Use of gypenosides mixed components in preparation of medicines for preventing and treating alzheimer's disease

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a use of gypenoside mixed components in preparing medicines for preventing and treating Alzheimer's disease based on a new mechanism. The application shows through experiments that the gypenoside has the functions of improving microbial community structure, regulating TMAO generation, reducing the amount of TMAO entering the brain, reversing the cognition of AD mice, and improving the histopathology of the hippocampus of AD model mice. Since the gypenoside simultaneously regulates related enzymes in the tricarboxylic acid cycle and has an impact on lipid metabolism, its multiple effects can improve the metabolism of the body, and in the case of resisting hyperlipidemia, the gypenoside can also prevent and treat AD through intestinal flora to improve AD symptoms. The gypenoside mixed components can be used in preparing medicines for delaying the occurrence of AD, and / or used in preparing medicines for improving AD symptoms, and / or used in preparing medicines for regulating intestinal flora to delay the progress of degenerative diseases, and / or used in preparing medicines for regulating intestinal flora to improve the symptoms of degenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the novel use of Gynostemma pentaphyllum saponins in pharmaceutical manufacturing, and more particularly to the use of a mixture of Gynostemma pentaphyllum saponins in the preparation of drugs for the prevention and treatment of Alzheimer's disease. In particular, it relates to the use of a mixture of Gynostemma pentaphyllum saponins based on a novel mechanism in the preparation of drugs for the prevention and treatment of Alzheimer's disease. Background Technology

[0002] According to literature, Alzheimer's disease (AD) is a primary degenerative disease of the central nervous system, with the main clinical symptom being dementia syndrome. The main manifestations of brain dysfunction in AD are cognitive impairment, decreased abilities, and mental and behavioral disorders. Its clinical characteristics include: (1) high prevalence, ranking alongside malignant tumors and cardiovascular diseases as one of the three major causes of death in the elderly; statistics show that it is more common in women than men; (2) progressive memory decline is a key feature, initially presenting as short-term memory impairment, gradually progressing to long-term memory impairment, cognitive, social activity, and daily living abilities decline, and even personality changes; (3) insidious onset, long course, and progressive aggravation. Studies have shown that abnormal mutations in the APP, PS1, and PS2 genes can trigger toxic AB. 42，43，40 Increased generation, Aβ 42，43，40The continuous accumulation and aggregation of amyloid plaques in the brain and cerebrospinal fluid gradually forms plaques. As these plaques increase in size, they activate microglia, releasing cytokines and inflammatory mediators, triggering a series of inflammatory responses that exacerbate nerve damage. Alterations occur in the activity of phosphorylated kinases / phosphatases, and tau protein becomes hyperphosphorylated, leading to the formation of fibrillary tangles. This results in extensive degeneration and death of neurons in the cerebral cortex, hippocampus, and other areas, causing them to lose their normal physiological functions. Research indicates that Alzheimer's disease (AD) is a heterogeneous disease syndrome caused by multiple factors, also known as a convergence syndrome. Its clinical diagnosis largely relies on clinical manifestations, but currently, auxiliary examinations such as measuring β-amyloid and tau protein levels in cerebrospinal fluid and ApoE genotyping can also be used. Current drug development largely focuses on developing causal treatments based on various hypotheses, including β-amyloid deposition, neurofibrillary tangles, neuroinflammation, and traumatic brain injury. According to data, most pharmaceutical companies worldwide have research pipelines centered around the two widely accepted theories: the β-amyloid hypothesis and the tau protein hypothesis. On June 7, 2021, the FDA approved Aducanumab (an AB protein monoclonal antibody) for marketing, making it the only FDA-approved AD treatment drug since 2003. Traditional Chinese medicine (TCM) considers Alzheimer's disease to be related to "forgetfulness," with its pathogenesis linked to kidney essence deficiency, insufficient marrow, spleen deficiency with phlegm turbidity, and blood stasis obstructing the meridians. Researchers have used a TCM inheritance computing platform to explore the patterns of TCM medication in treating Alzheimer's disease, primarily using tonifying, diuretic, and calming herbs. Frequently used herbs include ginseng, poria cocos, polygala tenuifolia, licorice, angelica sinensis, and pinellia ternata. Studies of the effective components of TCM have shown that saponins, alkaloids, phenolic acids, and flavonoids have good preventive and therapeutic effects on Alzheimer's disease. However, it remains difficult to clearly define how the effective components of TCM, especially those processed orally through the gastrointestinal tract and other internal environments, cross the blood-brain barrier (BBB) ​​to reach their target and ultimately exert their effects. Studies have also shown that the maximum blood concentration of chemical components in traditional Chinese medicine (TCM) is less than 100 ng / ml, far lower than the minimum effective blood concentration of Western medicine. In fact, modern research indicates that the gut microbiota not only significantly influences the metabolism and absorption of TCM components, but its mediated metabolites, such as polyunsaturated fatty acids, short-chain fatty acids, and hormones, can interact with the enteric and vagus nerve systems through blood circulation. This functional abnormality may be a contributing factor to neurodegenerative diseases. In recent years, the gut-myobiota-brain axis (MGBA) has been considered a potential target for neurodegenerative diseases. The gut microbiota is closely related to neurodegenerative diseases such as Alzheimer's disease (AD), forming a complex network through interactions with the immune system, neuroendocrine system, and vagus nerve. Some studies suggest that certain TCM components may participate in MGBA regulation through this network, thereby affecting brain function and behavior.Since Nature reported in 2011 that the gut microbiota metabolite trimethylamine oxide (TMAO) could promote atherosclerosis (AS), subsequent studies have found that TMAO causes varying degrees of damage in the cognitive and behavioral domains of mice, and so on.

[0003] Based on the current state and foundation of existing technology, the inventors of this application intend to provide new uses of Gynostemma pentaphyllum saponins in pharmaceuticals, specifically providing the use of a mixture of Gynostemma pentaphyllum saponins in the preparation of drugs for the prevention and treatment of Alzheimer's disease based on a new mechanism. Summary of the Invention

[0004] The purpose of this application is to provide new uses of Gynostemma pentaphyllum saponins in pharmaceuticals based on the current state and foundation of existing technology, specifically the use of a mixture of Gynostemma pentaphyllum saponins in the preparation of drugs for the prevention and delay of Alzheimer's disease based on a new mechanism.

[0005] The Gynostemma pentaphyllum saponin mixture described in this application is as follows:

[0006]

[0007] Gynostemma pentaphyllum saponin skeleton

[0008]

[0009]

[0010] Among them, R5

[0011]

[0012] This application used a mixture of Gynostemma pentaphyllum saponins to conduct animal experiments, especially an animal experiment on how the mixture of Gynostemma pentaphyllum saponins improved AD symptoms through the brain-gut axis. The experimental results showed that the contents of choline, L-carnitine, betaine, lecithin, TMA, TMAO, imidazole propionic acid, and creatinine, which are related substances in the choline-TMA-TMAO pathway, were significantly changed in various tissues of Alzheimer's mice, and could be regulated by Gynostemma pentaphyllum saponins administered via the gastrointestinal tract.

[0013] In this application, based on multi-omics technology, we further verified a new mechanism by which the mixed components of Gynostemma pentaphyllum saponins improve cognition and repair neuronal damage in a mouse model of Alzheimer's disease through MGBA.

[0014] Animal experiments have shown that the aforementioned Gynostemma pentaphyllum saponin mixture improves AD symptoms through the gut-brain axis; it improves gut microbiota structure, regulates TMAO production, reduces TMAO influx into the brain, reverses cognition in AD mice, and improves hippocampal histopathology in AD model mice. Because the Gynostemma pentaphyllum saponin mixture improves gut microbiota structure, regulates TMAO production, reduces TMAO influencing brain, reverses cognition in AD mice, and improves hippocampal histopathology in AD model mice, and because the Gynostemma pentaphyllum saponins simultaneously regulate related enzymes in the tricarboxylic acid cycle and affect lipid metabolism, its multiple effects contribute to improving metabolism. Even in cases of hyperlipidemia, it can also prevent and treat AD through gut microbiota, thus improving AD symptoms. The gypenosides mixture of this application can be further used in the preparation of drugs that delay the onset of Alzheimer's disease (AD); and / or, in the preparation of drugs that improve AD symptoms; and / or, in the preparation of drugs that regulate gut microbiota to delay the progression of degenerative diseases; and / or, in the preparation of drugs that regulate gut microbiota to improve the symptoms of degenerative diseases.

[0015] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease and to prevent pathological damage to the brain tissue of Alzheimer's mice.

[0016] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease, and to prevent behavioral changes in Alzheimer's mice such as Y-maze, water maze, and new object recognition.

[0017] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease: the changes in the content of choline, L-carnitine, betaine, lecithin, TMA, TMAO, imidazole propionic acid and creatinine in various tissues of Alzheimer's disease mice.

[0018] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease and to prevent changes in the intestinal flora structure of Alzheimer's mice.

[0019] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease. The proportion of TMA-producing bacteria in mice with Alzheimer's disease is reduced, and the conversion from choline to TMA is reduced. TMA lyase is inhibited, and the efficiency of choline degradation into TMA is also weakened.

[0020] More specifically, the mixed components of the above-mentioned structures of Gynostemma pentaphyllum saponins of the present invention can be used to prepare drugs for the prevention and treatment of Alzheimer's disease. These drugs can prevent the downregulation of MAOa levels in the brain tissue of Alzheimer's mice, reduce the conversion rate of TMA to TMA0 in the brain, and simultaneously restore various neuroinflammatory, synaptic plasticity, and neuronal excitability / inhibition-related proteins, thereby improving cognitive abnormalities and pathological damage in AD mice.

[0021] Furthermore, the present invention provides a pharmaceutical composition comprising various compounds as described above, or pharmaceutically acceptable salts or hydrates thereof, or a composition comprising a compound of formula I above, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to prepare drugs for the prevention and treatment of Alzheimer's disease. Attached Figure Description

[0022] Figure 1 Brain and liver microsomal enzymes could catalyze the oxidation of D9-TMA to D9-TMAO (mean ± SEM, n = 6) Figure 1-1.

[0023] Figure 2 Spontaneous alternation rate of the Y-maze in each group of mice (mean±SD, n=9)

[0024] Figure2 The spontaneous alternation rate of Y maze test in mice ineach group, where: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 3 Discriminant index (mean ± SD, n = 9) for novel object recognition in each group of mice.

[0026] Figure 3Discrimination index of new object recognition experiments in groups of mice, among which: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0027] Figure 4 The latency, number of platform crossings, and time spent in the target quadrant of the water maze were measured in each group of mice.

[0028] (mean ± SD, n = 9)

[0029] Figure 4. Escape latency, numbers of crossing the virtual platform, and time spent in the target quadrant of mice in each group. Where: A: Escape latency in the water maze for each group of mice; B: Number of times mice crossed the platform for each group of mice; C: Time spent in the target quadrant for each group of mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 5 HE staining pathological morphology of hippocampal tissue from each group of mice (magnification: 20×; scale bar = 200μm).

[0031] Figure 5 HE staining of mouse hippocampal tissue in each group (Magnification: 20×; bar=200μm). Detailed Implementation

[0032] Example 1

[0033] This invention utilizes blood-brain barrier permeation experiments of TMA and TMAO, FMO3 and MAO-catalyzed trimethylamine oxidation experiments, targeted metabolomics analysis of TMA / TMAO metabolites by Gynostemma pentaphyllum saponins in AD mice, and omics sequencing technology to validate the choline-TMA-TMAO circulation pathway in mice, confirming a novel mechanism by which Gynostemma pentaphyllum saponins intervene in the TMA / TMAO metabolic pathway in AD model mice via MGBA. This invention also assesses the efficacy of Gynostemma pentaphyllum saponins administered via the gastrointestinal tract in AD mice, evaluating its effects on cognitive improvement, neuronal repair, and cholinergic system. Proteomics technology further confirms the key targets of Gynostemma pentaphyllum saponins acting on the choline-TMA-TMAO pathway.

[0034] The experimental results are as follows:

[0035] Blood-brain barrier permeability test of TMA and TMA0

[0036] TMA and TMAO are present in both the plasma and brain of normal mice. The B / P ratio of TMA is 28.58±1.75, indicating that the concentration of TMA in the brain is much higher than that in plasma. The B / P ratio of TMAO is 0.303±0.035, meaning that, in contrast to TMA, the concentration of TMAO in plasma is higher. When mice were administered the isotope-labeled D9-choline, the concentration ratio of D9-TMA and D9-TMAO in the blood and brain was measured after administration. Both D9-TMAO and D9-TMA reached their peak blood concentrations after approximately 8 hours, after which the concentrations began to decrease. The isotope labeling experiment confirmed that the B / P ratio of D9-TMA was 23.34±5.51, and the B / P ratio of D9-TMA0 was 0.099±0.011, verifying their blood-brain barrier permeability.

[0037] Experiments on trimethylamine oxidation catalyzed by 2FM03 and MAO and verification of the choline-TMA-TMAO circulation pathway in mice.

[0038] 1. FMO3 is considered the main enzyme catalyzing the oxidation of TMA to TMAO in the liver. When D9-TMA is used as the reaction substrate, both brain and liver microsomal enzymes can catalyze the oxidation of D9-TMA to D9-TMAO. Furthermore, due to the significantly higher amount of FMO3 in the liver compared to brain tissue, the resulting D9-TMAO product also shows significant differences (P < 0.0001 vs. brain). Figure 1 However, when FMO3 and MAO inhibitors were added to the incubation system respectively, the oxidation reaction was significantly inhibited.

[0039] 2. In the validation of the choline-TMA-TMAO circulation pathway in mice, choline is first broken down into TMA by gut microbiota, and then oxidized into TMAO by FMO3 in the liver. The product TMAO crosses the blood-brain barrier and enters the brain tissue. Some TMA also enters the brain tissue and is oxidized into TMAO by MAO in the brain.

[0040] Experimental animals were divided into a normal group (Control group), a model group (Model group), a donepezil-positive drug group (DNP group), a low-dose gypenosides group (GYPL group), a high-dose gypenosides group (GYPH group), and a trimethylamine lyase inhibitor group (DMB group). LC-MS / MS was used to quantitatively analyze eight TMAO series intestinal flora metabolites (TMA, TMAO, choline, L-carnitine, betaine, phosphatidylcholine, imidazole propionic acid, and creatinine) in the plasma, brain tissue, and liver tissue of mice in each group. The results showed that gypenosides could, to some extent, reverse the changes in TMAO series metabolites in AD mice. The improvement effect was more significant with high-dose gypenosides, and the effect of high-dose gypenosides in reversing the changes in each compound was very similar to that of DMB, suggesting that gypenosides and DMB may have similar regulatory mechanisms on the intestinal flora.

[0041] Tri-omics sequencing technology and in vitro antibacterial experiments validated a novel mechanism by which Gynostemma pentaphyllum saponins intervene in the TMA / TMAO metabolic pathway in AD model mice via MGBA.

[0042] Analysis of the gut microbiota of AD mice treated with gypenosides showed that gypenosides could reduce the proportion of TMA-producing bacteria in the gut of AD mice. Metagenomic analysis revealed that gypenosides intervened in the choline-TMA-TMAO metabolic pathway by regulating trimethylamine lyase. In vitro bacterial incubation experiments further verified that gypenosides could inhibit choline trimethylamine lyase activity and reduce TMA production, and that this inhibitory effect was dose-dependent.

[0043] Efficacy evaluation of Gynostemma pentaphyllum saponins administered via the gastrointestinal tract in AD mice: cognitive improvement, neuronal repair, and effects on the cholinergic system.

[0044] 1. Both low-dose (400 mg / kg) and high-dose (600 mg / kg) gavage administration of Gynostemma pentaphyllum saponins for 8 weeks can improve the learning and memory abilities and cognitive levels of AD mice to a certain extent, reduce neuronal damage, and significantly increase acetylcholine levels.

[0045] Table 1. Effects of Gynostemma pentaphyllum saponins on plasma acetylcholinesterase and acetylcholine in cognitively impaired mice.

[0046] (mean ± SD, n = 5)

[0047] Tablel The effect of gypenosides on the contents of acetylcholinesterase and acetylcholine in plasma of mice with cognitive impairment (mean ± SD, n = 5)

[0048]

[0049] Note: Compared with the control group, a p<0.05, b P<0.01; compared with the model group, c P<0.05, d P<0.01.

[0050] 5. Proteomics Analysis of DIA: Targets and Pathways of Gynostemma pentaphyllum Total Saponins in Regulating AD Through the Gut-Brain Axis

[0051] Compared with the normal group, 46 significantly differentially expressed proteins were identified in the model group, mainly involving synaptic dysfunction, calcium signaling imbalance and abnormal cholinergic neurotransmitter transmission, suggesting that the establishment of AD model affected synaptic function, signal transduction and cholinergic system damage.

[0052] The trimethylamine lyase inhibitor DMB significantly affected protein expression in model mice, identifying 29 differentially expressed proteins. GO and KEGG enrichment analyses indicated that DMB primarily affects neurofilament protein stability, synaptic transmission, myelination, glycine-mediated inhibitory synaptic transmission, and immune inflammation regulation. Proteins such as Nefl, Nefm, Nefh, Prph, Slc6a5, Glrb, and Serpinb1b are likely key targets of DMB's effects through the gut-brain axis. PPI network analysis further confirmed that DMB may improve the pathological state of Alzheimer's disease (AD) by reducing TMA levels in the brain, thereby regulating synaptic function, reducing Aβ accumulation, and modulating immune responses.

[0053] The GYPL and GYPH groups had different effects on protein expression in the model group. The GYPL group identified 16 differentially expressed proteins, mainly enriched in chromatin modification and neuropeptide signaling pathways, while the GYPH group identified 22 differentially expressed proteins, primarily involved in synaptic plasticity, neuroinflammation, and neuronal cytoskeleton stability. Further comparison of differentially expressed proteins between GYPH and DMB revealed 15 shared differentially expressed proteins with consistent regulatory directions, including Prph, Nefl, Nefm, Nefh, Slc6a5, G1rb, and Serpinb1b. This suggests that high-dose Gynostemma pentaphyllum saponins may regulate brain tissue-related proteins through a DMB-like mechanism, intervening in the gut-brain axis to improve the neuropathological features of Alzheimer's disease (AD). In contrast, the protein overlap between GYPL and DMB was low, suggesting that low-dose Gynostemma pentaphyllum saponins may exert their effects through a different mechanism.

Claims

1. The use of a mixture of Gynostemma pentaphyllum saponins in the preparation of drugs for the prevention and delay of Alzheimer's disease, wherein the mixture of Gynostemma pentaphyllum saponins comprises: Gynostemma pentaphyllum saponin skeleton Among them, R5 2. The use according to claim 1, characterized in that: The Alzheimer's disease mentioned refers to the pathological damage to the brain tissue of Alzheimer's mice.

3. The use according to claim 1, characterized in that, The Alzheimer's disease described refers to behavioral changes in the Y-maze, water maze, and new object recognition of Alzheimer's mice.

4. The use according to claim 1, characterized in that, The Alzheimer's disease described refers to the changes in the content of choline, L-carnitine, betaine, lecithin, TMA, TMAO, imidazole propionic acid, and creatinine in various tissues of Alzheimer's mice.

5. The use according to claim 1, characterized in that, The Alzheimer's disease mentioned refers to the alteration of the gut microbiota structure in Alzheimer's mice.

6. The use according to claim 1, characterized in that, The Alzheimer's disease is characterized by a reduced proportion of TMA-producing bacteria in Alzheimer's mice, resulting in a decrease in the conversion of choline to TMA; TMA lysin is inhibited, and the efficiency of choline degradation into TMA is also weakened.

7. The use according to claim 1, characterized in that, The Alzheimer's disease described here involves downregulating the level of MAOa in the brain tissue of Alzheimer's mice, reducing the conversion rate of TMA to TMAO in the brain, and simultaneously restoring various neuroinflammatory, synaptic plasticity, and neuronal excitability / inhibition-related proteins, thereby improving cognitive abnormalities and pathological damage in AD mice.

8. A pharmaceutical composition comprising various compounds of the Gynostemma pentaphyllum saponin mixture as described in claim 1, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

9. The use of the pharmaceutical composition as described in claim 8 in the preparation of a drug for the prevention and treatment of Alzheimer's disease.

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