Application of cortex phellodendri in relieving minoxidil-induced autism-like toxicity
By regulating related signaling pathways and gene expression using cork or cork extracts, the problem of minoxidil inducing autism-like toxicity was solved, and a significant toxicity relief effect was achieved, and a new strategy for the treatment of hypertension or hair loss was provided.
Patent Information
- Application Number
- CN202510217677.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-04-15
AI Technical Summary
Neurotoxicity induced by minoxidil leads to autism-like behavioral manifestations. Existing antidotes cannot specifically regulate neurotransmitter imbalances, and behavioral models have problems with long detection cycles and high costs, and lack systematic analysis of the synergistic effects of multiple targets.
Using cork or cork extract, the expression or function of Neuroactive ligand-receptor interaction, Calcium signaling pathway, cAMP signaling pathway and genes such as ESR1, SLC6A4, EGFR, SLC6A3, EGF, minoxidil-induced autism-like toxicity was alleviated.
Cork or Cork extract significantly alleviates the autism-like toxicity induced by minoxidil, provides a new strategy for treating hypertension or hair loss and reducing the autism-like toxicity side effects induced by minoxidil, which has important clinical application value.
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Figure CN119818581A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to application of Phellodendron chinense or a Phellodendron chinense extract in alleviating autism-like toxicity induced by minoxidil, and belongs to the technical field of medicines. Background Art
[0002] Minoxidil is a commonly used vasodilator and is widely used to treat hair loss and hypertension. However, the inventor's latest research shows that, as shown in Example 1, minoxidil can cause neurotransmitter imbalance and induce autism-like behavior, indicating that it has relevant autism-like toxicity. Summary of the invention
[0003] Traditional antidotes (such as β-blockers) cannot specifically regulate neurotransmitter imbalance for minoxidil-induced neurotoxicity, and existing behavioral models (such as rodents) have problems such as long detection cycles and high costs. In addition, there is a lack of systematic analysis of multi-target synergistic effects in natural product research. Therefore, it is of great significance to develop a natural drug that can effectively alleviate minoxidil-induced autism-like toxicity.
[0004] The invention provides the use of Phellodendron chinense or Phellodendron chinense extract in alleviating autism-like toxicity induced by minoxidil.
[0005] The concentration range of Phellodendron chinense or Phellodendron chinense extract is 0.01-1 mg / mL, preferably 0.1 mg / mL. The weight ratio of Phellodendron chinense or Phellodendron chinense extract to minoxidil in minoxidil-induced autism-like toxicity is 1:5-1:20, preferably 1:10.
[0006] The above-mentioned Cortex Phellodendri or Cortex Phellodendri extract is one of oral preparations, injections, patches, and external preparations, wherein the oral preparation is one of tablets, capsules, granules, and oral liquids.
[0007] The present invention has found that Phellodendron chinense or Phellodendron chinense extract can alleviate the autism-like toxicity induced by minoxidil by regulating at least one of Neuroactive ligand-receptor interaction, Calcium signaling pathway and cAMP signaling pathway.
[0008] It was also found that Phellodendron amurense or Phellodendron amurense extract alleviates minoxidil-induced autism-like toxicity by regulating the expression or function of at least one gene among ESR1, SLC6A4, EGFR, SLC6A3, and EGF.
[0009] The present invention also provides a pharmaceutical composition for treating hypertension, which is used to reduce the side effects of autism-like toxicity induced by minoxidil. The composition comprises Phellodendron chinense or a Phellodendron chinense extract and minoxidil, and the weight ratio of the Phellodendron chinense or a Phellodendron chinense extract to minoxidil is 1:5-1:20, preferably 1:10.
[0010] The composition is one of an oral preparation and an injection, wherein the oral preparation is one of a tablet, a capsule, a granule, and an oral liquid.
[0011] The present invention also provides a pharmaceutical composition for treating hair loss, which is used to reduce the side effects of autism-like toxicity induced by minoxidil. The composition comprises Phellodendron chinense or a Phellodendron chinense extract and minoxidil, and the weight ratio of Phellodendron chinense or a Phellodendron chinense extract to minoxidil is 1:5-1:20, preferably 1:10.
[0012] The composition is one of an oral preparation, an injection, a patch, and an external preparation.
[0013] The oral preparation is one of tablets, capsules, granules and oral liquid.
[0014] The external preparation is one of ointment, cream, gel, patch, spray, solution, lotion, powder, aerosol, suppository, film and poultice.
[0015] The beneficial effects of the present invention are:
[0016] The present invention first discovered that Phellodendron chinense or Phellodendron chinense extract can significantly alleviate the autism-like toxicity induced by minoxidil, and exert its therapeutic effect by regulating the expression or function of related signal pathways and key genes. In addition, the pharmaceutical composition provided by the present invention provides a new strategy for treating hypertension or hair loss and reducing the autism-like toxicity induced by minoxidil, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is a minoxidil-autism target 66 cross-target;
[0018] Figure 2 The relationship between five genes (ESR1, SLC6A4, EGFR, SLC6A3, EGF) and minoxidil reveals its potential effects on the nervous system;
[0019] Figure 3 yes Figure 2 Genes and their related signaling pathways;
[0020] Figure 4It is the relationship between the main pathways related to hub targets and the pathways related to neuroactiveligand-receptor interaction in the process of Nordil affecting autism;
[0021] Figure 5 is the interaction relationship between minoxidil and five core target genes (ESR1, SLC6A4, EGFR, SLC6A3, and EGF);
[0022] Figure 6 There was no significant difference in the behavior of Caenorhabditis elegans leaving food after treatment with 0.1mg / ml minoxidil, but the behavior of Caenorhabditis elegans leaving food was significantly affected after treatment with 0.5mg / ml and 1mg / ml;
[0023] Figure 7 It is the behavior of leaving the food that continues to affect the offspring, indicating that the social behavior of the parents and offspring of C. elegans treated with high concentrations of minoxidil is impaired;
[0024] Figure 8 This is a comparison of the pharyngeal pumping rates of the parent generations of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0025] Fig. 9 This is a comparison of the body curvature of the parent generations of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0026] Fig.10 This is a comparison of the reproductive rates of the parents of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0027] Fig.11 This is a comparison of the pharyngeal pumping rates of the offspring of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0028] Fig.12 This is a comparison of the body curvature of the offspring of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0029] Fig.13 This is a comparison of the reproductive rates of the offspring of Caenorhabditis elegans treated with different high concentrations of minoxidil;
[0030] Fig.14 These are five herbs selected based on the common targets of autism and minoxidil - active ingredients - medicinal and food homologous medicinal materials;
[0031] Fig.15 This is a data chart showing the effects of five herbs on improving autistic behaviors caused by minoxidil;
[0032] Fig.16 This is the experimental data diagram of concentration screening of Phellodendron chinense;
[0033] Fig.17 This is a graph of toxicity test data of Phellodendron chinense;
[0034] Fig.18 After treatment with Phellodendron chinense, food leaving behavior was improved, but pharyngeal pumping rate was not affected;
[0035] Fig.19 After treatment with Phellodendron chinense, food leaving behavior was improved, but body bending was not affected;
[0036] Fig. 20 After treatment with Phellodendron chinense, food leaving behavior was improved, but reproductive rate was not affected. DETAILED DESCRIPTION
[0037] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.
[0038] Example 1
[0039] 1. Discovery of minoxidil-induced autism through network toxicology.
[0040] We searched for "minoxidil" in the PubChem database to determine the standard structure of minoxidil and related information, and then used the ChEMBL database to retrieve potential targets of minoxidil, limiting the species to "Homo sapiens". We sorted and removed duplicates from the retrieved results, and finally entered the results into the Uniprot database for standardization.
[0041] Relevant targets were obtained by searching the GeneCards and OMIM databases using the keywords "Autism Spectrum Disorder" and "autism". To ensure that the obtained genes were highly correlated with autism, the "score" threshold was set to the median, and genes with "score" values higher than the median were used to establish an autism target library. In addition, we used a Venn diagram to depict the crossover genes between minoxidil and autism, and regarded the overlapping parts as potential targets for minoxidil to affect autism.
[0042] The potential targets of minoxidil affecting autism were entered into the STRING database, which only contained "Homo sapiens" as the species, and "medium confidence (0.700)" was set as the minimum required interaction score, and any unconnected nodes in the network were not displayed. Subsequently, the results generated by STRING were introduced into Cytoscape (v3.10.1) to construct a protein-protein interaction (PPI) network diagram, and the cytoHubba plug-in was used to select core targets according to their degree values.
[0043] To examine the biological effects of potential targets of minoxidil affecting autism, information was collected using the DAVID database. It was used for gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Gene ontology (GO) analysis included the evaluation of BP, CC, and MF to reveal the main biological functions of the relevant targets. The significant pathways associated with minoxidil affecting autism were identified by KEGG enrichment analysis, and the significance level of the corrected p value <0.05 was used to determine the main pathways of the identified targets, and the SRplot platform was used for data visualization.
[0044] In order to further analyze the intermolecular interactions between the core target protein of minoxidil, molecular docking was performed. First, the ligand was prepared. The SDF structure file of the ligand was downloaded from PubChem, converted to a PDB file using OpenBabel, then hydrogenated and dehydrated using Autodock, and saved in pdbqt format. Secondly, Pymol was used to remove water molecules and ligands from the protein and saved in pdb format. Then, molecular docking was performed using Autodock, and finally, pymol was used for visualization analysis.
[0045] 2. Minoxidil-induced autism-like behavior experiment
[0046] The following experimental plan was designed according to the references "Neuroligin dependence of social behavior in Caenorhabditis elegans provides a model to investigate an autism-associated gene" and "Investigating autism associated genes in C.elegans reveals candidates with a role in social behavior":
[0047] Use lysis solution (3% NaClO solution, 1mol / L NaOH solution and water) to lyse the oviposition period of Caenorhabditis elegans to obtain enough eggs after synchronization. After culturing for 18-22 hours, the nematodes hatched to the L1 stage are collected with K solution (0.3g NaCl, 1g KCl and 500mL water), centrifuged and discarded the supernatant, added K solution, and inoculated into ordinary NGM and minoxidil-containing culture medium for culture. The NGM solid culture medium includes 3g sodium chloride, 1.5g tryptone, 18g agar, 1mL 1M magnesium sulfate, 1mL 1M calcium chloride, 25mL 1M potassium phosphate buffer, 1mL 3mg / L cholesterol and 1L water. The concentrations of minoxidil in the NGM culture dish are 0mg / ml, 0.1mg / ml, 0.5mg / ml, and 1mg / ml, respectively, and OP50 is used as food.
[0048] Method for inducing autistic behavior with minoxidil: nematodes were synchronized and hatched into L1 stage and placed in minoxidil and ordinary NGM. 20ul of OP50 was dropped in the center of 35mm NGM culture dish 18 hours in advance before the behavioral assay. Seven L4+1 day-old hermaphroditic nematodes were selected and picked to the center of the bacterial lawn. The plate was then incubated at 20C for 24 hours, during which seven nematodes laid eggs to produce colonies full of offspring. In all food departure assays, a stereo microscope was used during the 30-minute observation period. The departure of the entire body of the nematode from the colony was counted as a food departure event.
[0049] Body bending: The nematodes to be tested were transferred to a new NGM culture medium. After the nematodes crawled on the culture medium until there was no food on their bodies, the number of body bendings within 1 minute was recorded. The process of moving forward in a sinusoidal waveform with the S-shaped central axis of the nematode's body as the axis was defined as a successful body bending. Ten nematodes were tested in each group of experiments.
[0050] Pharyngeal pumping: Feeding behavior was quantified by counting pharyngeal pumping of 10 worms for 30 s per concentration, repeated three times, after measuring food left at the 24 hr time point. Worms selected for these measurements were present on food during the observation period. One pharyngeal pump was defined as one contraction-relaxation cycle of the pharyngeal muscle terminal bulb.
[0051] Reproductive measurements: After minoxidil treatment, nematodes were individually transferred to fresh NGM dishes before laying eggs, each containing a drop of 20 μL OP50 bacterial solution. The nematodes were left to lay eggs until they stopped laying eggs. The number of offspring of each nematode was observed and recorded, and data were collected from 30 nematodes in each concentration group.
[0052] result Figure 1 It can be seen that there are 66 cross-targets of minoxidil-autism targets. Figure 2The relationship between five genes (ESR1, SLC6A4, EGFR, SLC6A3, EGF) and minoxidil may reveal its potential effects on the nervous system. Figure 3 These genes are associated with multiple signaling pathways, including Neuroactive ligand-receptor interaction, Calciumsignaling pathway, cAMP signaling pathway and cancer-related pathways. Figure 4 It can be seen that in the process of minoxidil affecting autism, the main pathways related to the hub target are closely related to the pathways related to neuroactive ligand-receptor interaction. Figure 5 We found that minoxidil interacts with five core target genes (ESR1, SLC6A4, EGFR, SLC6A3, and EGF). All five docking results were generated and showed low binding energy. This indicates that minoxidil spontaneously binds to each core target protein, which means that they play an important role in the molecular mechanism of minoxidil's effect on autism.
[0053] Figure 6 It can be seen that after being treated with 0.1mg / ml minoxidil, there was no significant difference in the behavior of Caenorhabditis elegans leaving the food. However, after being treated with 0.5mg / ml and 1mg / ml, the behavior of Caenorhabditis elegans leaving the food was significantly affected. Figure 7 This behavior continues to affect the offspring. This shows that the social behavior of parents and offspring of C. elegans treated with high concentrations of minoxidil is impaired. Figure 8-Figure 10 conclude that defects in parental feeding and locomotor behavior and reproduction are unlikely to cause defects in food departure behavior, and Figure 11-13 It was concluded that defects in offspring feeding and locomotor behavior and reproduction were unlikely to cause defects in food departure behavior.
[0054] Example 2
[0055] 2.1. Network pharmacology revealed that Phellodendron chinense can alleviate minoxidil-induced autism
[0056] The core target protein names were queried in the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) to retrieve relevant chemical components. Then, using the relevant chemical components, active compounds with oral bioavailability (OB) ≥ 30%, drug similarity (DL) index ≥ 0.18, and half-life (HL) ≥ 4 were collected. These active ingredients were further explored using the TCMSP database to identify related herbal medicines. Fig.14 It was concluded that the five herbal plants with the highest marginal values were selected from the common targets of autism and minoxidil - active ingredients - medicine and food homologous medicinal materials for further research.
[0057] 2.2 Experiment on the alleviating effect of Phellodendron chinense
[0058] Use lysis solution (3% NaClO solution, 1mol / L NaOH solution and water) to lyse the oviposition period of Caenorhabditis elegans to obtain enough eggs after synchronization. After culturing for 18-22 hours, the nematodes hatched to the L1 stage are collected with K solution (0.3g NaCl, 1g KCl and 500mL water), centrifuged and discarded the supernatant, added K solution, and inoculated into ordinary NGM and culture medium containing minoxidil and minoxidil and minoxidil mixed with Phellodendron amurense. The NGM solid culture medium includes 3g sodium chloride, 1.5g tryptone, 18g agar, 1mL 1M magnesium sulfate, 1mL 1M calcium chloride, 25mL 1M potassium phosphate buffer, 1mL 3mg / L cholesterol and 1L water. The concentrations of minoxidil in the NGM culture dish are 0mg / ml, 0.1mg / ml, 0.5mg / ml, and 1mg / ml, respectively, and OP50 is used as food. The concentration of Phellodendron amurense is 0.1mg / ml.
[0059] Method for Phellodendron to alleviate autistic behavior induced by minoxidil: The nematodes were synchronized and then hatched into the L1 stage and placed in minoxidil and ordinary NGM. 20ul of OP50 was dropped in the center of a 35mm NGM culture dish 18 hours in advance before the behavioral assay. During this period, seven nematodes laid eggs to produce colonies full of offspring. In all food departure assays, a stereo microscope was used during the 30-minute observation period. The departure of the entire body of the nematode from the colony was counted as a food departure event.
[0060] Body bending: The nematodes to be tested were transferred to a new NGM medium. After the nematodes crawled on the medium until there was no food on their bodies, the number of body bendings within 1 min was recorded. The process of moving forward in a sinusoidal waveform with the S-shaped central axis of the nematode's body as the axis was defined as a successful body bending. Ten nematodes were tested in each group of experiments.
[0061] Pharyngeal pumping: Feeding behavior was quantified by counting pharyngeal pumping of 10 worms for 30 s per concentration, repeated three times, after measuring food left at the 24 hr time point. Worms selected for these measurements were present on food during the observation period. One pharyngeal pump was defined as one contraction-relaxation cycle of the pharyngeal muscle terminal bulb.
[0062] Reproductive measurements: After minoxidil treatment, nematodes were individually transferred to fresh NGM dishes before laying eggs, each containing a drop of 20 μL OP50 bacterial solution. The nematodes were left alone until they stopped laying eggs. The number of offspring of each nematode was observed and recorded, and data were collected from 30 nematodes in each concentration group.
[0063] The five traditional Chinese medicines obtained in Example 2.1 were preliminarily screened. Fig.15 It can be seen that Phellodendron chinense significantly improves the autistic behavior caused by minoxidil. Then the optimal concentration of Phellodendron chinense ( Fig.16 ) is 0.1 mg / ml. And Fig.17 It showed that Phellodendron chinense had no toxic effects. Figure 18-20 It was concluded that food leaving behavior was improved after Phellodendron chinense treatment, but not by enhancing feeding and locomotor behavior and reproductive rate.
[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Application of Phellodendron amurense or Phellodendron amurense extract in alleviating minoxidil-induced autism-like toxicity.
2. The use according to claim 1, characterized in that The use concentration of the Phellodendron chinense or Phellodendron chinense extract is in the range of 0.01-1 mg / mL, preferably 0.1 mg / mL.
3. The use according to claim 1, characterized in that The weight ratio of the Phellodendron chinense or the Phellodendron chinense extract to minoxidil in minoxidil-induced autism-like toxicity is 1:5-1:20, preferably 1:
10.
4. The use according to claim 1, characterized in that The Phellodendron chinense or Phellodendron chinense extract is one of an oral preparation, an injection, a patch, and an external preparation.
5. The use according to claim 4, characterized in that The oral preparation is one of tablets, capsules, granules and oral liquid.
6. The use according to claim 1, characterized in that The Phellodendron chinense or Phellodendron chinense extract alleviates the autism-like toxicity induced by minoxidil by regulating at least one of Neuroactive ligand-receptor interaction, Calcium signaling pathway and cAMP signaling pathway.
7. The use according to claim 1, characterized in that The Phellodendron chinense or Phellodendron chinense extract alleviates the autism-like toxicity induced by minoxidil by regulating the expression or function of at least one gene among ESR1, SLC6A4, EGFR, SLC6A3 and EGF.
8. A pharmaceutical composition for treating hypertension, used to reduce the side effects of minoxidil-induced autism-like toxicity, characterized in that: The composition comprises Phellodendron chinense or Phellodendron chinense extract and minoxidil, and the weight ratio of Phellodendron chinense or Phellodendron chinense extract to minoxidil is 1:5-1:20, preferably 1:
10.
9. The pharmaceutical composition according to claim 8, characterized in that The composition is an oral preparation or an injection.
10. The pharmaceutical composition according to claim 8, characterized in that The oral preparation is one of tablets, capsules, granules and oral liquid.
11. A pharmaceutical composition for treating hair loss, used to reduce the side effects of minoxidil-induced autism-like toxicity, characterized in that: The composition comprises Phellodendron chinense or Phellodendron chinense extract and minoxidil, and the weight ratio of Phellodendron chinense or Phellodendron chinense extract to minoxidil is 1:5-1:20, preferably 1:
10.
12. The pharmaceutical composition according to claim 11, characterized in that The composition is one of an oral preparation, an injection, a patch, and an external preparation.
13. The pharmaceutical composition according to claim 12, characterized in that The oral preparation is one of tablets, capsules, granules and oral liquid.
14. The pharmaceutical composition according to claim 12, characterized in that The external preparation is one of ointment, cream, gel, patch, spray, solution, lotion, powder, aerosol, suppository, film and poultice.