Application of tangerine seed extract in preparation of medicine for treating Parkinson's disease

Abstract

The invention relates to the technical field of medicines, in particular to application of a tangerine seed extract in preparation of a medicine for treating Parkinson's disease. The research on a Parkinson's disease model mouse finds that CRSE6 # of a 90% ethanol part of tangerine seeds can significantly improve the neuron function of the mouse and improve the athletic ability of the mouse, so that the invention provides the application of the tangerine seed extract in preparation of the medicine for treating the Parkinson's disease.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of a citrus serrulata extract in preparing a medicine for treating Parkinson's disease. Background Art

[0002] Parkinson's disease, abbreviated as PD, is one of the fastest growing neurodegenerative diseases in the world, which seriously affects the quality of life of patients and imposes a huge burden on society and the medical system. Currently, the treatment of Parkinson's disease mainly relies on dopamine drugs. However, long-term use of these drugs often leads to tolerance in patients, the efficacy gradually decreases, and the drug dose needs to be increased to control symptoms. This treatment strategy not only increases the economic burden on patients, but also comes with a higher risk of drug toxicity. Although there are some auxiliary treatments, there is still a lack of effective therapies that can delay the progression of the disease.

[0003] Existing technologies in the field of Parkinson's disease treatment mainly rely on dopamine replacement therapy, such as levodopa and dopamine receptor agonists, which control the motor symptoms of PD by replenishing the lack of dopamine in the brain. However, with the progression of the disease and long-term drug use, the efficacy of existing drugs gradually decreases, and patients often need to increase the dose to maintain the treatment effect, which will lead to drug toxicity and tolerance problems, seriously affecting the quality of life of patients.

[0004] In addition to traditional chemical drugs, plant extracts have attracted widespread attention as natural drug alternatives in the treatment of neurodegenerative diseases in recent years. The application of Vitex ethyl acetate extract is a typical example of the existing traditional Chinese medicine for the treatment of Parkinson's disease. In this patent, Vitex ethyl acetate extract demonstrated significant neuroprotective effects, reducing neuroinflammation and neuronal damage, and improving the motor ability of Parkinson's disease model mice. However, although Vitex ethyl acetate extract showed good effects in preclinical studies, there is a lack of research on drug specificity and drug targets. In addition, its active ingredients and mechanism of action still need to be further clarified, which limits the possibility of its clinical transformation.

[0005] At present, there are many medicinal effects of Chinese herbal extracts on Parkinson's disease. For example, Vitex rotundifolia extract has anti-inflammatory and neuroprotective effects, but its mechanism of action is relatively broad and lacks specificity. Existing studies have not yet clarified its specific targeting effect on α-syn, a key pathological protein in Parkinson's disease, making it difficult to accurately intervene in the progression of Parkinson's disease at the molecular level. Vitex rotundifolia has a wide range of effects, but for Parkinson's disease, a disease involving α-syn aggregation, the existing technology lacks effective targeted treatment methods and cannot fundamentally solve α-syn-induced neuronal dysfunction. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of a citrus aurantium extract in preparing a drug for treating Parkinson's disease.

[0007] The invention discloses an application of a tangerine seed extract in preparing a drug for treating Parkinson's disease. The tangerine seed extract is an alcohol extract obtained by alcohol extraction of a tangerine seed material.

[0008] Preferably, the Citrus aurantium extract contains Zapoterin.

[0009] Preferably, the drug contains the Citrus aurantium extract as the only active ingredient.

[0010] Preferably, the medicament reduces neuronal dysfunction.

[0011] Preferably, the medicament improves motor performance.

[0012] Preferably, the drug regulates calcium homeostasis.

[0013] Preferably, the preparation method of the core tangerine extract is: soaking the tangerine seed medicinal material with a first alcohol solvent, using 5 mL to 10 mL of the first alcohol solvent for hot reflux extraction per gram of the medicinal material, filtering the obtained liquid, recovering the alcohol solvent to obtain a crude extract, loading the crude extract onto a polystyrene-divinylbenzene filler on a column after activation, eluting with a second alcohol solvent, recovering the eluate, concentrating, and drying to obtain the core tangerine extract; The first alcohol solvent is ethanol with a volume fraction of 70% by volume; The second alcohol solvent is ethanol with a volume fraction of 20% to 90%.

[0014] Preferably, soak the orange seed medicinal material for 10 h to 20 h Preferably, the recovered eluent is ethanol with a volume fraction of 90%.

[0015] Preferably, the pore size of the polystyrene-divinylbenzene filler is 300Å and the particle size is 100 μm to 150 μm.

[0016] Preferably, the temperature of the heat reflux is 65°C to 75°C.

[0017] The present invention uses 70% by volume ethanol during soaking and extraction to simultaneously dissolve polar and weak polar compounds, thereby achieving extraction of multiple components.

[0018] Through the study of Parkinson's disease model mice, the present invention found that the 90% ethanol part of orange seeds (CRSE6#) can significantly improve the neuronal function of mice and enhance their motor ability. This provides a new natural drug option for the treatment of Parkinson's disease, which is expected to reduce the dependence and toxic side effects of existing drugs and show great potential in future clinical applications.

[0019] The present invention is different from the existing Vitex rotundifolia treatment methods, and focuses on exploring the specific therapeutic potential of CRSE6# for Parkinson's disease. Through the study of the A53T-αSyn-Tg mouse model, the present invention found that CRSE6# can improve neuronal function and motor ability. Different from the non-specific neuroprotective effect of Vitex rotundifolia, the present invention reveals the potential mechanism of CRSE6# by studying the IP3Rs-MCU calcium ion pathway. CRSE6# may regulate calcium homeostasis by improving the interaction between IP3Rs, GRP75 and VDAC1, thereby alleviating neuronal dysfunction and significantly improving the motor ability of Parkinson's disease model mice.

[0020] In addition, the present invention screened out specific components in CRSE6# using BLI technology and mass spectrometry analysis, and found that its specific binding effect with α-syn was significant. This means that CRSE6# not only has a neuroprotective effect, but also can provide a more accurate treatment plan by directly targeting α-syn related to Parkinson's disease pathology. In contrast, the 90% ethanol part of the orange seed of the present invention can not only improve neuronal function and improve its motor ability. At the same time, it can directly target α-syn related to Parkinson's disease pathology. This greatly increases the possibility of clinical transformation of active ingredients of traditional Chinese medicine.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. Specific targeting of natural drugs: The present invention screened out the key active ingredient Zapoterin in CRSE6# by combining biotinylated α-syn protein with biolayer interferometry (BLI) technology, and verified its specific binding ability with α-syn, a protein related to Parkinson's disease pathology. This specific binding means that CRSE6# not only has a neuroprotective effect, but can also directly target the key pathological protein α-syn, delaying the progression of Parkinson's disease at the molecular level. Unlike the existing Chinese medicines such as Vitex Manjiu, which have a wide range of anti-inflammatory effects, the present invention has more precise targeting.

[0022] 2. Significantly improve neuronal function and motor ability: Through the rotarod test, Y-maze test and TUNEL test of A53T-αSyn-Tg mouse model, it was verified that CRSE6# can significantly improve the neuronal function and motor ability of Parkinson's disease mice and has a good neuroprotective effect. This provides an alternative or auxiliary treatment for existing Parkinson's disease drugs, which can reduce the dependence on drugs such as levodopa and prolong the effective period of patients' medication.

[0023] 3. Balance between safety and efficacy: The orange seed extract in the present invention is derived from natural plants and has higher safety and acceptability than chemically synthesized drugs. Its specific component Zapoterin's targeting effect on α-syn reduces nonspecific intervention and potential drug toxicity, making it particularly suitable for long-term treatment.

[0024] 4. Economic benefits: Tangerine seeds are common Chinese medicinal materials. The preparation process of their extracts is relatively simple and low-cost, and they have the potential for large-scale production. Compared with existing dopamine replacement therapies, the present invention provides a more cost-effective treatment option. Due to its significant efficacy and low toxicity, this technology has broad application prospects in the future market, which can reduce the treatment costs of patients and alleviate the burden on society and the medical system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1-Figure 3 The binding status of CRSE 6# and α-syn and the binding components analyzed by mass spectrometry.

[0026] Figure 1 This is the chromatogram of sample 2 components. Figure 2 This is the chromatogram of sample 3 components. Figure 3 This is the spectrum of the components specifically binding to α-syn after deducting the components non-specifically binding to the probe by CRSE#6.

[0027] Figure 4-Figure 5 This is the structural spectrum of the components that specifically bind to CRSE 6# and α-syn.

[0028] Figure 4 This is the liquid phase diagram of Zapoterin. Figure 5 This is the mass spectrum of Zapoterin.

[0029] Figure 6 The effects of different concentrations of CRSE6# and Rotenone on PC-12 cell viability are shown. The effects of gradient concentrations of CRSE6# (A) and Rotenone (B) on cell viability were detected using CCK8 (Cell Counting Kit-8) technology. C shows the protective effect of different concentrations of CRSE6# on cells at a Rotenone concentration of 2μM.

[0030] Figure 7The results show that the active extract of orange seed improves rotenone-induced apoptosis of PC12 cells. A is Western Blot analysis of the protein expression levels of Bcl-2, Bax, Cytochrome c and β-actin in different treatment groups (control group, model group, low concentration group (80μg / mL), medium concentration group (160μg / mL), and high concentration group (320μg / mL)). B is the statistical graph of the Bcl-2 / Bax ratio in different treatment groups. C is the statistical graph of the Cytochrome c / β-actin ratio in different treatment groups.

[0031] Figure 8 The results show that the active extract of orange seed improves rotenone-induced PC12 cell apoptosis. A is the flow cytometer analysis result of PC12 cell apoptosis in the control group, B is the flow cytometer analysis result of PC12 cell apoptosis in the model group, C is the flow cytometer analysis result of PC12 cell apoptosis in the low-dose CRSE6# group, D is the flow cytometer analysis result of PC12 cell apoptosis in the medium-dose CRSE6# group, E is the flow cytometer analysis result of PC12 cell apoptosis in the high-dose CRSE6# group, and F is a statistical analysis bar graph of PC12 cell apoptosis rates in different treatment groups.

[0032] Fig. 9 The results show the regulatory effect of CRSE6# on the behavioral function of A53T-α-syn-Tg mice. A is the trajectory of the Y-maze spontaneous alternation test, B is the mouse alternation index, and C is the rotarod test data.

[0033] Fig.10 It shows that the active extract of orange seed improves mouse apoptosis. A is a TUNEL staining image showing the distribution of apoptotic cells in mouse brain tissue, and B is a statistical graph of the proportion of TUNEL-positive cells.

[0034] Fig.11 It shows that the active extract of orange seed reduces the expression of a-syn protein in mice. A is an α-syn staining image showing the expression of α-synuclein (α-syn) in mouse brain tissue, and B is a statistical graph of α-syn fluorescence signals.

[0035] Fig.12 It is a heat map for the analysis of differential gene expression, chr (chromosome), biotype (gene type). The heat map shows the differential patterns of gene expression in different sample groups (A53T-α syn-Tg group and CRSE treatment group). Each row represents a gene, and each column represents a sample. Red indicates high gene expression, and blue indicates low gene expression. The color intensity reflects the expression level.

[0036] Fig.13This is a GO enrichment analysis bar chart, which is used to show the significant enrichment results of the gene expression differences between the CRSE treatment group and the A53T-α syn-Tg group in the Gene Ontology (GO) classification.

[0037] Fig.14 This is a KEGG pathway enrichment analysis bar chart, which shows the enrichment results of the gene expression differences between the CRSE-treated group and the A53T-αsyn-Tg group in the KEGG pathway. The bar chart is sorted according to the number of genes and significance (adjusted p-value), and describes the biological pathways in which the differential genes are significantly enriched.

[0038] Fig.15 GSEA analysis: Calcium signaling pathway. This figure shows the enrichment of calcium signaling pathway genes in gene set enrichment analysis (GSEA). The calcium signaling pathway is a key biological pathway that is closely related to neural function and cell signaling.

[0039] Fig.16 The expression of genes related to the calcium ion signaling pathway is displayed to confirm whether CRSE6# acts through the calcium ion pathway. A is Cav1, B is Cav2, C is Cav3, D is TRPC4, E is Adcy8, F is Pdela, G is Grm2, H is Gamk2d, I is Cacnalg, and J is Cacng6.

[0040] Fig.17 To further verify the protective effect of CRSE6# in Rotenone-induced cell damage by regulating the IP3Rs-MCU calcium axis, A showed the expression of GRP78 detected by immunofluorescence staining, B showed the expression of GRP78 protein by Western blotting, C statistically analyzed the fluorescence intensity of GRP78, D statistically analyzed the ratio of GRP78 to β-actin by wb results, E showed the expression of IP3R, GRP75 and VDAC1 proteins, F statistically analyzed the relative expression levels of IP3R, GRP75 and VDAC1 proteins, G used Rhod-2 and Mito-Tracker dyes to detect intracellular calcium ion flow and mitochondrial function. DETAILED DESCRIPTION

[0041] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0042] Example 1. Preparation of tangerine seed extract CRSE6#.

[0043] After the orange seed medicinal material was crushed into coarse powder, it was soaked in 70% ethanol by volume for 12 hours. According to the weight of the orange seed medicinal material before soaking, 7 mL of 70% ethanol by volume was used for hot reflux extraction at 70°C for 2 hours / time, and the extraction was repeated 3 times. The combined extracts were filtered and the ethanol was recovered by rotary evaporation to obtain a crude extract. The crude extract was then loaded onto the activated polystyrene-divinylbenzene filler on the column with a pore size of 300Å and a particle size of 100-150μm. It was eluted with ethanol at twice the column volume fraction of 20%, 50%, 70%, and 90%, and the 90% ethanol eluate was collected. The 90% ethanol eluate was then rotary evaporated at 40°C to recover ethanol, concentrated in a water bath, and freeze-dried to obtain a 90% ethanol extract of orange seed, recorded as CRSE6#. Among them, polystyrene-divinylbenzene is abbreviated as PS-DVB.

[0044] 2. Research on the drug targeting of CRSE6#.

[0045] (1) Biotinylated α-syn, α-syn is α-synuclein According to the operation procedures of the biotinylation kit, the target α-syn solution was first prepared with a concentration of 1 mg / mL and a volume of 50 μL.

[0046] The molecular weight of α-syn is 14 kDa. According to the concentration and molecular weight of the target, the molar ratio of biotinylation reagent to α-syn during biotinylation is 1:1. According to the calculation formula, 70 μL of biotinylation reagent NHS-biotin was added to 50 μL of α-syn solution, mixed evenly and incubated at room temperature for 60 minutes. After the reaction was completed, 100 μL of the sample after the reaction was completed was added to the desalting column balanced with PBS. After the sample was freely adsorbed to the column, 400 μL of PBS balance solution was added for elution. After the elution was completed, 400 μL of PBS balance solution was added for elution, and the eluate was collected, which was the biotinylated α-syn for use on the BLI machine.

[0047] (2) Biolayer interferometry experiment After the preparation of biotinylated α-syn, the Streptavidin sensor was first immersed in the biotinylated α-syn solution and the blank control solution, respectively, to obtain the sensor carrying biotinylated α-syn and the blank sensor. Then, the sensor carrying biotinylated α-syn and the blank sensor were placed in the PBST buffer for baseline measurement to ensure the stability of the captured protein amount. Subsequently, the sensor carrying biotinylated α-syn was immersed in different concentrations of drug CRSE6# solution in turn to monitor the binding of the drug to α-syn in real time, and the signal of the blank sensor was recorded as a control. After the binding reaction was completed, the sensor carrying biotinylated α-syn was moved into the PBST buffer for dissociation reaction, the dissociation process of the drug from the α-syn protein was monitored, and the corresponding BLI signal was recorded. Finally, the binding and dissociation curves were analyzed using BLI software, and the blank control signal was subtracted to evaluate the binding affinity and kinetic properties of the drug and protein.

[0048] In order to explore the specific components that bind to α-syn in CRSE6#, the present invention immerses the sensor carrying biotinylated α-syn and the blank sensor in the CRSE6# solution respectively to bind the components in CRSE6#. After binding, the sensors are moved into the blank control solution for dissociation. The binding and dissociation steps are repeated overnight in the same plate wells. The dissociation solutions are collected separately the next day. The components dissociated from the sensor carrying biotinylated α-syn are specific binding components, recorded as dissociation solution a, and the components dissociated from the blank sensor are non-specific binding components, recorded as dissociation solution b. For mass spectrometry analysis

[0049] (3) Mass spectrometry analysis Take dry CRSE6#, dissolve it in chromatographic pure methanol to a concentration of 0.1 mg / mL, centrifuge at 12000 rap / min for 15 min, take the supernatant to obtain sample 1, and similarly centrifuge dissociation solution a and dissociation solution b at 12000 rap / min for 15 min, take the supernatant to obtain sample 2 and sample 3. Use Waters BEH-C18 column, a mixture of water and formic acid with a volume ratio of 100:0.1 as mobile phase A, acetonitrile as mobile phase B, and chemically analyze the main components of orange seeds at a column temperature of 40°C. The flow rate of the mobile phase is 0.2 mL / min, and the injection volume is 3 μL. The specifications of the Waters BEH-C18 column are 150 mm×2.1 mm, 1.7 μm, 130A, and the gradient separation process is:

[0050] The Thermo Fisher Q-Exactive Orbitrap MS system was used, and the mass spectrometry parameters were set as follows: spray voltage, 3500 V; capillary temperature, 350°C; auxiliary gas heating temperature, 350°C. The scanning range was 50 m / z to 2000 m / z, and the resolution was 70000. The mass spectrometry data were analyzed using Xcalibur.

[0051] Mass spectrometry results showed that CRSE6# had both specific and non-specific binding compounds to α-syn ( Figure 1 and Figure 2 ), the present invention uses sample 3 as the background of sample 2, and obtains the chromatogram of the specific binding component after deducting the background ( Figure 3 ). Zapoterin, a component of Rutaceae plants, was found in the background-subtracted chromatogram ( Figure 4 is the Zapoterin chromatogram; Figure 5 The second mass spectrum of Zapoterin is shown in Figure 2). This component can specifically bind to α-syn and has the highest content.

[0052] (4) The active extract of orange seed improves rotenone-induced apoptosis of PC12 cells.

[0053] In the in vitro experiment, Western Blot technology and flow cytometry were used to analyze cell apoptosis ( Figure 6 , 7 , 8). The CCK8 experiment screened the most suitable CRSE6# concentration gradient (low concentration group (80μg / mL), medium concentration group (160μg / mL), high concentration group (320μg / mL)), and determined the optimal Rotenon model concentration (2μM). WB results showed the Bcl-2 / Bax ratio. Under the combined treatment of CRSE6# and Rotenon, the ratio was significantly higher than that of the group treated with Rotenone alone (p<0.01). Quantitative analysis of the Cyto-c / β-actin ratio showed that the expression of Cyto-c was significantly increased in the combined treatment group of CRSE6# and Rotenone (p<0.001). Flow cytometry results also showed that the active extract of orange seed could improve the apoptosis of PC12 cells induced by rotenone (p<0.001).

[0054] (5) Animal models In this experiment, the present invention selected 10 10-month-old B6129SF2 / J mice as a control group, recorded as WT, and used 40 A53T-αSyn-Tg mice as a Parkinson's disease model for experimental grouping. A53T-αSyn-Tg mice were divided into a model group A53T-αSyn-Tg, a low-dose orange seed group CRSE-L (100 mg / kg), a high-dose orange seed group CRSE-H (500 mg / kg) and a positive drug group L-DOPA (50 mg / kg). The orange seed group and the L-DOPA group were treated with different doses of orange seed extract and L-DOPA orally for 8 weeks. After the modeling was completed, subsequent experiments were carried out.

[0055] (6) Mouse Rotarod Test In the rotating rod test, a rotating rod with a diameter of 3 cm and a length of 50 cm was used. At the beginning of the experiment, the mouse was placed on the rotating rod and the rotation speed was gradually increased from 4 revolutions per minute to 40 revolutions per minute. The total test duration was 300 seconds. During the experiment, the time when the mouse fell from the rotating rod was recorded, and the retention time (Time to fall) was calculated. Each mouse was tested three times and the average value was taken for statistical analysis. After the test, the rotating rod was cleaned with 75% ethanol to ensure that it did not affect the subsequent testing of mice.

[0056] In the rotating rod test, the falling time of mice in different treatment groups was compared. The experimental results showed that the CRSE group could significantly improve the motor coordination of the effect mice ( Fig. 9 ). Compared with the control group (WT), the falling time of A53T-asyn-Tg mice was significantly shortened (p<0.001), showing poor motor coordination. Compared with the A53T-asyn-Tg model group, the motor coordination of mice in the CRSE group was significantly improved (p<0.05).

[0057] (7) Mouse Y-maze experiment In the Y-maze experiment, a maze consisting of three arms was used, each arm was 35 cm long, and the angle between the arms was 120 degrees. The mice were placed in the central area of ​​the Y-maze and allowed to explore freely for 5 minutes. The order and frequency of the mice entering each arm were recorded by the behavioral video analysis system, and the alternation percentage (Alternation) was calculated, that is, the proportion of the number of consecutive entries of the mice into different arms to the total number of times. After each mouse experiment, the maze was cleaned with 75% ethanol to prevent residual odor from affecting the behavior of the next mouse.

[0058] The Y-maze test evaluated the memory ability of mice in different treatment groups, indicating that CRSE6# treatment helped improve the working memory of mice ( Fig. 9). Using the alternation rate as an indicator, the CRSE group showed a significant improvement compared to the A53T-asyn-Tg group (p<0.01).

[0059] (8) Effect of CRSE6# on brain tissue apoptosis in Parkinson's disease model mice and TUNEL detection experimental scheme After 8 weeks, the mice were anesthetized and the brains were removed. The brains were fixed with 4% paraformaldehyde for 24 hours, and then the brain tissues were dehydrated and embedded in paraffin. After dewaxing and hydration, the brain tissue sections were stained with TUNEL to detect cell apoptosis. The sections were also stained with DAPI to mark the cell nuclei. After staining, the images were observed and taken using a fluorescence microscope. TUNEL-positive cells were apoptotic cells, and DAPI-positive cells were all cells. The number of apoptotic cells in each sample was counted in 5 fields of view, and the percentage of apoptotic cells in the total number of cells was calculated. TUNEL staining refers to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and the Chinese name of DAPI is 4',6-diamidino-2-phenylindole.

[0060] The experimental results showed that CRSE could significantly inhibit apoptosis in the brain tissue of A53T-a syn-Tg mice, indicating that it has a neuroprotective effect. Fig.10 ) The number of apoptotic cells in the A53T-asyn-Tg group was significantly higher than that in the WT group (p<0.001). Compared with the A53T-asyn-Tg group, CRSE6# significantly reduced the level of cell apoptosis (p<0.001).

[0061] (9) Study on the inhibitory effect of CRSE6# on α-syn protein expression in PD mouse model As above, after the brain tissue was embedded in paraffin, the brain tissue sections were dewaxed and hydrated, and the protein expression level was detected by α-syn protein immunofluorescence staining. The sections were stained with DAPI to mark the cell nucleus, and fluorescence detection was performed in combination with α-syn specific antibody staining. After staining, the images were observed and taken using a fluorescence microscope. DAPI-positive cells were all cells, and α-syn-positive cells were cells expressing α-syn protein. Fluorescence images were obtained from 5 randomly selected fields of view for each sample, and the fluorescence intensity of α-syn was analyzed to compare the expression of α-syn in the brains of each group of mice to evaluate the effect of CRSE6# on the expression of α-syn protein in Parkinson's disease model mice.

[0062] CRSE can effectively reduce α-synuclein aggregation in the brain tissue of A53T-asyn-Tg mice, suggesting that it may have a neuroprotective effect ( Fig.11The fluorescence intensity of α-Syn in the A53T-asyn-Tg group was significantly higher than that in the WT group (p<0.001). Compared with the A53T-asyn-Tg group, the fluorescence intensity of α-Syn in the CRSE group was significantly reduced (p<0.001).

[0063] (11) Mechanism verification based on transcriptomics The transcriptome sequencing heat map results of mouse striatum tissue show the differential gene expression between the A53T-αsyn-Tg group and the CRSE-treated group ( Fig.12 ). The color gradient represents the gene expression level, red represents high expression, and blue represents low expression. The results showed that CRSE treatment significantly affected the expression of multiple genes, involving different types such as protein-coding genes, lncRNA, and miRNA. Further research can explore the functions and signaling pathways of these differentially expressed genes through GO and KEGG enrichment analysis.

[0064] Fig.13 The results of GO enrichment analysis of differentially expressed genes (DEGs) between the CRSE-treated group and the A53T-αsyn-Tg group are shown. The horizontal axis represents the significantly enriched GO terms (GOterms), and the vertical axis represents the number of genes enriched in the GO terms. GO terms are classified by biological process (BP, blue), cellular component (CC, orange), and molecular function (MF, green). The results showed that CRSE treatment mainly affected multiple biological processes such as ion transport, neuronal signaling, and transmembrane channel activity, suggesting that CRSE may exert its neuroprotective effect by regulating these key biological functions.

[0065] KEGG heat map ( Fig.14 ) shows the results of KEGG signaling pathway enrichment analysis of differentially expressed genes (DEGs) between the CRSE-treated group and the A53T-αsyn-Tg group. The horizontal axis represents the number of genes enriched in the pathway, and the vertical axis represents the significantly enriched KEGG signaling pathway. The color gradient represents the p-value, with red representing a more significantly enriched pathway and blue representing a relatively lower significance. The analysis results showed that CRSE treatment mainly affected multiple neural-related pathways, including neuroactive ligand-receptor interactions, calcium signaling pathways, cAMP signaling pathways, and glutamatergic, cholinergic, and GABAergic synapses. In addition, circadian rhythm regulation, addiction pathways (morphine, nicotine), and immune-related signaling pathways (chemokines, GnRH secretion) were also significantly enriched. These findings suggest that CRSE may play an important role in neuroprotection and improvement of neurodegenerative diseases by regulating neurotransmitter signaling and cell signaling pathways. Among them, the calcium signaling pathway has attracted our attention and interest.

[0066] GSEA curve chart ( Fig.15) shows the enrichment analysis results of the calcium signaling pathway, which was performed using the gene set enrichment analysis (GSEA) method. The green curve in the figure represents the enrichment score of the calcium signaling pathway in the entire gene ranking data, showing that the pathway is strongly enriched in specific gene regions. The ranking list indicators below show the genes related to the pathway, and the red area represents that these genes are closely related to the calcium signaling pathway. The analysis results show that the pathway has a significant degree of enrichment, with a p-value of 0.0042 and a normalized enrichment score (NES) of 1.4522, indicating that the calcium signaling pathway plays an important role in this dataset.

[0067] The active ingredients of orange seeds may prevent PD through the IP3Rs-MCU calcium ion axis, p<0.05. The present invention then verified the relevant genes in mouse brain tissue by RT-QPCR technology ( Fig.16 ). The experimental results showed the changes in mRNA expression levels (Foldchange) of different genes (Cav1, Cav2, Cav3, TRPC4, Adcy8, Pde1a, Grm2, Camk2d, Cacna1g, Cacng6) in the WT group, A53T-αsyn-Tg group, CRSE-treated group and L-DOPA-treated group. The results showed that in the A53T-αsyn-Tg group, the expression of multiple genes (such as Cav1, Cav3, TRPC4, etc.) was significantly upregulated, and CRSE treatment could regulate their expression levels to varying degrees, making them close to the WT group, suggesting its potential role in regulating calcium signaling-related genes. These results indicate that CRSE may improve neuropathological features in the A53T-αsyn-Tg mouse model by regulating calcium signaling pathway genes.

[0068] Subsequently, it was further verified in cells that orange seed prevents PD by regulating the IP3Rs-MCU calcium axis ( Fig.17). CRSE6# regulates the protective effect of Rotenone-induced cell injury through the IP3Rs-MCU calcium axis. The expression of GRP78 was detected by immunofluorescence staining to evaluate the endoplasmic reticulum stress response. The Rotenone-treated group showed higher GRP78 expression, while different concentrations of CRSE6# helped to reduce the expression of GRP78, indicating that CRSE6# could alleviate endoplasmic reticulum stress. The results of Western blot analysis of GRP78 protein expression verified the results of immunofluorescence staining. Subsequently, Western blot analysis of IP3R, GRP75 and VDAC1 (these proteins are related to intracellular calcium homeostasis and mitochondrial function) protein expression results showed that CRSE6# could significantly regulate the levels of these proteins. Subsequently, Rhod-2 and Mito-Tracker immunofluorescence results showed that CRSE6# could reduce Rotenone-induced calcium overload and mitochondrial damage. Overall, CRSE6# has a protective effect at a certain concentration and regulates Rotenone-induced endoplasmic reticulum stress, calcium imbalance and mitochondrial dysfunction through the IP3Rs-MCU calcium axis.

[0069] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0070] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0071] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of a citrus aurantium extract in the preparation of a drug for treating Parkinson's disease, characterized in that: The tangerine seed extract is an alcohol extract obtained by alcohol extraction of tangerine seed material.

2. The use according to claim 1, characterized in that: The citrus fruit extract contains Zapoterin.

3. The use according to claim 1, characterized in that: The medicine uses the citrus aurantium extract as the only active ingredient.

4. The use according to claim 1, characterized in that: The drug reduces neuronal dysfunction.

5. The use according to claim 1, characterized in that: The drug improves motor performance.

6. The use according to claim 1, characterized in that: The drug regulates calcium homeostasis.

7. The use according to claim 1, characterized in that: The preparation method of the tangerine seed extract comprises: soaking tangerine seed medicinal materials with a first alcohol solvent, using 5 mL to 10 mL of the first alcohol solvent for hot reflux extraction per gram of the medicinal materials according to the weight of the tangerine seed medicinal materials before soaking, filtering the obtained liquid, recovering the alcohol solvent to obtain a crude extract, loading the crude extract onto a polystyrene-divinylbenzene filler on a column after activation, eluting with a second alcohol solvent, recovering the eluate, concentrating, and drying to obtain the tangerine seed extract; The first alcohol solvent is ethanol with a volume fraction of 70% by volume; The second alcohol solvent is ethanol with a volume fraction of 20% to 90%.

8. The use according to claim 7, characterized in that: Soak the orange seed medicinal materials for 10 h~20 h.

9. The use according to claim 7, characterized in that: The recovered eluent is 90% ethanol by volume.

10. The use according to claim 7, characterized in that: The temperature of the heat reflux is 65℃~75℃.

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