Application of ganoderic acid A and Erinacine A in preparation of medicine for treating neurodegenerative diseases

Abstract

The invention provides application of ganoderic acid A and Erinacine A in preparation of a medicine for treating neurodegenerative diseases, and belongs to the technical field of biological medicines. According to the invention, the combined application of ganoderic acid A and Erinacine A is provided for the first time, and multi-channel regulation of neurodegenerative diseases is realized through complementary and overlapped target networks. The invention further discusses the application of the ganoderic acid A and the Erinacine A in preparing the medicine for treating the neurodegenerative diseases. Metabonomics proves that ganoderic acid A and Erinacine A can significantly inhibit ferroptosis of nerve cells by adjusting pathways such as cobalt amic acid metabolism and fatty acid metabolism, and the mechanism is not fully developed in existing clinical drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of ganoderic acid A and erinacine A in preparation of a medicament for treating neurodegenerative diseases. BACKGROUND

[0002] Neurodegenerative diseases are a class of complex diseases characterized by progressive dysfunction or death of neurons, and clinically manifested as cognitive decline, motor dysfunction and autonomic nervous function impairment, covering major disease types such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In recent years, it has been found that excitotoxicity and aging-associated neurotoxicity constitute the core pathological mechanism of such diseases.

[0003] Under physiological conditions, glutamate, as the main excitatory neurotransmitter in the central nervous system, regulates synaptic plasticity and learning and memory function through pathways such as activation of NMDA receptors. However, mitochondrial dysfunction, oxidative stress accumulation and calcium homeostasis imbalance during aging process can lead to abnormal glutamate metabolism and excessive accumulation in the synaptic cleft, triggering persistent calcium influx, and then activating downstream cascade reactions, including reactive oxygen species (ROS) burst, protein misfolding, DNA damage and mitochondrial membrane potential collapse, ultimately driving neuronal death through multiple pathways such as apoptosis, necroptosis and ferroptosis. This process is particularly significant in aging-related neurodegenerative diseases (such as AD / PD), and is positively correlated with disease progression.

[0004] Current clinical interventions for neuroexcitotoxicity (such as memantine, riluzole, etc.) are mainly based on single target (such as NMDA receptor antagonism), which has limitations in efficacy and significant side effects. Therefore, the development of natural product combinations with multi-target synergistic regulation characteristics, especially active ingredients that can simultaneously antagonize glutamate toxicity, repair metabolic disorders and inhibit novel cell death pathways (such as ferroptosis), has become a frontier direction in this field.

[0005] Medicinal higher fungi extracts have been proved to have good effects on AD through multi-targets. Extracts of Ganoderma lucidum (rich in ganoderic acid A) have protective effects on the degenerative changes of hippocampal neurons in the brain tissue of AD model rats, significantly reduce the inflammatory response of the brain tissue, and increase the SOD activity and reduce the MDA content of the hippocampal tissue of the rats. After the extracts are administered by gavage for 14 days, the learning and memory abilities of the AD mice can be obviously enhanced, and the contents of 5-hydroxytryptamine and dopamine neurotransmitters in the brain of the mice are significantly increased. Erinacines in Hericium erinaceum promote the growth of PC-12 cell and 1321N1 cell processes, and after erinacine A is added to the feed of the mice (8 mg / kg of body weight) and fed for 3 weeks, the norepinephrine and homovanillic acid levels are increased, and the secretion of NGF in the locus coeruleus and hippocampus is increased. SUMMARY

[0006] The application aims to provide the application of ganoderic acid A and erinacine A in the preparation of medicines for treating neurodegenerative diseases, which can significantly inhibit the ferroptosis of neural cells by regulating the cobalamin metabolism and fatty acid metabolism.

[0007] In order to achieve the above application purposes, the application provides the following technical solutions. The application provides the application of ganoderic acid A and erinacine A in the preparation of medicines for treating neurodegenerative diseases.

[0008] Preferably, the mass ratio of the ganoderic acid A and the erinacine A is 2:1.

[0009] Preferably, the effective concentration of the ganoderic acid A is 5 mg / kg / day, the effective concentration of the erinacine A is 5 mg / kg / day, and the effective concentration of the medicine is 2-5 mg / kg / day.

[0010] Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

[0011] The application also provides the application of ganoderic acid A and erinacine A in the preparation of medicines for improving the amino acid metabolism and lipid metabolism of the brain.

[0012] The application also provides the application of ganoderic acid A and erinacine A in the preparation of medicines for inhibiting the ferroptosis of neural cells.

[0013] The application also provides the application of ganoderic acid A and erinacine A in the preparation of medicines for neuroprotection.

[0014] Preferably, the mass ratio of Ganoderic acid A and Erinacine A is 2:1.

[0015] Preferably, the effective concentration of the drug is 2-5 mg / kg / day.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application provides the use of Ganoderic acid A and Erinacine A in the preparation of a drug for treating neurodegenerative diseases. Network pharmacology analysis results show that the combination of Ganoderma and Hericium erinaceus covers multiple pathways such as anti-inflammatory, antioxidant, anti-apoptotic and immune regulation through complementary exposure (18 intersection target points), breaking through the limitations of single target drugs. At the level of monomer compounds, the combination of Ganoderic acid A and Hericium erinaceus A is better than single drug in improving memory and learning function; at the level of extracts, Ganoderic acid A and Erinacine A can synergistically improve brain amino acid metabolism (such as glutamic acid toxicity regulation) and lipid metabolism disorder, and block the core pathological process of neuronal damage from the metabolic level. In addition, metabolomics confirms that Ganoderma and Hericium erinaceus extract can significantly inhibit Ferroptosis of neural cells by regulating cobalamin metabolism and fatty acid metabolism, and this mechanism has not been fully developed in existing clinical drugs. At the same time, the present application first proposes the combined application of triterpenes and bird nest xanthene diterpenes, which indeed has better effect than single drug in improving memory and learning ability. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0018] Figure 1 The target point network relationship between Hericium erinaceus and Ganoderma in resisting AD is complementary exposure; Figure 2 The intersection gene of Hericium erinaceus-Ganoderma-AD is 18; Figure 3 The protein-protein interaction (PPI) network model is constructed according to the intersection gene; Figure 4 The biological process graph in which Ganoderic acid A and Erinacine A are involved together; Figure 5 The positioning cruise experiment trajectory graph of mice in each group: wherein, Figure 5 A in the above formula represents the positioning cruise experiment trajectory graph of mice in the control group; Figure 5 B in the above formula represents the positioning cruise experiment trajectory graph of mice in the EA group;Figure 5 In the diagram, C represents the trajectory of mice in the high-concentration drug administration group during the localization and navigation experiment. Figure 5 In the diagram, D represents the trajectory of the mice in the model group during the localization and navigation experiment. Figure 5 In the diagram, E represents the trajectory of the mice in the GA group during the localization and navigation experiment. Figure 5 In the diagram, F represents the trajectory of mice in the low-concentration drug administration group during the localization and navigation experiment. Figure 6 Spatial exploration trajectory diagrams for each group of mice: (The diagrams are not included in the provided text.) Figure 6 In the diagram, A represents the spatial exploration trajectory of mice in the control group. Figure 6 In this diagram, B represents the spatial exploration trajectory of mice in group EA. Figure 6 In this diagram, C represents the spatial exploration trajectory of mice in the high-concentration drug administration group. Figure 6 In this diagram, D represents the spatial exploration trajectory of the mice in the model group. Figure 6 In this context, E represents the spatial exploration trajectory of mice in the GA group; Figure 6 In this context, F represents the spatial exploration trajectory of mice in the low-concentration drug administration group; Figure 7 The Venn diagrams for each sample are shown, where "C" represents the blank group, "M" represents the glutamate model group, "H" represents the high concentration group (Ganoderma lucidum-Hericium erinaceus 5 mg / kg / day), and "L" represents the low concentration group (Ganoderma lucidum-Hericium erinaceus 2 mg / kg / day). Figure 8 The PCA scatter plot is obtained for all samples; Figure 9 The scatter plot is obtained from the PCA model of group M versus group H (high concentration group); Figure 10 Volcano plot for screening differential metabolites in group M versus group H (high concentration group); Figure 11 The heatmap shows the hierarchical cluster analysis of group M against group H (high concentration group); Figure 12 The matchstick diagram for group M versus group H; Figure 13 This is a KEGG heatmap of group M versus group H. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these intervening values are also specifically included within the scope of the present application. The stated ranges are also intended to encompass any and all sub-ranges thereof. These sub-ranges are specifically included within the scope of the present application, as are all the individual values lying within the sub-ranges. For example, a range from 1 to 10 should be read to include any number from 1 to 10, including the end values in the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0022] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Rather, the intent is to embrace all changes and modifications that are within the spirit and scope of the application.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0024] The present application provides the use of Ganoderic acid A and Erinacine A in the preparation of a medicament for treating neurodegenerative diseases.

[0025] In the present application, the mass ratio of Ganoderic acid A and Erinacine A is preferably 2:1, the effective concentration of Ganoderic acid A is preferably 5 mg / kg / day, and the effective concentration of Erinacine A is preferably 5 mg / kg / day; the effective concentration of the medicament is preferably 2-5 mg / kg / day, further preferably 3-4 mg / kg / day, and more preferably 3.5 mg / kg / day. The combination of the two drugs at a concentration of 5 mg / kg / day is superior to the single drug. The neurodegenerative diseases preferably include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

[0026] Herculeum (H. erinaceus) Hericium erinaciumErinacines series in Ophiocordyceps sinensis (Berk.) G.H.Sugita, which can promote the neurite outgrowth of PC-12 cells and 1321N1 cells, and the norepinephrine and homovanillic acid levels are increased after Erinacine A is added into the mouse feed (8 mg / kg body weight) for 3 weeks, and the secretion of NGF in the locus coeruleus and hippocampus is increased.

[0027] Ganoderma is recorded in Compendium of Materia Medica: "increasing wisdom, not forgetting, long-term service light body, not old, prolonging life and becoming a god of immortality. From the commonalities of aging and Alzheimer's disease, "not old" can reduce the probability of AD occurrence, and "increasing wisdom, not forgetting" is related to the process and result of AD. Ganoderma lucidum (Leyss. ex Fr.) Karst. G. lucidum Ganoresinoid A can reduce the levels of inflammatory factors and pro-inflammatory factors NO, IL-1β, IL-6 and TNF-α by inhibiting the TLR-4 / NF-κB pathway and the MAPK pathway, thereby playing an anti-neurogenic role; effectively maintaining the stability of the mitochondrial membrane induced by LPS, reducing the content of ROS, activating the Akt / GSK-3β / Nrf2 signaling pathway to play an antioxidant activity, and protecting the oxidative damage of SH-SY5Y cells induced by hydrogen peroxide.

[0028] The application also provides application of ganoderic acid A and Erinacine A in preparation of a medicine for improving brain amino acid metabolism and lipid metabolism.

[0029] The application also provides application of ganoderic acid A and Erinacine A in preparation of a medicine for inhibiting ferroptosis of nerve cells.

[0030] The application also provides application of ganoderic acid A and Erinacine A in preparation of a medicine for inhibiting ferroptosis of nerve cells.

[0031] Preferably, the mass ratio of ganoderic acid A and Erinacine A is preferably 2:1; the effective concentration of the medicine is preferably 2-5 mg / kg / day, further preferably 3-4 mg / kg / day, and more preferably 3.5 mg / kg / day.

[0032] Example 1 Exposure mode of ganoderic acid A in Ganoderma lucidum (Berk.) G.H.Sugita and ophiocordyceps sinensis (Berk.) G.H.Sugita Erinacine A Through screening and analysis of Ganoderma lucidum and Hericium erinaceus by TCMSP, TCMIP and ETCM three databases, Ganoderma lucidum (the database records the main active ingredients as triterpenes) was obtained as a potential target for treating diseases as a traditional Chinese medicine, and after merging and deleting duplicate values, 366 potential targets were obtained; Hericium erinaceus as a potential target for treating diseases as a traditional Chinese medicine (the database records the main active ingredient structure as bird nest anhydride type diterpene), and after merging and deleting duplicate values, 181 potential targets were obtained.

[0033] With "Alzheimer's disease", "AD", "dementia", "Alzheimer's" as keywords, the potential targets of AD in GeneCard and disgenet databases were mined. In the Genecards database, the higher the Score value, the closer the target is related to the disease. If there are too many targets, set the target with Score greater than the median value as the potential target of AD abnormality. After merging the target points of the two disease databases, delete the duplicate values to obtain the AD target points. Through the above screening, 12828 Alzheimer's disease targets were obtained from the Genecards database, and Score>11 was taken as the potential target of Alzheimer's disease, and finally 1728 Alzheimer's disease related targets were obtained. 3398 Alzheimer's disease targets were obtained from the DISGENET database. After merging the results of the two databases and deleting the duplicate values, 4067 (ADT) AD related targets were obtained.

[0034] The target set of the three was intersected (as shown in Figure 1 ) using Venny2.1.0 platform, and a Venn diagram (as shown in Figure 2 ) was drawn, and 18 intersection genes were obtained. Further, the intersection targets were submitted to the STRING 11.5 database to construct a protein interaction (PPI) network model (as shown in Figure 3 ). And using its built-in functional enrichment analysis module, with Gene Ontology biological process as the background database, the core targets were functionally annotated and enrichment analyzed. The significance level was set to P value<0.05 or FDR<0.05. The analysis results showed that these core targets were significantly enriched in the following biological processes: inflammatory response (inflammatory response), intracellular receptor signaling pathway (intracellular receptor signaling pathway) and phagocytosis (phagocytosis), and the visualization results (as shown in Figure 4 ) were shown. Network analysis showed that triterpenes and bird nest anhydride type diterpenes mainly relied on complementary exposure in the fight against AD targets.

[0035] Therefore, we plan to design an in vivo experiment to combine ganoderic triterpenes (such as ganoderic acid A) and stolon-type diterpenes (such as hericin A) from our compound library. Erinacine A As a synergistic combination, we verified its synergistic effect against AD in vivo.

[0036] Experiment Example 1: Cognitive Impairment Experiment Forty male 6-month-old APP / PS1 transgenic mice (purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were selected and subjected to one week of environmental acclimatization. They were then randomly divided into 5 groups of 8 mice each: the model group (M), the EA group (…), and the… Erinacine A Mice were fed a combination of three diets: a GA group (Ganoderic Acid A, 5 mg / kg / day), a high-dose H group (GA+EA group, 2:1 mass ratio, 5 mg / kg / day), and a low-dose L group (GA+EA group, 2:1 mass ratio, 2 mg / kg / day). Normal wild-type B6C3F1 mice (purchased from the aforementioned Spefol Corporation) served as the control group (C). Administration was performed on a normal diet and a diet rich in the compound (purchased from Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd.). After 3 months of feeding, the mice underwent 4 days of training, and the escape latency was recorded on day 5. The experiment was conducted twice a day, once in the morning and once in the afternoon, with four trials in each session. In each trial, mice were placed in the pool facing the pool wall from one of four different entry points. The time it took for the mice to find the hidden platform (escape latency) was recorded. If the platform was not found within 60 seconds, the mice were gently guided to the platform and allowed to stay there for 15 seconds to form spatial memory. Each trial within the same session was spaced 15 minutes apart. After training, a spatial exploration test was conducted on the fifth day: the platform was removed, and the mice were placed in the pool from the target quadrant. They swam freely for 60 seconds, and the number of times they crossed the original platform location, the time spent in the target quadrant, and their swimming trajectory were recorded to assess their spatial memory retention ability. The results are shown in Tables 1 and 2. Figure 5 and Figure 6 As shown.

[0037] Table 1. Latency of escape from the Morris water maze in each group of mice (x±s)

[0038] Note:" "P < 0.01 compared to the control group" indicates that the value of ...

[0039] Table 2. Number of times mice in each group crossed the platform (x±s)

[0040] Note:" "P < 0.01 compared to the control group" indicates that the value of ...

[0041] The escape latency time of group M was significantly increased compared with group C, the space exploration period after removing the platform (representing the spatial memory ability), the latency time of group M mice to find the platform was significantly increased, and the target quadrant residence time ratio was less. It is proved that the spatial learning ability and memory ability of mice in group M in this experiment decreased significantly, that is, the cognitive function was impaired. Compared with the model group, the latency time of group GA and group L was significantly decreased (p<0.01), especially group H and group L showed excellent properties of improving the spatial learning ability of mice. At the same time, the number of times of crossing the platform of group M was significantly reduced compared with group C (p<0.001). The number of times of crossing the platform of group H was significantly increased compared with group GA and group L. It is proved that group H and group L show improvement in spatial memory ability, and are superior to the single drug group.

[0042] From the above, the effect of combination of hericium A and ganoderic acid A on improving the cognitive dysfunction of APP / PS1 transgenic mice is more significant than that of single drug.

[0043] Test example 2 metabolomics experiment 32 male 6-month-old APP / PS1 transgenic mice (purchased from Spire Biotechnology (Beijing) Co., Ltd.) were selected for one week of environmental adaptive feeding, and randomly divided into 4 groups, 8 in each group. They are model group (M), ganoderic acid A-erinacine A combined high dose group (5 mg / kg / day, the ganoderic acid A and erinacine A are mixed in a mass ratio of 2:1, H group), ganoderic acid A-erinacine A combined low dose group (2 mg / kg / day, the ganoderic acid A and erinacine A are mixed in a mass ratio of 2:1, L group), and normal wild type B6C3F1 mice (purchased from the above-mentioned Spire Biotechnology Co., Ltd.) are used as control group (C). The feeding method is to feed normal feed and feed rich in extract (purchased from Jiangsu Cooperation Pharmaceutical Biological Engineering Co., Ltd.).

[0044] After 3 months of feeding, the mice were sacrificed after anesthesia, and the whole brain tissue was taken (6 biological replicates were set in each group, n=6 / group), and immediately transferred to a-80℃ ultra-low temperature refrigerator after being frozen in liquid nitrogen. After freeze-drying treatment, the sample was precisely weighed, 80% methanol solution and internal standard (2-bromophenylalanine) were added for tissue homogenization treatment. The homogenate was centrifuged at 4℃ for 15min at 12000xg, and the supernatant was transferred to a liquid phase sample bottle. After the extraction of metabolites was completed, the third party testing agency Shanghai Baqu Biological Technology Co., Ltd. carried out non-targeted metabolomics analysis based on ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS).

[0045] To analyze the metabolic characteristics of each group, the sPLS-DA score plot was used to distinguish the metabolic profiles of the high-dose group (5 mg / kg / day) and the low-dose group (2 mg / kg / day) from the glutamate model group. The results are shown in Figure 7 to Figure 9 .

[0046] The VIP values of the OPLS-DA model were used to identify the differentially expressed metabolites (DEMs) between Ganoderma lucidum and Hericium erinaceus and the glutamate model group. The Venn diagram shows the overlapping area of the element set, which shows the relationship between the differential metabolites of each group. Metabolites with VIP > 1 and P < 0.05 were focused on. Significant changes were observed in the volcano plot of metabolites after extraction intervention. Most of the DEMs belong to amino acids and their derivatives, organic acids, benzene and its substituted derivatives, heterocyclic compounds, nucleotides and their derivatives, and alcohols and amines, etc. (as shown in Figure 10 ). According to VIP ≥ 1, the key small molecule metabolites of the high concentration group were further screened. The changes of the top 20 differential metabolites in the high concentration group and the glutamate model group are shown in Figure 11 .

[0047] For each group comparison, we calculated the corresponding ratio of the quantitative value of the differential metabolites and took the logarithmic transformation with base 2; the top 10 up-regulated and down-regulated fold changes were selected for result display. The horizontal axis of the graph shows the logarithmic transformation of the change fold, and the color depth of the points represents the size of the VIP value. This analysis shows that the differential metabolites with larger change degree may have strong activation or strong inhibition of corresponding enzyme gene expression, and some metabolite regulation enzyme gene expression related to the topic can be verified accordingly. representing significance. (Note: “ ” represents 0.01 < p < 0.05, “ ” represents 0.001 < p < 0.01, and “ ” represents p < 0.001), here we take the high-dose group (H) compared with the M group as an example, and select the top 10 significantly up-regulated and down-regulated differential substances (as shown in Figure 12 ).

[0048] According to the enrichment results of the differential metabolites in the KEGG metabolic pathways, the neuroprotective activity of the extract may be due to the fact that the extract affects the brain metabolism of amino acids, especially cobalamin metabolism, fatty acid metabolism, improves immune-related pathways and ferroptosis, and inhibits the death of neural cells ( Figure 13 ).

[0049] In summary, Ganoderic acid A and Erinacine A can improve brain amino acid metabolism and lipid metabolism, and can inhibit the ferroptosis of neural cells, thereby exerting a neuroprotective effect.

[0050] From the above examples, the application provides the use of ganoderic acid A and Erinacine A in the preparation of drugs for treating neurodegenerative diseases, which can synergistically improve brain amino acid metabolism (such as glutamic acid toxicity regulation) and lipid metabolism disorder, and block the core pathological process of neuronal damage from the metabolic level.

[0051] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Application of ganoderic acid A and erinacine A in the preparation of drugs for treating neurodegenerative diseases.

2. The application according to claim 1, characterized in that, The mass ratio of ganoderic acid A to erinacine A is 2:

1.

3. The application according to claim 1, characterized in that, The effective concentration of ganoderic acid A is 5 mg / kg / day, the effective concentration of erinacine A is 5 mg / kg / day, and the effective concentration of the drug is 2-5 mg / kg / day.

4. The application according to claim 1, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

5. Application of ganoderic acid A and erinacine A in the preparation of drugs that improve brain amino acid metabolism and lipid metabolism.

6. Application of ganoderic acid A and erinacine A in the preparation of drugs that inhibit ferroptosis of nerve cells.

7. Application of ganoderic acid A and erinacine A in the preparation of neuroprotective drugs.

8. The application as described in any one of claims 5 to 7, characterized in that, The mass ratio of ganoderic acid A to erinacine A is 2:

1.

9. The application as described in any one of claims 5 to 7, characterized in that, The effective concentration of the drug is 2-5 mg / kg / day.

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