Preparation method of typhaneoside and protective effect of typhaneoside on relieving parkinson nerve injury

Abstract

The application provides a preparation method of typhaneoside and a protective effect of the typhaneoside on relieving Parkinson nerve injury, and belongs to the technical field of medicines.The application comprises the following steps: (1) mixing typha powder and ethanol, ultrasonic-assisted extraction, and collecting the extraction liquid, which is a typha extract; and (2) separating and purifying the typha extract to obtain typha total flavones.The typha total flavones obtained by the application have high purity, and the typha total flavones can relieve PD nerve injury by reducing PD oxidative stress and intestinal flora composition and functional disorder.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a method for preparing Typhae Flavonoids and its protective effect in alleviating nerve damage in Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a chronic, progressive neurodegenerative disease whose incidence and prevalence have risen rapidly over the past 20 years, currently averaging 1-2% globally, making it a global health problem and a significant public health challenge. The main clinical manifestations of PD include bradykinesia, muscle rigidity, resting tremor, and postural and gait disturbances, which gradually worsen as the disease progresses. In addition to motor disorders, PD patients also experience a range of non-motor symptoms, such as cognitive impairment, gastrointestinal dysfunction, sleep disturbances, and autonomic dysfunction. The pathological features of PD include degeneration and loss of dopaminergic neurons in the substantia nigra (SNc) and other brain regions, decreased striatal dopamine levels, and the formation of Lewy bodies within neurons. These pathological changes lead to neuronal dysfunction in the brain, resulting in the typical clinical symptoms of PD. Although PD has been extensively studied, its pathogenesis remains not fully understood. Numerous studies have shown that the occurrence and development of Parkinson's disease involves multiple factors, including oxidative stress, mitochondrial dysfunction, and abnormal aggregation of α-synuclein. In addition, with the development of the "gut origin theory" of PD, intestinal disorders are also considered to be one of the pathogenesis mechanisms of PD.

[0003] The gut microbiota, as the core of the gut microecological system, has evolved alongside the host and is an integral part of the human body. One of the most significant characteristics of the gut microbiota is its stability; imbalance can lead to various intra- and extra-gut diseases. Therefore, maintaining gut microecological balance is crucial for humans to resist infectious diseases caused by intestinal pathogens. Furthermore, the gut microbiota participates in multiple neurochemical pathways through the highly interconnected "gut-brain axis," also known as the microbiota-gut-brain axis. In recent years, increasing research has found that gut microbiota dysbiosis may trigger or exacerbate the progression of Parkinson's disease (PD). Early studies found that PD patients commonly experience various gastrointestinal symptoms such as constipation and abnormal intestinal motility, often appearing before disease diagnosis. Subsequent epidemiological studies have shown that these gastrointestinal symptoms not only increase the risk of PD but also worsen existing symptoms. With further research, it has become increasingly clear that PD is not merely a brain disease but a systemic disease closely related to the gut microbiota.

[0004] Typha pollen, a traditional Chinese medicine, has a long history and abundant resources. Its main active components are flavonoids, which have been shown to possess various pharmacological activities with relatively low side effects. However, there are no reports on whether total flavonoids from Typha pollen can be used for Parkinson's disease (PD) and dopaminergic neuron damage. This suggests that its potential value deserves further exploration and development, and it may demonstrate excellent therapeutic effects in more fields. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for preparing Typhae pollen flavonoids and their protective effect in alleviating Parkinson's disease-related nerve damage. The total Typhae pollen flavonoids obtained by this method can reduce oxidative stress and gut microbiota composition and function disorders in Parkinson's disease, thereby alleviating nerve damage.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing total flavonoids from Typha pollen, comprising the following steps:

[0008] (1) Mix cattail pollen powder with ethanol, extract with ultrasonic assistance, and collect the extract to obtain cattail pollen extract;

[0009] (2) The extract of Typha pollen was separated and purified to obtain total flavonoids from Typha pollen.

[0010] Preferably, the material-to-liquid ratio of the ethanol extraction in step (1) is 1:55-65, and the volume fraction of the ethanol is 30%-40%.

[0011] Preferably, the ultrasonic power in step (1) is 500-700W, the ultrasonic time is 20-30min, and the ultrasonic temperature is 30-45℃.

[0012] Preferably, the separation and purification in step (2) is performed using a macroporous resin method.

[0013] Preferably, the separation and purification steps of the macroporous resin method are as follows: dilute the cattail pollen extract to obtain a cattail pollen extract solution, add the cattail pollen extract solution to a macroporous resin column, elute, collect the eluent, concentrate, and dry to obtain total cattail pollen flavonoids.

[0014] Preferably, the concentration of the cattail pollen extract solution is 20-30 mg / mL.

[0015] Preferably, the elution is carried out sequentially using water and 45%–70% ethanol, and the drying is performed by freeze drying.

[0016] Preferably, the total flavonoids in the Typha pollen mainly include apigenin, hesperidin, quercetin, quercetin-3-O-glucoside, kaempferol-3-O-rutin, rutin, narcissin, isoquercetin, and typhain.

[0017] The present invention also provides total flavonoids from Typha pollen obtained by the preparation method described above.

[0018] This invention also provides the application of total flavonoids from Typhae pollen in the preparation of drugs to alleviate motor dysfunction and dopaminergic neuron damage in Parkinson's disease.

[0019] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a method for preparing Typhae pollen flavonoids and their protective effect in alleviating Parkinson's disease-related neurological damage. The total Typhae pollen flavonoids obtained by the present invention have high purity. Utilizing the obtained total Typhae pollen flavonoids to combat oxidative stress in PD mice, the results show that the protective intervention of total Typhae pollen flavonoids alleviates MPTP-induced oxidative stress in the brain tissue of PD mice, thereby promoting neuronal cell activity and improving motor dysfunction in PD mice. Utilizing the obtained total Typhae pollen flavonoids to regulate MPTP-induced gut microbiota dysbiosis in PD mice, the results show that total Typhae pollen flavonoids can effectively regulate the diversity, structure, and function of the gut microbiota in MPTP-induced PD mice. The results of the present invention indicate that total Typhae pollen flavonoids alleviate PD neurological damage by reducing PD oxidative stress and gut microbiota composition and function disorders. Attached Figure Description

[0020] Figure 1 These are the main compounds extracted and purified from total flavonoids of Typha pollen;

[0021] Figure 2 This refers to the effect of total flavonoids from Typhae pollen on antagonizing serum oxidative stress in PD mice, as shown in Experiment Example 1.

[0022] Figure 3 This refers to the effect of total flavonoids from Typhae pollen on oxidative stress in the brain tissue of PD mice in Experiment Example 1.

[0023] Figure 4 This is a diagram showing how total flavonoids from Typhae pollen alleviated motor function impairment caused by Parkinson's disease in Experiment Example 1.

[0024] Figure 5 This refers to the effect of total flavonoids from Typhae pollen in alleviating dopaminergic neuron damage in PD mice, as shown in Experiment Example 1.

[0025] Figure 6 The results of the effect of total flavonoids from Typha pollen on SCFAs levels in Experiment Example 2;

[0026] Figure 7 This is the result of total flavonoids from Typhae pollen regulating MPTP-induced gut microbiota dysbiosis in PD mice in Experiment Example 3;

[0027] Figure 8 This is an example of the effect of total flavonoids from Typha pollen on the differences in gut microbiota composition among different groups of mice in Experiment Example 3.

[0028] Figure 9 This is a KEGG enrichment diagram of total flavonoids from Typhae pollen for differential intestinal metabolites among different groups of mice in Experiment Example 4.

[0029] Figure 10 This study examines the effects of total flavonoids from Typha pollen on intestinal metabolic pathways in different groups of mice in Experiment Example 4. Detailed Implementation

[0030] This invention provides a method for preparing total flavonoids from Typha pollen, comprising the following steps:

[0031] (1) Mix cattail pollen powder with ethanol, extract with ultrasonic assistance, and collect the extract to obtain cattail pollen extract;

[0032] (2) The extract of Typha pollen was separated and purified to obtain total flavonoids from Typha pollen.

[0033] In this invention, cattail pollen powder is mixed with ethanol, and ultrasonic-assisted extraction is performed. The extract is collected to obtain cattail pollen extract. The cattail pollen is selected from high-altitude areas, preferably from the Lalu Wetland. After pulverizing, the cattail pollen is passed through a 100-140 mesh sieve, preferably a 110-130 mesh sieve, and more preferably a 120 mesh sieve. The ratio of cattail pollen to ethanol for extraction is 1:55-65, preferably 1:57-63, and more preferably 1:60. The volume fraction of ethanol is 30%-40%, preferably 33%-36%, and more preferably 34.9%. The ultrasonic power is 500-700W, preferably 550-650W, and more preferably 600W. The ultrasonic time is 20-30 min, preferably 22-23 min, and more preferably 23.2 min. The ultrasonic temperature is 30-45℃, preferably 35-40℃, and more preferably 38.4℃.

[0034] In this invention, the extract of Typha pollen is separated and purified to obtain total Typha pollen flavonoids. The separation and purification are carried out using a macroporous resin method. The steps of the macroporous resin method for separation and purification are as follows: the Typha pollen extract is diluted with ultrapure water to obtain a Typha pollen extract solution with a concentration of 20-30 mg / mL, preferably 23-28 mg / mL, and more preferably 25 mg / mL. The Typha pollen extract solution is added to a macroporous resin column, and ultrapure water and 45%-70% ethanol, preferably 50%-60% ethanol, and more preferably 55% ethanol are used for elution. The elution method is as follows: after elution with ultrapure water to remove non-specifically adsorbed impurities, elution is carried out with 55% ethanol, controlling the flow rate at 1-2 mL / min, until the effluent is colorless and transparent. Collect the ethanol eluent, concentrate the ethanol eluent to 1 / 4 to 1 / 3 of its original volume by rotary evaporation at 35-45℃ to obtain a concentrated extract, pre-freeze the concentrated extract at -80℃ for 1 day, and then dry it in a freeze dryer at -50℃ and a vacuum of 10-100Pa to obtain total flavonoids from Typha pollen.

[0035] In this invention, the total flavonoids in Typha pollen mainly include apigenin, hesperidin, quercetin, quercetin-3-O-glucoside, kaempferol-3-O-rutin glycoside, rutin, narcissin, isoquercetin, and typhain, the structural formula of which is shown below. Figure 1 .

[0036] The present invention also provides total flavonoids from Typha pollen obtained by the preparation method described above.

[0037] This invention also provides the application of total flavonoids from Typhae pollen in the preparation of drugs to alleviate motor dysfunction and dopaminergic neuron damage in Parkinson's disease.

[0038] In this invention, total flavonoids from Typhae pollen exert an ameliorative effect on motor dysfunction in PD mice by alleviating MPTP-induced motor function impairment and regulating intestinal flora and metabolic disorders in MPTP-induced PD mice.

[0039] In this invention, cattail pollen from a high-altitude region (Lalu Wetland) is selected as the raw material for the extraction of total cattail pollen flavonoids. Cattail pollen from high-altitude regions has a higher flavonoid content and antioxidant properties. As a prebiotic, total cattail pollen flavonoids can also enrich beneficial intestinal bacteria (Muribaculum, Turicimonas, Bacteroides, Muribaculaceae, Rodentibacter, and Parabacteroides, etc.) and inhibit potentially harmful bacteria (Acutalibacter, Firmicutes, and Helicobacter, etc.). It effectively promotes intestinal homeostasis by regulating the structure and function of intestinal flora, and mediates the "gut-brain axis" through the enrichment of key intestinal flora (Parabacteroides and Bacteroides), thereby exerting its neuroprotective effect on PD mice.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] After being pulverized, the pollen was passed through a 120-mesh sieve to obtain pollen powder. The pollen powder was mixed with 34.9% ethanol at a ratio of 1:60 and ultrasonically extracted for 23.2 minutes at an ultrasonic power of 600W and an ultrasonic temperature of 38.4℃. The extract was collected to obtain pollen extract. Under these conditions, the average yield of pollen extract was 13.34 mgRE / g.

[0043] The extract of *Typha orientalis* was diluted with ultrapure water to obtain a crude flavonoid solution with a concentration of 25 mg / mL. First, the crude flavonoid solution was eluted with ultrapure water to remove non-specifically adsorbed impurities. Then, it was eluted with 55% ethanol at a flow rate of 1–2 mL / min until the eluent was colorless and transparent. The ethanol eluent was collected and concentrated to 1 / 4–1 / 3 of its original volume by rotary evaporation at 40℃±5℃ to obtain a concentrated extract. The concentrated extract was pre-frozen at -80℃ for 1 day and then freeze-dried at -50℃ under a vacuum of 40 Pa to obtain purified total flavonoids from *Typha orientalis*. The results showed that the flavonoid content in the purified total flavonoids increased from 0.04 gRE / g to 0.42 gRE / g.

[0044] Example 2

[0045] After being pulverized, the pollen was passed through a 130-mesh sieve to obtain pollen powder. The pollen powder was mixed with 30% ethanol at a ratio of 1:55 and ultrasonically extracted for 30 minutes at a power of 500W and a temperature of 30℃. The extract was collected to obtain pollen extract. Under these conditions, the average yield of pollen extract was 13.13 mgRE / g.

[0046] The extract of *Typha orientalis* was diluted with ultrapure water to obtain a crude flavonoid solution with a concentration of 20 mg / mL. First, the crude flavonoid solution was eluted with ultrapure water to remove non-specifically adsorbed impurities. Then, it was eluted with 45% ethanol at a flow rate of 1–2 mL / min until the eluent was colorless and transparent. The ethanol eluent was collected and concentrated to 1 / 4–1 / 3 of its original volume by rotary evaporation at 40℃±5℃ to obtain a concentrated extract. The concentrated extract was pre-frozen at -80℃ for 1 day and then freeze-dried at -50℃ under a vacuum of 10 Pa to obtain purified total flavonoids from *Typha orientalis*. The results showed that the flavonoid content in the purified total flavonoids increased from 0.04 gRE / g to 0.38 gRE / g.

[0047] Example 3

[0048] After being pulverized, the pollen was passed through a 140-mesh sieve to obtain pollen powder. The pollen powder was mixed with 40% ethanol at a ratio of 1:65 and ultrasonically extracted for 20 minutes at a power of 700W and a temperature of 45℃. The extract was collected to obtain pollen extract. Under these conditions, the average yield of pollen extract was 12.98 mgRE / g.

[0049] The extract of *Typha orientalis* was diluted with ultrapure water to obtain a crude flavonoid solution with a concentration of 30 mg / mL. First, the crude flavonoid solution was eluted with ultrapure water to remove non-specifically adsorbed impurities. Then, it was eluted with 70% ethanol at a flow rate of 1–2 mL / min until the eluent was colorless and transparent. The ethanol eluent was collected and concentrated to 1 / 4–1 / 3 of its original volume by rotary evaporation at 40℃±5℃ to obtain a concentrated extract. The concentrated extract was pre-frozen at -80℃ for 1 day and then placed in a freeze dryer at -50℃ and a vacuum of 100 Pa for freeze drying to obtain purified total flavonoids from *Typha orientalis*. The results showed that the flavonoid content in the purified total flavonoids increased from 0.04 gRE / g to 0.40 gRE / g.

[0050] Experimental Example 1

[0051] The total flavonoids from Typha pollen obtained by the preparation method in Example 1 were used as samples. The effects of total flavonoids from Typha pollen on oxidative stress response in mice, damage to dopaminergic neurons, and changes in gut microbiota were evaluated.

[0052] A mouse model of PD induced by MPTP was established: Mice were acclimatized for 7 days and then intraperitoneally injected with MPTP (20 mg / kg / d) once daily for 7 days. The flavonoid control group and the normal control group were simultaneously intraperitoneally injected with the same amount of PBS. Mice were then divided into groups (n=15 per group): flavonoid control group (total flavonoids from Typha orientalis) by gavage (FLN); normal control group (PBS by gavage) (CON); PD model group (PBS by gavage) (MPTP); flavonoid intervention group (total flavonoids from Typha orientalis) by gavage (MPTP+FLN); and fecal microbiota transplantation group (fecal microbiota suspension prepared from MPTP+FLN group by gavage) (FMT). Prophylactic administration of flavonoids was given at 200 mg / kg / d by gavage (starting from day 7 in the FMT group) from day 1 of the experiment until the end of the experiment. Starting from day 7 of the experiment, fresh feces were collected from the flavonoid intervention group, homogenized with sterile PBS (1g:5mL), and then filtered after centrifugation (1000rpm, 4℃, 1min) to obtain bacterial solution for fecal microbiota transplantation (prepared fresh for each use).

[0053] 1.1 Effects of total flavonoids from Typha pollen on antioxidant stress in PD mice

[0054] The activities of MDA, SOD and GPX antioxidant enzymes, and total antioxidant capacity (T-AOC) in the serum and brain tissue of the five groups of mice were measured.

[0055] MDA assay: The thiobarbituric acid (TBA) method was used. A red complex was formed by the reaction with TBA, and the absorbance was measured at a wavelength of 532 nm. The sample (serum or brain tissue homogenate supernatant) was mixed with SDS, acetic acid and TBA, heated in a 95°C water bath for 60 minutes, cooled, and then a mixed solution of n-butanol and pyridine was added. The mixture was shaken, centrifuged, and the supernatant was used to measure the absorbance.

[0056] Superoxide dismutase (SOD) activity assay: The WST-1 method was used, utilizing the SOD-catalyzed superoxide anion dismutation to generate oxygen and hydrogen peroxide, while inhibiting the formation of yellow compounds by WST-1. A mixed solution of xanthine, xanthine oxidase, and WST-1 was added to the sample (serum or brain tissue homogenate supernatant), and the absorbance was measured at 450 nm to calculate the SOD activity.

[0057] GPX activity assay: The amount of GSH consumed was measured. After adding H2O2 and GSH solution to the sample (serum or brain tissue homogenate supernatant) and reacting, DTNB solution was added to react with the remaining GSH to generate a yellow product. The absorbance was measured at a wavelength of 412 nm to calculate the GPX activity.

[0058] Total antioxidant capacity (T-AOC) assay: The FRAP method was used to reduce Fe under acidic conditions. 3+ - Tripyridine triazine (Fe 3+-TPTZ) produces blue Fe 2+ -TPTZ, by measuring absorbance at a wavelength of 593nm, can obtain the total antioxidant capacity in a sample (serum or brain tissue homogenate supernatant).

[0059] like Figure 2 As shown, compared with the control group, the MPTP group showed significantly increased serum MDA levels and significantly decreased SOD and GPX enzyme activities. Compared with the MPTP group, the flavonoid intervention group showed significantly decreased serum MDA levels and significantly increased SOD enzyme activity and T-AOC levels; while the FMT group showed significantly increased serum SOD and GPX enzyme activities and T-AOC levels. These results indicate that both total flavonoid intervention and fecal microbiota transplantation intervention can effectively increase antioxidant enzyme activity, thereby reducing MPTP-induced systemic oxidative stress in PD mice. The results of oxidative stress indicators in mouse brain tissue in this study are as follows: Figure 3 As shown, compared with the control group mice, the T-AOC level and SOD enzyme activity in the brain tissue of MPTP group mice were significantly reduced, while the MDA content was significantly increased, indicating that oxidative stress damage occurred in the brain tissue of PD mice. Flavonoid intervention increased the GPX and SOD enzyme activity and decreased the MDA level in the brain tissue of PD mice, indicating that the protective intervention of total flavonoids from Typhae pollen alleviated the oxidative stress in the brain tissue of PD mice induced by MPTP, thereby promoting neuronal cell activity and improving motor dysfunction in PD mice.

[0060] 1.2 Total flavonoids from Typhae pollen alleviate motor function impairment in MPTP-induced PD mice

[0061] Three behavioral experiments were used to assess motor function deficits in PD mice: open field test, pole climbing test, and string hanging test.

[0062] Open field experiment: A camera was installed 40-60 cm above the open field, and video recording was turned on. The mouse was gently and smoothly removed from the cage by holding it in the palm of the hand and placed in the center of the open field. The experimental animal was allowed to move freely in the experimental box, and the activity of the mouse in the open field was recorded for 10 minutes.

[0063] Pole climbing test: Place the mouse head-up on the top of a vertical wooden pole (50cm high, 2cm in diameter). Record the time it takes for the mouse to turn head-down (T-turn) and to reach the floor (T-total) (three tests were conducted at 30-minute intervals, and the average time was taken).

[0064] Hanging Experiment: Install a camera 40-60cm away from the suspension device, ensuring the entire device is clearly visible through the camera feed. Place the mouse in the metal frame. Gently and smoothly flip the frame over (allowing the mouse to grip the frame with all four limbs). After fully flipping, hold the frame with one hand and move it back and forth in a straight line. End the experiment when the mouse can no longer maintain its grip and falls from the frame into the box (repeat the experiment three times at 30-minute intervals, and take the average time).

[0065] like Figure 4 As shown, compared with the control group, the MPTP group mice exhibited significantly reduced movement distance in the open field test, longer turning time and crawling time from the top to the bottom of the pole in the pole climbing test, and shorter persistence time in the string hanging test, indicating that MPTP successfully induced motor function impairment in mice. Behavioral experiments in the flavonoid intervention group showed that total flavonoids from Typhae pollen had a significant protective effect against MPTP-induced motor function impairment in mice, while the FMT group mice also showed some improvement, but the difference was not significant. This suggests that total flavonoids from Typhae pollen may have neuroprotective effects, significantly alleviating or reversing motor function impairment in PD mice, and this protective effect may be related to gut microbiota.

[0066] 1.3 Total flavonoids from Typhae pollen alleviate MPTP-induced dopaminergic neuron damage in PD mice

[0067] Mouse brain tissue was obtained via cardiac perfusion and fixed in 4% paraformaldehyde, embedded in paraffin, and then subjected to immunohistochemical staining. Images of the sections were collected and analyzed. Results are as follows: Figure 5 As shown, compared with the control group, the TH region of the SNc region in the MPTP group mice was significantly higher. + Neuronal levels decreased by 22.35%, while compared with the MPTP group, the SNc region TH level in mice in the flavonoid intervention group and FMT group was significantly higher. + Neuronal recovery rates were 37.12% and 12.67%, respectively. These results indicate that MPTP successfully induced damage to dopaminergic neurons in PD mice and validated the feasibility of the PD model establishment method used in this experiment. Furthermore, total flavonoids from Typhae pollen exhibit a protective effect against dopaminergic neurons, providing evidence for the previous findings that total flavonoids from Typhae pollen alleviated MPTP-induced motor dysfunction in mice. In addition, fecal microbiota transplantation alleviated dopaminergic neuronal damage to some extent, suggesting that regulating gut microbiota may be one of the pathways by which flavonoids exert their neuroprotective effects.

[0068] Experiment Example 2 evaluates the effect of total flavonoids from Typha pollen on intestinal SCFA levels.

[0069] Mouse fecal samples frozen at -80℃ were thawed on ice. 100 mg of the sample was weighed and added to 400 μL of ultrapure water and one steel ball. The mixture was then homogenized using a high-throughput cryogenic grinder (60 Hz, 1 min, 4℃) and allowed to stand at 4℃ for 1 h for extraction. After centrifugation (12000 rpm, 15 min, 4℃), 200 μL of the supernatant was added to 0.02 mL of 25% metaphosphoric acid solution containing the internal standard 2-ethylbutyric acid. The mixture was placed on ice for 30 min and then centrifuged again (12000 rpm, 15 min, 4℃). The supernatant was filtered through a 0.22 μm filter and then placed into GC vials for analysis.

[0070] SCFAs are the main metabolites produced by the gut microbiota and play a crucial role in maintaining intestinal function. For example... Figure 6 As shown, compared with the control group, the levels of acetic acid, propionic acid, isobutyric acid, and isovaleric acid in the feces of mice in the MPTP model group were significantly decreased (p<0.05). Compared with the MPTP group, the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in the feces of mice in the flavonoid intervention group were restored (p<0.05); while the results of the FMT group showed that the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in their feces slightly increased, but the difference was not significant (p>0.05).

[0071] Experimental Example 3: Total flavonoids from Typhae pollen regulate MPTP-induced gut microbiota dysbiosis in PD mice

[0072] Metagenomic sequencing was performed on the feces of the five groups of mice in Experiment 1, and the sequencing results were analyzed. This study analyzed the structure and function of the gut microbiota in each group of mice using metagenomics. Figure 7 The results showed that flavonoid intervention could effectively regulate the diversity, structure, and function of the gut microbiota in MPTP-induced PD mice. Compared with the control group, the Shannon index and Simpson index of MPTP group mice were increased to some extent, but the difference was not significant (p>0.05).

[0073] Figure 8 The data shows bacterial abundance at the species level. At the genus level, compared with the control group, the relative abundance of Parabacteroides, Muribculaceae, Rodentibacter, Muribculum, Bacteroides, Turicimonas, Firmicutes, and Clostridium in the MPTP group showed a decreasing trend, while the relative abundance of inflammation-related bacteria such as Proteus, Helicobacter, Acetatifactor, Mucispirillum, and Acutalibacter showed an increasing trend.

[0074] Experiment Example 4: Total Flavonoids from Typhae Pollen Regulate Metabolic Disorders in MPTP-Induced PD Mice

[0075] Metabolites are often key factors affecting nervous system function and are also considered important mediators of communication between the host and the microbiome. Furthermore, metabolites can reflect the overall physiological state of the body. Non-targeted metabolomics analysis was performed on the feces of mice in each group. Based on the results of enrichment analysis, combined with correlation analysis of some pathological symptoms of PD, KEGG enrichment maps of differentially expressed metabolites were constructed to further explore changes in these metabolic pathways. The results are as follows: Figure 9 As shown in the figure, the effects of total flavonoids from Typhae pollen on metabolic pathways in PD mice were mainly concentrated in: tyrosine biosynthesis and metabolism, valine, leucine, and isoleucine biosynthesis and degradation, fatty acid biosynthesis and metabolism, arachidonic acid metabolism, phenylalanine, oxidative phosphorylation, the AMPK signaling pathway, the ferroptosis signaling pathway, and the lysosomal signaling pathway. The significant enrichment of these metabolic pathways suggests that total flavonoids from Typhae pollen may act through multiple pathways, thereby improving the symptoms of PD mice. Furthermore, the AMPK and ferroptosis pathways are interconnected and mutually influential with other pathways besides amino acid metabolism pathways. Figure 10 This correlation and interaction may be an important mechanism by which total flavonoids from Typhae pollen exert a comprehensive effect on PD mice.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of total flavonoids from Typhae pollen in the preparation of drugs to alleviate motor dysfunction and dopaminergic neuron damage in Parkinson's disease, characterized in that, The method for preparing the total flavonoids from Typha pollen includes the following steps: (1) Mix cattail pollen powder with ethanol, extract with ultrasonic assistance, and collect the extract to obtain cattail pollen extract; (2) The extract of cattail pollen was separated and purified to obtain total flavonoids of cattail pollen.

2. The application according to claim 1, characterized in that, In step (1), the material-to-liquid ratio for ethanol extraction is 1:55~65, and the volume fraction of ethanol is 30%~40%.

3. The application according to claim 1, characterized in that, The ultrasonic power in step (1) is 500~700W, the ultrasonic time is 20~30min, and the ultrasonic temperature is 30~45℃.

4. The application according to claim 1, characterized in that, The separation and purification in step (2) uses the macroporous resin method.

5. The application according to claim 4, characterized in that, The separation and purification steps of the macroporous resin method are as follows: dilute the cattail pollen extract to obtain a cattail pollen extract solution, add the cattail pollen extract solution to a macroporous resin column, elute, collect the eluent, concentrate, and dry to obtain total cattail pollen flavonoids.

6. The application according to claim 5, characterized in that, The concentration of the cattail extract solution is 20~30 mg / mL.

7. The application according to claim 5, characterized in that, The elution was carried out sequentially using water and 45%~70% ethanol, and the drying was carried out by freeze drying.

8. The application according to any one of claims 1 to 7, characterized in that, The total flavonoids in Typha pollen mainly include apigenin, hesperidin, quercetin, quercetin-3-O-glucoside, kaempferol-3-O-rutin, rutin, narcissin, isoquercetin, and typhain.

Images