based on this, the anti-ciliate flavonoid compounds mentioned in the present invention provide candidate compounds for the development of fishery drugs targeting ciliates. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Screening of 22 Chinese herbal medicine extracts for antiparasitic effects in vitro

[0009] Figure 2 A-Normal phase preparative chromatogram of ethyl acetate layer of Psoralea corylifolia;

[0010] B- minimum insecticidal concentration of fraction against Tetrahymena pyriformis;

[0011] Figure 3 A- Three-dimensional normal phase analysis chromatogram of active fractions F3, F4, F5, F6, F10, F11 and F12;

[0012] B- Three-dimensional reverse phase analysis chromatogram of active fractions F3, F4, F5, F6, F10, F11 and F12;

[0013] Figure 4 Total ion chromatograms of reversed-phase liquid chromatography-tandem high-resolution mass spectrometry of active fractions F3, F4, F5, F6, F10, F11, and F12;

[0014] Figure 5 High-resolution mass spectrometric identification of compounds in active fractions;

[0015] Figure 6 The in vitro insecticidal effects of psoralen, psoralen, neopsoralen and psoralen A against Tetrahymena pyriformis;

[0016] Figure 7 Acute toxicity of psoralen to guppies after 24 and 48 h;

[0017] Figure 8 After 48h, the toxic dose curve of psoralen to guppies;

[0018] Fig. 9 After 24 h of exposure, psoralen had an antiparasitic effect on Tetrahymena pyriformis in vivo;

[0019] Fig.10 Chemical structures of psoralen, neopsoralen, psoralen A and psoralen flavonoid methyl ether;

[0020] Fig.11 Compounds having a C6-C3-C6 structure; DETAILED DESCRIPTION

[0021] The present invention is further described with reference to examples, which are intended to illustrate the present invention but not to limit the present invention.

[0022] Example 1: In vitro antiparasitic screening of 22 Chinese herbal medicine extracts

[0023] Materials: 22 kinds of Chinese medicinal materials were purchased from Yichuntang Pharmacy in Dalian, Liaoning Province, and stored in Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University. Ethyl acetate, ethanol, isopropanol, and dichloromethane were purchased from Komiou Company.

[0024] The screened Chinese herbal medicine extracts include ethyl acetate extracts and water extracts. The ethyl acetate extracts were extracted by Soxhlet extraction. 5g of each Chinese herbal medicine was crushed, wrapped with filter paper, placed in a sealed jar, and soaked in ethyl acetate for 4 hours. The ethyl acetate soaking solution was placed in a Soxhlet extractor and refluxed at 90°C for extraction. The reflux extraction process was repeated five times. The obtained solution was concentrated to 5×10 5 mg / L The ethyl acetate extracts of 22 kinds of Chinese medicine were first dissolved in ethanol and then diluted with aerated tap water. The water extracts of 22 kinds of Chinese medicine were directly diluted with tap water. Approximately 1×10 4 1000 Trypanosoma pyriformis were exposed to ethyl acetate extracts of plants at concentrations of 5000, 2500, 1000, 800, 600, 400, 200, 100, 50 and 0 (negative control) mg / L. The water extract of each plant was diluted to 5000, 2500, 1000 and 500 mg / L. After incubation at 20.0-21.0℃ for 3h, Tetrahymena pyriformis lost its ability to move, had abnormal morphology and died. The extracts with the highest antiparasitic activity were selected for further experiments. Observation and photography were performed under 10x, 20x and 40x magnification. All experiments were repeated three times.

[0025] The in vitro antiparasitic effects of ethyl acetate and water extracts of 22 traditional Chinese medicines on Tetrahymena pyriformis were determined. Figure 1 The antiparasitic effects of most ethyl acetate extracts were significantly better than those of water extracts. Among them, the antiparasitic effect of ethyl acetate extract of Psoralea corylifolia was the best, with the lowest concentration of 100 mg / L within 3 hours. The antiparasitic effects of ethyl acetate extracts of Melia azedarach and Hammerwort at 600 and 800 mg / L, respectively, were second. On this basis, ethyl acetate extract of Psoralea corylifolia was selected for further study.

[0026] Example 2: Preparation of Psoralea corylifolia ethyl acetate layer by normal phase chromatography and screening of insecticidal activity of fractions

[0027] Materials: Psoralea corylifolia was purchased from Yichuntang Pharmacy in Dalian, Liaoning Province, and stored in the Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University.

[0028] 1kg of Psoralea corylifolia medicinal material was extracted with ethyl acetate, and the extract was added to a distillation flask for distillation. The distilled ethyl acetate was recycled for the extraction of Psoralea corylifolia. After repeated extraction and distillation, an asphalt-like Psoralea corylifolia extract was obtained, followed by a normal phase liquid chromatography column: NH2 (10×250mm, 10μm). Mobile phase A was propanol, and mobile phase B was dichloromethane. The gradient elution was set as: 0min (0% B)-10min (40% B)-20min (60% B)-30min (100% B), with a flow rate of 3.3mL / min. The chromatogram was recorded at 280nm. The column temperature was maintained at 30°C. The Psoralea corylifolia ethyl acetate extract was dissolved to 210mg / mL with a dichloromethane solution. The injection volume was 70μL, and the solid content was about 15mg per injection. Figure 2 A is the chromatogram when the sample load is 15 mg, and a total of 915 mg of sample was prepared. 12 fractions were collected by peak and vacuum dried for later use.

[0029] Ciliate culture: A strain of Tetrahymena pyriformis was isolated from a guppy in the Anshan ornamental fish market and cultured in guppy broth medium, which was obtained by filtering the liquid from the cooked guppy meat residue.

[0030] Add a small amount of anhydrous ethanol to each of the 12 fractions and dissolve them by ultrasonication. Place approximately 1×10 4 Each fraction was diluted with saline to 60, 50, 40, 30, 20, 10 and 0 mg / L (negative control) and added to a 24-well plate for observation. The 100% lethal concentration of parasites within 2 hours was recorded. Figure 2 In B, F4, F5 and F10 had the best insecticidal effect, with the lowest insecticidal concentration within 2 hours being 10 mg / L, and the minimum insecticidal concentrations of F3, F6, F11 and F12 were all below 40 mg / L. It can be inferred that these seven fractions contain components that inhibit ciliates.

[0031] Example 3: Active fraction separation and analysis

[0032] Materials: NH2 (4.6×250mm, 10μm) was used for normal phase analysis. Mobile phase A was isopropanol and mobile phase B was dichloromethane. Flow rate was 0.7mL / min. Chromatograms were recorded at 254nm and 280nm. In both systems, the column temperature was maintained at 30℃. The ethanol dissolution concentration was 5-50mg / mL and the injection volume was 10μL.

[0033] Reverse phase liquid chromatography (RPLC) analysis was performed on C18HCE (4.6×250 mm, 7 μm). Mobile phase A was acetonitrile and mobile phase B was 0.1% FA (v / v) in water. The gradient elution program was set as: 0 min (0% B)-15 min (50% B)-30 min (95% B). The flow rate for both NPLC and RPLC analysis was 0.7 mL / min. Chromatograms were recorded at 280 nm. The column temperature was maintained at 30°C. Each component was dissolved in ethanol at a concentration of 5-50 mg / mL. The injection volume of each component was 10 μL.

[0034] In order to track the active ingredients, the active components were further separated and analyzed. Since the normal phase × reversed phase separation system has good orthogonality and the resolution of reversed phase chromatography is better than other separation methods, reversed phase chromatography was used to separate and screen the active components. Figure 3 A and Figure 3 As can be seen in B, there are fewer chromatographic peaks in normal phase mode, and more chromatographic peaks in reverse phase mode, and the distribution is more uniform. Many trace components are displayed, and the peak shape is better than that in normal phase mode.

[0035] High-resolution mass spectrometry (HRMS) was performed on an Agilent 1290 Infinity LC instrument (Agilent, USA) and an Agilent 6540 series QTOF-MS (Agilent, USA), which was equipped with an ESI source, a diode array detector, an autosampler, a degasser, and a column thermostat. The chromatographic column was C18HCE (2.1×150 mm, 5 μm). The flow rate was 0.2 mL / min. Other chromatographic conditions were the same as for RPLC analysis. The mass spectrometer conditions were: nebulizer gas pressure (35 psi), drying gas flow rate (8 L / min), gas temperature (350°C), capillary voltage (3500 V), and collision energy (25 eV). The primary mass spectrometer scan range was 100-1000 m / z, and the secondary mass spectrometer scan range was 50-500 m / z.

[0036] The chemical components of the seven antiparasitic active parts were characterized by high-resolution Q-TOF mass spectrometry coupled with optimized RPLC, which can provide accurate molecular weight, molecular formula and fragmentation information for each peak. Due to the crossover phenomenon in the NPLC preparation process, the same component may enter the adjacent component. Therefore, the RPLC peaks with the same retention time, accurate molecular weight and fragmentation information in the adjacent components were identified as the same component and were more likely to be insecticidal active components. The results showed Figure 4 Reversed phase liquid chromatography tandem high resolution mass spectrometry total ion chromatogram of active components in Figure 4Compound 1 is a common component of F5 and F6, with the same retention time, accurate molecular weight and fragment, and was identified as 3'-hydroxybavachinin (Wang, PL, Yao, ZH, Zhang, FX, Shen, XY, Dai, Y., Qin, L., Yao, XS, 2015. Identification of metabolites of psoraleae fructus in rats by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis. Journal of Pharmaceutical and Biomedical Analysis. 112, 23-35.). Similarly, compound 2 is a common component of F5 and F6 and was identified as psoralea corylifolia chalcone (Tewari, A., Bhakuni, RS, 2010. New constituents from psoralea corylifolia. Indian Journal of Chemistry Section B-Organic Chemistry Including Medicinal Chemistry. 49, 256-259.); compounds 3 and 4 are common components of F3, F4, F5, F6 and F5, F6, and were both identified as psoralea corylifolia methyl ether or psoralea corylifolia chalcone (Gupta, N., Qayum, A., Singh, S., Mujwar, S., Sangwan, PL, 2022. Isolation, anticancer evaluation, molecular docking, drug likeness and admet studies of secondary metabolites from psoralea corylifolia seeds. Chemistry select. 7, 41.). Compounds 5 to 9 are the common components of F11 and F12, F10, F11 and F12, F11 and F12, F10 and F11, F10 and F11, and F10, F11 and F12, respectively, and are identified as dihydroxyisopsoralen (Gupta, GK, Dhar, KL, Atal, CK, 1977.Isolation and constitution of corylidin - new coumestrol from fruits of psoralea - corylifolia. Phytochemistry. 16, 403 - 404.), psoralenol or psoralenol D (Suri, J.L., Gupta, G.K., Dhar, K.L., Atal, C.K., 1978. Psoralenol - new isoflavone from seeds of psoralea - corylifolia. Phytochemistry. 17, 2046 - 2046.), neobavaisoflavone (Gupta, N., Qayum, A., Singh, S., Mujwar, S., Sangwan, P.L., 2022. Isolation, anticancer evaluation, molecular docking, drug likeness and admet studies of secondary metabolites from psoralea corylifolia seeds. Chemistryselect. 7, 41.), licoagrochalcone A / corylifolinin (Luan, L., Shen, X., Liu, X., Wu, Y., Tan, M., 2018. Qualitative analysis of psoraleae fructus by hplc - dad / tof - ms fingerprint and quantitative analysis of multiple components by single marker. Biomedical Chromatography. 32, 2.) and corylifolinin A (Gupta, N., Qayum, A., Singh, S., Mujwar, S., Sangwan, P.L., 2022. Isolation, anticancer evaluation, molecular docking, drug likeness and admet studies of secondary metabolites from psoralea corylifolia seeds. Chemistryselect. 7, 41. It should be noted that there seem to be some formatting and citation issues in the original text which might need to be further clarified for a more accurate and proper presentation.All the identified components are flavonoid compounds, which are speculated to be the parasiticidal components of Psoralea corylifolia. High-resolution mass spectrometry can accurately give the molecular weight and secondary fragments of chemical components (. Figure 5 ), to achieve the derivation and identification of parasiticidal components, and finally identified 9 flavonoid compounds. Due to a certain amount of crossover in the normal phase preparation process, the same component will enter the adjacent component. We believe that among the adjacent active components, the same component with the same retention time, accurate molecular weight and consistent secondary fragments is more likely to be an anti-ciliate active component. Therefore, the chemical components psoralen dihydroflavonoid methyl ether, psoralen B, new psoralen isoflavones, and psoralen isoflavone A in the active fraction are likely to be anti-ciliate active components.

[0037] Example 4: Evaluation of in vitro anti-ciliate activity of active compounds

[0038] Psoralea corylifolia methyl ether (CAS No. 19879-30-2), psoralea corylifolia isoflavone (CAS No. 41060-15-5), psoralea corylifolia ethyl (CAS No. 20784-50-3) and psoralea corylifolia isoflavone A (CAS No. 775351-88-7) were purchased from Chengdu Munster Biotechnology Co., Ltd. (Chengdu, China). All compounds were dissolved in dimethyl sulfoxide (DMSO). Finally, a stock solution of 5000 mg / L of each compound was obtained. About 100 Tetrahymena pyriformis were placed in each well of a 24-well cell culture plate and exposed to compounds at concentrations of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0 mg / L (control, 1% DMSO). All experiments were repeated three times. After exposure for 2 h, the 100% lethal concentration of the parasites within 2 h of each compound was recorded. The insecticidal activity evaluation results of flavonoids are shown in Figure 6. Psoralen B had the best activity, with a minimum insecticidal concentration of 3.125 mg / L at 2 h. Neopsoralen isoflavones and psoralen isoflavone A were second in activity, with a minimum insecticidal concentration of 12.5 mg / L at 2 h. However, psoralen dihydroflavonoid methyl ether was poor, and could not kill Tetrahymena pyriformis even at a concentration of 25 mg / L. The antiparasitic activity of neopsoralen, psoralen B and psoralen isoflavone A was the same as that of the corresponding components, but the minimum insecticidal concentration of psoralen dihydroflavonoid methyl ether was higher than that of the corresponding components (it could not kill half of Tetrahymena pyriformis even at 25 mg / L). This indicated that there were other more active compounds. Flavonoids are an important class of plant secondary metabolites, which are widely present in various natural products. According to the structure of the three carbon atoms between the two benzene rings and the different substituents, flavonoids can be divided into 15 subclasses. Based on the identification results, the antiparasitic effects of five flavonoids from three subclasses were evaluated. Among them, the chalcone subclass psoralen B has the best in vitro insecticidal activity. The antiparasitic activity of psoralen isoflavone A and neopsoralen isoflavone, which are also isoflavone subclasses, is second. The activity of psoralen dihydroflavone methyl ether, which belongs to the dihydroflavone subclass, is weaker. In summary, the chalcone subclass with an opening between the 1st and 2nd positions of the flavonoid skeleton may enhance the antiparasitic activity against Tetrahymena pearformis. For the other unopened flavonoid compounds, the lack of a double bond between the 2nd and 3rd positions of the dihydroflavone subclass may reduce the activity against Tetrahymena pearformis. On this basis, psoralen B with the best insecticidal activity was selected for the acute toxicity test on guppies and the in vivo anti-insect evaluation.

[0039] Example 5: Acute toxicity of psoralen to guppies

[0040] The most effective antiparasitic compound, psoralen, was tested for tolerance in guppies. 3 The experiment was carried out in a glass aquarium (5 fish in a glass tank). Each fish was placed in 3L of aerated tap water. In the preliminary experiment, the concentrations of psoralen were 18.75 and 75mg / L, respectively, and the mortality rate of guppies was 0% and 100%. On this basis, 90 healthy guppies were randomly placed in 18 glass tanks (5 / glass tank) and exposed to psoralen at concentrations of 75, 56.25, 37.5, 28.125, 18.75 and 0mg / L (control) for 48h. The experiment was repeated 3 times and compared. When the gill cover and tail of the fish stopped moving and did not respond to mechanical stimulation, the fish was considered dead. The median lethal concentration (LC) of psoralen for guppies at 24h and 48h was determined. 50 ).from Figure 7 and Figure 8 It can be seen that the concentration of psoralen that can cause death of guppies within 24 hours and the median lethal concentration (LC) of psoralen at 48 hours 50) were 75mg / L and 46.18mg / L respectively. All treated fish showed decreased vitality and body imbalance before death. These results ensure the safety of psoralen in controlling Tetrahymena pyriformis infection and suggest that it can be used as a lead compound with the potential to develop new fishery drugs.

[0041] Example 6: In vivo insect resistance evaluation

[0042] Healthy guppies were infected by scratch inoculation. Tetrahymena pyriformis infection was by scratch inoculation, with an infection dose of 50 ciliates / mL. The infected fish and healthy fish were placed in a 1-m 3 75 guppies moderately infected with Tetrahymena pyriformis (about 20 per fin) were randomly placed in 15 1500 cm 3 Glass aquarium (5 fish / glass aquarium) and 3-L water. The infected fish were exposed to psoralen at concentrations of 10, 5, 2.5, 1.25 and 0.625 mg / L for 24 h. The test was performed three times and a control (under the same test conditions, without chemicals) was performed. Each concentration was repeated 3 times. The in vivo anti-insect efficacy of psoralen was calculated by the following formula.

[0043] E=(CT)×100 / C

[0044] Where, E is the antiparasitic effect, C is the average number of Tetrahymena pyriformis in the negative control, and T is the average number of Tetrahymena pyriformis in each treatment group.

[0045] from Fig. 9 It can be seen that when the concentration of psoralen is 10, 5, 2.5, 1.25 and 0.625 mg / L, the E is 92.5%, 88.9%, 78.9%, 10.3% and 10.1% respectively. The 24h median effective concentration (EC 50 ) value was 1.916mg / L, which also significantly increased the survival rate of guppies. The concentration of psoralen that can cause death of guppies within 24 hours is 24h-EC 50 It can be seen that the compound psoralen B exhibits good effects in killing ciliates and expelling parasites in vivo.

Claims

1. A flavonoid molecule with anti-ciliate activity in psoralea corylifolia, characterized in that: It refers to a class of compounds with a C6-C3-C6 structure formed by two benzene rings with phenolic hydroxyl groups connected to each other through three central carbon atoms.

2. A flavonoid molecule with anti-ciliate activity according to claim 1, characterized in that: It also includes pharmaceutically acceptable carriers or excipients.

3. The anti-ciliate active flavonoid molecule according to claim 1, characterized in that: The order of the killing effect of the compounds on Tetrahymena pyriformis is psoralen B, neopsoralen isoflavones, and psoralen isoflavone A.

4. The anti-ciliate active flavonoid molecule according to claim 1, characterized in that: The minimum killing concentrations of the compounds psoralen, neopsoralen and psoralen A against Tetrahymena pyriformis in vitro are 3.125 mg / L, 15 mg / L and 15 mg / L respectively.

5. The anti-ciliate active flavonoid molecule according to claim 1, characterized in that: The compound psoralen B at the animal level starts to cause death in guppies at 24h and 48h-LC 50 The values ​​are 75mg / L and 46.18mg / L respectively.

6. The anti-ciliate active flavonoid molecule according to claim 1, characterized in that: The 24h anthelmintic efficacy EC of the compound psoralen at the animal level 50 The value is 1.916mg / L.

7. The use of the anti-ciliate active flavonoid molecules in Psoralea corylifolia according to any of the above claims, characterized in that: The applications described mainly relate to ciliates.

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