Use of bakuchiol in the preparation of a medicine for treating helminthiasis

Abstract

The application discloses application of bakuchiol in preparation of a medicine for treating helminthiasis, and in-vitro effectiveness experiment results of the bakuchiol on the protoscoleces of multilocular hydatid cysts show that the killing effect of maduramicin ammonium on the protoscoleces is concentration-dependent, the mortality of the protoscoleces can reach 90.63%+ / -0.96% under the treatment of 50 muM concentration for 6 hours, and the mortality of the protoscoleces can directly reach 100% under the treatment of 100 muM concentration for 6 hours; meanwhile, the in-vitro effectiveness experiment results of the bakuchiol on different developmental stages of trichinella spiralis show that the mortality of muscle larvae is 89.44%+ / -0.81%, 96.29%+ / -1.30% and 96.68%+ / -1.06% respectively under the treatment of 50, 80 and 100 muM concentration for 72 hours, and the mortality of adult worms is 100% after 48 hours of treatment, and the killing effect is remarkable. Especially importantly, the applicant observes by optical microscope and electron microscope, and for the first time, confirms that the IBC can directly destroy the epidermal structure and cell integrity of the worm, and the discovery provides intuitive morphological evidence for the anti-parasitic effect.

Description

Technical Field

[0001] This invention belongs to the field of antiparasitic drug development and application technology, specifically involving the application of psoralen in the preparation of drugs for treating helminthiasis. Background Technology

[0002] Helminthiasis is a group of zoonotic diseases caused by multicellular parasites belonging to the phyla Platyhelminthes, Nematodes, and Acanthocephala. Widely distributed globally, it poses a serious threat to public health and animal husbandry. Among the many helminth infections, alveolar echinococcosis (AE) and trichinosis are two representative highly pathogenic parasitic diseases that have received widespread attention.

[0003] Alveolar echinococcosis (AE) is caused by *Echinococcus multilocularis*, a tapeworm belonging to the class Tapeworms of the phylum Platyhelminthes. Echinococcus multilocularis, This disease is caused by the larval stage of *E. emblica*, primarily affecting organs such as the liver and lungs. The larvae proliferate asexually, forming infiltrative lesions that continuously damage the host's tissue structure and physiological functions. The clinical progression is insidious, and in later stages, it can lead to organ failure and even death. The disease is mainly prevalent in livestock-developed regions of the Northern Hemisphere, with an estimated 18,000 new cases globally each year, resulting in a disease burden of up to 666,000 disability-adjusted life years.

[0004] Trichinosis is caused by the genus Trichinella ( Trichinella Caused by nematodes (spp.), their life cycle includes stages such as adult worm reproduction in the intestines, hematogenous spread of newborn larvae, and eventual cyst formation in skeletal muscle. Humans are usually infected by consuming undercooked meat containing live larvae. Clinical manifestations are characterized by fever, myalgia, and a significant increase in eosinophils. Severe cases can lead to serious complications such as myocarditis or encephalitis, posing a continued challenge to food safety and human health.

[0005] In terms of treatment, albendazole (ABZ) and mebendazole (MBZ) are currently the main chemotherapy drugs for acute helminthiasis (AE) and trichinosis. However, the clinical application of these two types of drugs has significant limitations: for AE, although ABZ can inhibit the proliferation of worms, it is difficult to completely eliminate the worms, requiring patients to take the medication for a long time, and significantly increasing the risk of adverse drug reactions such as hepatotoxicity; for trichinosis, ABZ is only effective against adult worms in the intestine and migrating larvae, and has limited effect on larvae that have formed cysts in skeletal muscle, thus its optimal therapeutic window is usually limited to the early stage of infection. In summary, addressing the shortcomings of existing drugs for the treatment of AE and trichinosis, the development of novel anti-helmintic drugs with highly effective insecticidal activity, low toxicity and side effects, and the ability to completely eliminate worms in tissues has become an urgent need in the field of parasitic disease prevention and control.

[0006] Isobacachalcone (IBC) is a flavonoid active ingredient derived from the traditional Chinese medicine Psoraleacorylifolia L. Existing research has revealed its broad pharmacological potential, including anticancer effects through activation of the Akt / Nrf2 pathway and anti-inflammatory activity through inhibition of the NF-κB signaling pathway. Furthermore, isobacachalcone has been mentioned in pharmacological studies for its antiparasitic activity; for example, patent CN 117384125 A discloses an anti-ciliary active ingredient in Psoraleacorylifolia and its application. However, ciliates are unicellular protozoa, not multicellular worms. Currently, there are no reports on isobacachalcone's role in regulating host-worm interactions. Summary of the Invention

[0007] Based on the above, the purpose of this invention is to provide the application of psoralen in the preparation of a medicament for treating helminthiasis.

[0008] In this invention, the helminth disease is tapeworm disease or nematode disease.

[0009] Furthermore, the tapeworm is a multilocular echinococcosis larva, and the nematode is a trichinella spiralis.

[0010] Furthermore, the *Echinococcus multilocularis* larvae are *Echinococcus multilocularis* prostomia or cysts. The *Trichinella spiralis* is a muscle larva, adult, or newly formed larva.

[0011] The present invention also provides a method for killing worms in vitro, which involves dissolving psoralen in DMSO to prepare a DMSO stock solution, then diluting it with DMEM medium and adding it to the worm culture medium.

[0012] As a further preferred embodiment of the technical solution of the present invention, the concentration of the DMSO mother liquor is 10 mM.

[0013] Furthermore, the concentration of the DMSO mother liquor after dilution is 50-100 μM.

[0014] Furthermore, the worm culture conditions are 37 °C, 5% CO2, 12 h.

[0015] Furthermore, the worm is a tapeworm or a nematode.

[0016] The beneficial effects of this invention are as follows: The in vitro efficacy test results of psoralen against the protoscolex of *Echinococcus multilocularis* showed that the killing effect of psoralen on the protoscolex was concentration-dependent. At a concentration of 50 μM, the mortality rate of the protoscolex reached 90.63% ± 0.96% after 6 hours of treatment, while at a concentration of 100 μM, the mortality rate directly reached 100%. Simultaneously, the in vitro efficacy test results of psoralen against *Trichinella spiralis* at different developmental stages showed that after 72 hours of treatment at concentrations of 50, 80, and 100 μM, the mortality rates of the myolarvae reached 89.44% ± 0.81%, 96.29% ± 1.30%, and 96.68% ± 1.06%, respectively; after 48 hours of treatment, the mortality rate of the adults was 100%, demonstrating a significant killing effect. Most importantly, the applicant, through observation using optical and electron microscopes, has for the first time confirmed that IBC can directly destroy the epidermal structure and cellular integrity of the parasite, providing direct morphological evidence for its antiparasitic effect. Attached Figure Description

[0017] Figure 1 Results of the effect of IBC on the protoscolex of *E. m.*; A: Lethal effect of IBC on protoscolex; B: EC after 6 h of treatment with different concentrations of IBC. 50 C: Morphology of protocephalon before and after staining in the DMSO and IBC groups (100×); D: SEM images of protocephalon in the DMSO and IBC groups; *: P <0.05 indicates a significant difference compared to the DMSO group (the same applies below); **: P <0.01 indicates a highly significant difference; ***: P <0.001 indicates an extremely significant difference; ****: P <0.0001 indicates a highly significant difference; the same applies below. Figure 2 SEM image of the Em capsule; Figure 3 Results of the effects of IBC on Trichinella spiralis; Figure 4 Results of the cytotoxic effect of IBC on JS1 cells. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] All reagents and instruments used in this invention were purchased commercially.

[0020] 1. Materials and Methods 1.1 Biomaterials The Em protoscolex and cysts (Jiuzhi strain) and Trichinella spiralis (Henan strain) used in this invention were all preserved and passaged by the Animal Important Zoonotic Diseases Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The Em protoscolex and cysts were collected from Balb / c rats; Trichinella spiralis larvae at various stages were collected from Kunming rats.

[0021] 1.2 Acquisition and in vitro culture of Em protoscolex and cysts This invention uses Balb / c mice, a subculture of mice preserved at the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, as an experimental animal model. Transparent cysts approximately 3-5 mm in diameter are isolated from infected mice using aseptic surgical techniques, and the protoscolex contained within them is collected simultaneously. The obtained cysts and protoscolex are washed five times with phosphate-buffered saline (PBS) containing 1% penicillin-streptomycin (100 U / mL) to remove impurities. Subsequently, the washed cysts are seeded at a density of 3-5 per well in 12-well cell culture plates, with 2 mL of DMEM medium added to each well; the protoscolex are seeded at a density of approximately 200 per well in 96-well cell culture plates, with 200 μL of DMEM medium added to each well. All seeded culture plates are incubated at 37 ℃ and 5% CO2 for 12 h to allow the parasites to acclimatize to the in vitro culture environment, laying a stable initial foundation for subsequent drug treatment experiments.

[0022] 1.3 Evaluation of the in vitro killing effect of IBC on Em protoscolex and cysts This invention uses DMEM medium to dilute 10 mM IBC stock solution, and sets up three concentration gradients of 50, 80, and 100 μM to treat protoscolex, while the cyst group is uniformly treated with 100 μM IBC. A solvent control group containing an equal amount of DMSO was set up simultaneously in the experiment. The samples in each group were cultured at 37 ℃ and 5% CO2 for 6, 12, and 24 h, respectively. The viability of protoscolex was assessed by the 0.1% methylene blue staining method, with structural collapse and a deep blue color indicating death. At the same time, the integrity of the cyst wall and the state of the cyst fluid were observed, and the metabolic activity was judged by the change of phenol red indicator in the medium from red to yellow. The protoscolex and cyst samples after IBC treatment were fixed, dehydrated, dried, and sputter-coated with gold, and then their ultrastructural changes were observed using scanning electron microscopy (SEM). All experiments were performed in triplicate and independently repeated three times.

[0023] 1.4 Acquisition and in vitro culture of Trichinella spiralis muscle larvae, adults, and newborn larvae Muscle and bone tissues were isolated from breeding mice euthanized by cervical dislocation. Trichinella spiralis larvae were collected and counted after pepsin digestion. One thousand thousand larvae were orally inoculated into Kunming mice to establish infection. On day 3 post-infection, the small intestine of mice was longitudinally dissected, thoroughly rinsed with physiological saline, and incubated at 37 °C on gauze soaked in physiological saline for 4 h to collect adult worms. This process was repeated on day 5 post-infection to obtain another batch of adult worms. The obtained adult worms were placed in DMEM complete medium and cultured at 37 °C and 5% CO2 for 24 h. They were then filtered through a 200-mesh sieve and centrifuged to collect the released larvae. All worms were washed five times with sterile PBS to ensure purity, and then inoculated at a density of 100-200 larvae per well in 96-well plates. 200 μL of DMEM medium was added to each well, and the plates were pre-cultured under standard conditions for 12 h to allow them to fully adapt to the in vitro environment before drug intervention experiments.

[0024] 1.5 Evaluation of the in vitro killing effect of IBC on Trichinella spiralis muscle larvae, adults, and newborn larvae 10 mM IBC stock solution was diluted to the desired concentration using DMEM medium, with three concentration gradients of 50, 80, and 100 μM established to intervene in the myolarval, adult, and newly formed larval stages of Trichinella spiralis. A solvent control group containing an equal volume of DMSO was included in the experiment. Based on preliminary observations of IBC treatment at 24, 48, and 72 h, the time gradient was further refined, with a 10-minute treatment time point set for newly formed larvae to assess its dynamic lethal effect. Morphological changes in the worms were dynamically observed using an optical microscope. The mortality criteria included: abnormal worm structure, "C"-shaped rigidity or linear relaxation, body wall damage, and no spontaneous movement for 30 seconds. The mortality rate was calculated using the formula (number of dead worms / total number of worms) × 100%. All experiments were performed in triplicate and independently repeated three times.

[0025] 1.6 Evaluation of the toxicity of IBCs to mouse hepatic stellate cells This invention uses laboratory-preserved mouse hepatic stellate cells JS1, at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 10% fetal bovine serum (FBS) in 96-well plates and cultured in DMEM complete medium at 37 °C with 5% CO2 for 24 h to allow for full cell adhesion and entry into the logarithmic growth phase. Subsequently, cells were treated with gradient concentrations of IBC (0.3125–160 μM), with an equal volume of DMSO as a negative control. After 24 h of drug intervention, cell viability was assessed using the CCK-8 assay, and after 40 min of incubation, absorbance (A) was measured at 450 nm using a microplate reader. 450Cell viability was calculated using the following formula: Viability (%) = (Experimental Group A) / (Experimental Group A) 450 - Blank Group A 450 ) / (Control Group A) 450 - Blank Group A 450 ) × 100%.

[0026] 1.7 Statistical Analysis Statistical analysis was performed using Graphpad Prism 9.5 software, and results are expressed as mean ± standard deviation (x̅ ± SD). One-way ANOVA (IBC) was used to analyze the significance of differences in the effects of *E. m. protoscolex* and *Trichinella spiralis* muscle larvae, adults, and newborn larvae. P The difference is significant when <0.05.

[0027] 2. Experimental Results 2.1 In vitro efficacy of IBC for Em protostomy This invention systematically evaluated the in vitro killing effect of IBC on Em protoscolex. Results showed that the survival rate of the DMSO control group remained at 98% ( Figure 1 A), both before and after staining maintained intact morphology and normal calcium particle deposition. Figure 1 C). The mortality rates of 50, 80, and 100 μM IBC treatment for 6 h were 90.63% ± 0.96%, 93.86% ± 1.92%, and 100%, respectively (all...). P <0.0001), its 6-hour effective concentration (EC50) is 50. 50 The value was 17.31 μM. Figure 1 B). Morphological observation further revealed that IBC treatment could cause extensive structural damage to the protoscolex. Under an optical microscope, sucker collapse, detachment of surface grooves, and necrosis of parenchymal tissue were observed, and the insect body was generally deeply stained with 0.1% methylene blue (B). Figure 1 C), indicating that it lost its activity. SEM results further showed that after IBC treatment, the microvilli on the surface of the parasite were disordered and largely shed, the apical structure was deformed, the suckers were damaged, and multiple cracks and depressions appeared in the cuticle. Figure 1 D). The above results indicate that IBC may exert its protoscolex-killing effect by directly disrupting the structural integrity of the insect body and its surface barrier system.

[0028] 2.2 In vitro efficacy of IBCs for Em capsules This invention further evaluated the in vitro killing effect of IBC on Em cysts. 100 μM IBC was used to treat cysts for 72 h, with an equal volume of DMSO as a control. Macroscopic morphological observation showed that the IBC-treated cysts exhibited significant atrophy and structural collapse, while the phenol red indicator in the culture medium did not change color, indicating that its metabolic activity was significantly inhibited. SEM results further revealed structural changes: the IBC-treated keratinocytes showed multiple irregular cracks and pores, the germinal layer was discontinuous, the number of microvilli was significantly reduced, and germinal cells degenerated and disappeared. Figure 2 The above results indicate that IBC may effectively kill Em capsules by directly disrupting the structural integrity of the capsule's keratinocyte and germinal layer, thereby interfering with their barrier function and homeostasis maintenance.

[0029] 2.3 In vitro efficacy of IBC against Trichinella spiralis This invention systematically evaluated the in vitro killing effect of IBC on trichinella myolarvae, adults, and newborn larvae. The results showed that after treatment with concentrations of 50, 80, and 100 μM for 72 h, the mortality rates of myolarvae were 89.44% ± 0.81%, 96.29% ± 1.30%, and 96.68% ± 1.06%, respectively. P (All <0.0001) Figure 3 A), 72 h EC 50 The concentration was 17.59 μM. After 48 h of treatment, the mortality rate of adults was 100%. P (All <0.0001) Figure 3 C). After 10 minutes of treatment, the mortality rate of newly hatched larvae was 100% ( P (All <0.0001) Figure 3 E). Dead worms exhibit a typical "C" shape or rigid morphology, showing no signs of movement after 30 seconds of continuous observation. The survival rate of worms in each control group remained above 98%, with intact morphology and normal activity.

[0030] 2.4 The toxic effect of IBCs on JS1 cells in mouse hepatic stellate cells This invention systematically evaluated the toxic effects of IBC on JS1 cells using in vitro experiments. The results showed that after treatment with gradient concentrations of IBC for 24 h, the survival rate of JS1 cells decreased significantly in a dose-dependent manner. The half-maximal inhibitory concentration (IC50) was [not specified in the original text]. 50 The value was 32.72 μM. Figure 4 This suggests that the compound has relatively low toxicity to mammals, providing a reference for subsequent studies on its biological effects and the setting of safe concentration ranges.

[0031] As important zoonotic parasitic diseases, AE and trichinosis have always faced significant challenges in the development of drugs for their prevention and treatment. This invention systematically evaluated the antiparasitic activity of the natural active ingredient IBC through in vitro experiments, finding that it has significant in vitro killing effects on both Echinococcus multilocularis and Trichinella spiralis. Most importantly, through optical and electron microscopy, the applicant has for the first time demonstrated that IBC can directly disrupt the epidermal structure and cellular integrity of the parasites, providing direct morphological evidence for its antiparasitic effect.

[0032] IBCs differ fundamentally from traditional antiparasitic drugs. Compared to benzimidazole drugs (ABZs) which exert their effects by inhibiting β-tubulin polymerization, or macrolide drugs which produce effects by activating chloride ion channels, IBCs may exert their antiparasitic effects through a more complex multi-target mechanism. Based on the structural characteristics of its flavonoid compounds, the applicant speculates that its mechanism of action may involve the following levels: First, the unique phenolic hydroxyl structure of IBCs may induce a large accumulation of reactive oxygen species (ROS) in the parasite, triggering oxidative stress and leading to lipid peroxidation and protein dysfunction; second, IBCs may interfere with the mitochondrial electron transport chain in the parasite, inducing mitochondrial membrane potential collapse and thus initiating apoptosis; in addition, the known regulatory role of the GPR84-PI3K-AKT signaling pathway of IBCs may also indirectly affect the survival microenvironment of the parasite in the host.

[0033] Regarding safety assessment, IBC showed positive effects on the IC50 of mouse hepatic stellate cells. 50 Reaching a concentration of 32.72 µM, it exhibits relatively low systemic toxicity, a characteristic that provides an important safety advantage for its subsequent development. Compared with currently used antiparasitic drugs in clinical practice, IBC significantly reduces its impact on mammalian cells at effective concentrations, suggesting a potentially wider therapeutic window.

[0034] In the development of anti-helminthic drugs, optimizing the formulation of natural compounds with clear pharmacological activity, such as IBC, has significant advantages over developing entirely new chemical entities from scratch, including shorter development cycles, lower costs, and higher success rates. This "repurposing existing drugs" strategy is particularly relevant and valuable given the current slow progress in antiparasitic drug development.

[0035] In summary, the broad-spectrum antiparasitic activity and low cytotoxicity exhibited by IBC provide a solid foundation for its application in the treatment of helminthiasis.

Claims

1. The application of psoralen in the preparation of drugs for treating helminthiasis, characterized in that, The worms are either Echinococcus multilocularis or Trichinella spiralis.

2. The use of psoralen as described in claim 1 in the preparation of a medicament for treating helminthiasis, characterized in that, The multilocular echinococcosis is the prostomium or cyst of the multilocular echinococcosis.

3. The use of psoralen B as described in claim 1 in the preparation of a medicament for treating helminthiasis, characterized in that, The trichinella mentioned refers to trichinella muscle larvae, adults, or newborn larvae.

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