CN120000722A - Application of citrus aurantium extract in preparing drugs for treating Parkinson's disease - Google Patents

Abstract

The invention relates to the technical field of medicines, in particular to application of a tangerine extract in preparation of medicines for treating parkinsonism. According to the invention, through research on a parkinsonism model mouse, CRSE6# at 90% ethanol position of tangerine seed can obviously improve neuron function of the mouse and improve motor capacity of the mouse, so that the invention provides application of a tangerine seed extract in preparation of a medicament for treating parkinsonism.

Description

Application of tangerine extract in preparation of medicine for treating parkinsonism

Technical Field

The invention relates to the technical field of medicines, in particular to application of a tangerine extract in preparation of medicines for treating parkinsonism.

Background

Parkinson's disease, abbreviated as PD, is one of the fastest growing neurodegenerative diseases worldwide, severely affecting the quality of life of patients and imposing a great burden on social and medical systems. Currently, treatment of parkinson's disease is primarily dependent on dopamine drugs. However, long-term use of these drugs often results in tolerance in the patient, gradual decline in efficacy, and increased doses of the drug are required to control symptoms. Such treatment strategies not only increase the economic burden on the patient, but also are accompanied by a higher risk of drug toxicity. Despite the existence of some adjuvant therapies, there is currently a lack of effective therapies that can delay disease progression.

The current state of the art in the field of parkinson's disease treatment relies mainly on dopamine replacement therapies, such as levodopa and dopamine receptor agonists, which control the motor symptoms of PD by supplementing dopamine deficient in the brain. However, with the development of disease and long-term drug use, the curative effect of the existing drugs gradually declines, and patients often need to increase the dosage to maintain the therapeutic effect, which can cause problems of drug toxicity and tolerance, and seriously affect the life quality of the patients.

In addition to traditional chemicals, plant extracts have attracted considerable attention in recent years as natural drug substitutes in the treatment of neurodegenerative diseases. The application of the fructus viticis ethyl acetate extract is a typical example of the existing traditional Chinese medicine for treating the parkinsonism. In this patent, the fructus viticis extract exhibits remarkable neuroprotection, can reduce neuroinflammation and neuronal damage, and can improve motor ability of mice in a parkinsonism model. However, although fructus viticis extract shows good effects in preclinical studies, studies on drug specificity and drug targets are lacking. In addition, the effective components and action mechanism of the drug are still further defined, which limits the possibility of clinical transformation.

At present, the traditional Chinese medicine extract has a plurality of drug effects on Parkinson diseases, for example, the fructus viticis extract has anti-inflammatory and neuroprotective effects, but the action mechanism is wider and the specificity is lacking. The specific targeting effect of the specific targeting agent on the key pathological protein-alpha-syn in the parkinsonism is not clear in the prior study, and the progress of the parkinsonism is difficult to accurately intervene on the molecular level. The fructus viticis has a wider range of action, but for parkinsonism, which is a disease involving alpha-syn aggregation, the prior art lacks an effective targeting treatment method, and can not fundamentally solve the neuronal dysfunction induced by alpha-syn.

Disclosure of Invention

In order to solve the problems, the invention provides application of the tangerine extract in preparing a medicament for treating parkinsonism.

The application of the tangerine seed extract in preparing the medicine for treating the parkinsonism is that the tangerine seed extract is an alcohol extract obtained by alcohol extraction of a tangerine seed material.

Preferably, the extract of citrus reticulata contains Zapoterin.

Preferably, the medicament comprises the extract of the citrus reticulata as the only active ingredient.

Preferably, the medicament alleviates neuronal dysfunction.

Preferably, the medicament improves exercise capacity.

Preferably, the medicament modulates calcium homeostasis.

Preferably, the preparation method of the tangerine seed extract comprises the steps of soaking tangerine seed medicinal materials in a first alcohol solvent, performing hot reflux extraction on each g of medicinal materials by using 5-10 mL of the first alcohol solvent according to the mass of the tangerine seed medicinal materials before soaking to obtain liquid, filtering, recovering the alcohol solvent to obtain a crude extract, loading the crude extract into activated polystyrene-divinylbenzene filling materials on a column, eluting with a second alcohol solvent, recovering eluent, concentrating and drying to obtain the tangerine seed extract;

The first alcohol solvent is ethanol with the volume fraction of 70%;

the second solvent is ethanol with the volume fraction of 20% -90%.

Preferably, the orange core medicinal material is soaked for 10-20 h

Preferably, the recovered eluent is 90% ethanol by volume.

Preferably, the aperture of the polystyrene-divinylbenzene filler is 300A, and the particle size is 100-150 mu m.

Preferably, the temperature of the thermal reflux is 65-75 ℃.

The invention can dissolve polar and weak polar compounds simultaneously by using 70% ethanol by volume fraction during soaking and extraction, thereby realizing extraction of various components.

According to the invention, through research on a parkinsonism model mouse, the 90% ethanol part (CRSE 6#) of the tangerine seed can obviously improve the neuron function of the mouse and improve the motor capacity of the mouse. The preparation method provides a new natural medicine choice for treating the Parkinson's disease, is expected to reduce the dependence and toxic and side effects of the existing medicines, and has great potential in future clinical application.

The invention is different from the existing fructus viticis treatment method, and focuses on exploring the specific treatment potential of CRSE6# on parkinsonism. According to the invention, through research on an A53T-alpha Syn-Tg mouse model, CRSE6# can improve neuron function and motor capacity. Unlike the nonspecific neuroprotection of Vitex agnus-castus, the invention reveals the potential mechanism of CRSE6# by studying the calcium ion pathway of IP3 Rs-MCU. CRSE6# may regulate calcium homeostasis by improving the interactions between IP3Rs, GRP75 and VDAC1, thereby alleviating neuronal dysfunction and significantly improving motor capacity in parkinson's disease model mice.

In addition, the invention screens out specific components in CRSE6# by using BLI technology and mass spectrometry analysis, and finds that the specific binding effect of the CRSE6# and alpha-syn is obvious. This means that CRSE6# not only has neuroprotective effects, but also can provide a more accurate therapeutic regimen by directly targeting α -syn associated with parkinson's disease pathology. In contrast, the 90% ethanol part of the tangerine seed can improve the neuron function and the motor capacity. And simultaneously can directly target alpha-syn related to parkinsonism pathology. This greatly improves the clinical conversion possibility of the active ingredients of the traditional Chinese medicine.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention screens key active components Zapoterin in CRSE6# through biotinylation alpha-syn protein binding biological layer interference technology (BLI), and verifies the specific binding capacity of the CRSE6# and parkinsonism pathology related protein alpha-syn. This specific binding means that CRSE6# not only has neuroprotective effect, but also can directly target the pathological key protein α -syn, and delay the progress of parkinson's disease at molecular level. Unlike the wide anti-inflammatory effect of traditional Chinese medicines such as fructus viticis, the invention has more accurate targeting.

2. The neural function and the motor ability of the mice with parkinsonism can be obviously improved by the CRSE6# through the stick rotating experiment, the Y maze experiment and the TUNEL detection of the A53T-alpha Syn-Tg mouse model, and the neural protection effect is good. The medicine provides an alternative or auxiliary treatment scheme for the existing parkinsonism medicine, can reduce the dependence of medicines such as levodopa and the like, and prolongs the effective period of the medicine taking of patients.

3. The orange core extract is a natural plant source, and has higher safety and acceptability compared with chemical synthetic medicines. The targeting effect of the specific component Zapoterin on the alpha-syn reduces non-specific intervention, reduces potential drug toxicity, and is particularly suitable for long-term treatment.

4. The orange core is a common traditional Chinese medicine, and the preparation process of the extract is relatively simple and has low cost, and the orange core has the potential of large-scale production. The present invention provides a more cost effective treatment option than existing dopamine replacement therapies. Due to the remarkable curative effect and low toxicity characteristics, the technology has wide application prospect in the future market, and can reduce the treatment cost of patients and reduce the burden of society and medical systems.

Drawings

FIGS. 1 to 3 show the binding of CRSE 6# to alpha-syn and the analysis of the binding components by mass spectrometry.

FIG. 1 shows a component chromatogram of sample 2, FIG. 2 shows a component chromatogram of sample 3, and FIG. 3 shows a component spectrum specifically binding to α -syn after binding CRSE 6# to a probe nonspecific binding component.

FIGS. 4 to 5 show the structural spectra of the components specifically binding to alpha-syn by CRSE 6#.

Fig. 4 is a Zapoterin liquid phase diagram and fig. 5 is a Zapoterin mass spectrum.

FIG. 6 shows the effect of different concentrations of CRSE6# and Rotenone on PC-12 cell viability, the effect of CRSE6# (A) and Rotenone (B) on cell viability at gradient concentrations was examined by CCK8 (Cell Counting Kit-8) technique, respectively, and C shows the protective effect of different concentrations of CRSE6# on cells at Rotenone concentration of 2. Mu.M.

FIG. 7 shows that the orange nucleus active extract improves rotenone induced apoptosis of PC12 cells, A is Western Blot analysis of protein expression levels of Bcl-2, bax, cytochrome C and beta-actin in different treatment groups (control group, model group, low concentration group (80. Mu.g/mL), medium concentration group (160. Mu.g/mL), high concentration group (320. Mu.g/mL)), B is Bcl-2/Bax ratio statistical graph of different treatment groups, C is Cytochrome C/beta-actin ratio statistical graph of different treatment groups.

FIG. 8 shows that the extract of the orange nucleus activity improves the rotenone induced apoptosis of PC12 cells, A is the flow cytometry analysis result of the apoptosis of PC12 cells of the control group, B is the flow cytometry analysis result of the apoptosis of PC12 cells of the model group, C is the flow cytometry analysis result of the apoptosis of PC12 cells of the low dose CRSE6# group, D is the flow cytometry analysis result of the apoptosis of PC12 cells of the medium dose CRSE6# group, E is the flow cytometry analysis result of the apoptosis of PC12 cells of the high dose CRSE6# group, F is the statistical analysis histogram of the apoptosis rate of PC12 cells of different treatment groups.

FIG. 9 shows the effect of CRSE6# on the regulation of the behavioral functions of A53T-alpha-syn-Tg mice, A is a Y maze spontaneous alternation experimental trace graph, B is a mouse alternation index, and C is a rotating rod experimental data.

FIG. 10 shows that the extract of orange core activity improved apoptosis in mice, A is TUNEL staining chart showing apoptotic cell distribution in brain tissue of mice, and B is a statistical chart of the proportion of TUNEL positive cells.

FIG. 11 shows that the orange nucleus active extract reduces the expression of mouse a-syn protein, A is an alpha-syn staining chart showing the expression of mouse brain tissue alpha-syn protein (alpha-syn), and B is a statistical chart of alpha-syn fluorescent signals.

FIG. 12 is a thermal diagram of analysis of differential gene expression, chr (chromosome), biotype (genotype), showing the differential pattern of gene expression in different sample groups (A53T-. Alpha.syn-Tg group and CRSE treatment group), each row representing one gene, each column representing one sample, red representing high gene expression, blue representing low gene expression, and color intensity reflecting the level of expression.

FIG. 13 is a histogram of GO enrichment analysis showing the significant enrichment results of Gene expression differences in Gene Ontology (GO) classification in the CRSE treated group versus the A53T- αsyn-Tg group.

FIG. 14 is a bar graph of KEGG pathway enrichment analysis showing the results of enrichment of gene expression differences in KEGG pathways in the CRSE treated group versus the A53T- αsyn-Tg group, the bar graph being ordered by gene number and significance (adjusted p-value), depicting biological pathways in which the differential genes are significantly enriched.

FIG. 15 is a graph of the GSEA assay, calcium SIGNALING PATHWAY, which shows the enrichment of the Calcium signaling pathway gene in the Genset enrichment assay (GSEA), a key biological pathway, closely related to neural function and cell signaling.

FIG. 16 shows the expression of genes related to the calcium ion signal pathway, confirming whether CRSE6# acts through the calcium ion pathway, A is Cav1, B is Cav2, C is Cav3, D is TRPC4, E is Adcy, F is Pdela, G is Grm2, H is Gamk D, I is Cacnalg, J is Cacng6.

FIG. 17 further demonstrates the protective effect of CRSE6# in cell damage induced by IP3Rs-MCU calcium ion axis modulation Rotenone, A shows the detection of GRP78 expression by immunofluorescent staining, B shows the analysis of GRP78 protein expression by Western blot, C statistically analyzes GRP78 fluorescence intensity, D statistically analyzes wb results GRP78 to beta-actin ratio, E shows IP3R, GRP75 and VDAC1 protein expression, F statistically analyzes the relative expression levels of IP3R, GRP75 and VDAC1 proteins, G uses Rhod-2 and Mito-Tracker dyes to detect intracellular calcium ion flow and mitochondrial function.

Detailed Description

The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified.

Examples

1. Preparation of orange core extract CRSE6 #.

Pulverizing semen Citri Reticulatae into coarse powder, moistening with 70% ethanol by volume fraction, soaking for 12 hr, reflux-extracting with 7 mL% ethanol by volume fraction at 70deg.C for 2h times per g of the medicinal material, repeatedly extracting for 3 times, mixing extractive solutions, filtering, and recovering ethanol by rotary evaporation to obtain crude extract. And loading the crude extract into activated polystyrene-divinylbenzene packing with a pore size of 300A and a particle size of 100-150 mu m on a column, eluting with 20%, 50%, 70% and 90% ethanol twice the volume fraction of the column in sequence, and collecting 90% ethanol eluent. Subsequently, the 90% ethanol eluent is subjected to rotary evaporation at 40 ℃ to recover ethanol, and concentrated in a water bath and freeze-dried to obtain an orange core 90% ethanol extract, which is designated as CRSE No. 6. Wherein, polystyrene-divinylbenzene is abbreviated as PS-DVB.

2. Study of CRSE6# drug targeting.

(1) Biotinylated alpha-syn, alpha-syn is alpha-synuclein

According to the operational procedure of the biotinylation kit, firstly, a target substance alpha-syn solution is prepared, the concentration is 1 mg/mL, and the volume is 50 mu L.

The molecular weight of α -syn is 14 kDa. The molar ratio of biotinylation reagent to alpha-syn upon biotinylation was 1:1, depending on target concentration and molecular weight. By calculation formula, 70. Mu.L of the biotinylation reagent NHS-biotin was added to 50. Mu.L of the alpha-syn solution, and after mixing well, incubated at room temperature for 60 minutes. After the reaction is finished, 100 mu L of the sample is added into a desalting column balanced by PBS, 400 mu L of PBS balance solution is added for eluting after the sample is freely adsorbed to the column, 400 mu L of PBS balance solution is added for eluting after the elution is finished, and the eluent is collected, namely the biotinylated alpha-syn is used for BLI.

(2) Experiment of biological layer interference technique

After the preparation of the biotinylated alpha-syn is completed, firstly, the strepitavidin sensor is respectively immersed in the biotinylated alpha-syn solution and the blank control solution to obtain a sensor carrying the biotinylated alpha-syn and the blank sensor. Next, the sensor carrying biotinylated α -syn and blank sensor were placed in PBST buffer for baseline measurement, ensuring that the amount of protein captured was stable. Subsequently, the sensor carrying the biotinylation alpha-syn is immersed in CRSE6# solutions with different concentrations in sequence, the combination condition of the medicine and the alpha-syn is monitored in real time, and the signal of the blank sensor is recorded as a control. After the binding reaction is completed, the sensor carrying the biotinylated alpha-syn is transferred into a PBST buffer solution for dissociation reaction, the dissociation process of the drug from the alpha-syn protein is monitored, and the corresponding BLI signal is recorded. Finally, binding and dissociation curves were analyzed using BLI software, and the blank signal was subtracted to evaluate the binding affinity and kinetic properties of the drug to the protein.

In order to explore specific components in CRSE6# which are combined with alpha-syn, the invention immerses a sensor carrying biotinylated alpha-syn and a blank sensor in CRSE6# solution respectively to combine the components in CRSE 6#. After binding, the sensors were each removed into a blank solution for dissociation. The binding dissociation step was repeated overnight in the same plate wells. The following day, the dissociation solutions were collected separately. The component dissociated by the sensor carrying biotinylated alpha-syn is a specific binding component and is denoted as dissociation solution a, and the component dissociated by the blank sensor is a non-specific binding component and is denoted as dissociation solution b. For analysis of quality and plain

(3) Mass spectrometry analysis

Dissolving dried CRSE6# with chromatographic pure methanol to 0.1 mg/mL concentration solution, centrifuging at 12000 rap/min for 15 min, collecting supernatant to obtain sample 1, centrifuging dissociation solution a and dissociation solution b at 12000 rap/min for 15 min, and collecting supernatant to obtain sample 2 and sample 3. A Waters BEH-C18 chromatographic column is adopted, a mixed solution of water and formic acid with the volume ratio of 100:0.1 is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, chemical analysis is carried out on main components of the orange core at the column temperature of 40 ℃, the flow rate of the mobile phase is 0.2 mL/min, and the sample injection amount is 3 mu L. The specification of the Waters BEH-C18 chromatographic column is 150 mm X2.1 mm,1.7 μm,130A, and the gradient separation process is as follows:

The Q-Exactive Orbitrap MS system of the Siemens is selected, and the mass spectrum parameters are set to be spray voltage of 3500: 3500V, capillary temperature of 350 ℃ and auxiliary gas heating temperature of 350 ℃. Scan interval 50 m/z to 2000 m/z, resolution 70000. Mass spectral data were analyzed using Xcalibur.

Mass spectrometry results showed that CRSE6# had specific and non-specific binding compounds to α -syn (fig. 1 and 2), and that the present invention used sample 3 as the background of sample 2 and subtracted the background to obtain a chromatogram of the specific binding component (fig. 3). A background-subtracted chromatogram was found to contain components Zapoterin from the Rutaceae plant (FIG. 4 is Zapoterin chromatogram; FIG. 5 is Zapoterin secondary mass spectrum). The component can specifically bind to alpha-syn, and has highest content.

(4) The tangerine seed active extract can improve rotenone-induced PC12 apoptosis.

In vitro experiments, cells were analyzed for apoptosis using Western Blot technique and flow cytometry (fig. 6, 7, 8). CCK8 experiments screened the most suitable CRSE6# concentration gradient (low concentration group (80. Mu.g/mL), medium concentration group (160. Mu.g/mL), high concentration group (320. Mu.g/mL)), and determined the optimal Rotenon model concentration (2. Mu.M). WB results showed a Bcl-2/Bax ratio that was significantly higher in the CRSE6# and Rotenon combination treatment than in the Rotenone alone treatment group (p < 0.01). Quantitative analysis of the Cyto-c/beta-actin ratio showed that Cyto-c expression was significantly increased (p < 0.001) in the CRSE6# and Rotenone combination treatment group. The results of the flow analysis also show that the orange nucleus active extract can improve the rotenone induced apoptosis of PC12 cells (p < 0.001).

(5) Animal model

In this experiment, 10 month old B6129SF2/J mice were selected as control group, designated as WT, and 40A 53T-alpha Syn-Tg mice were used as Parkinson's disease model for experimental grouping. The A53T-alpha Syn-Tg mice were divided into model group A53T-alpha Syn-Tg, low dose orange core group CRSE-L (100 mg/kg), high dose orange core group CRSE-H (500 mg/kg) and positive drug group L-DOPA (50 mg/kg), and orange core group and L-DOPA group were treated by oral administration of different doses of orange core extract and L-DOPA, respectively, for 8 weeks. After the molding is completed, the subsequent experiments are carried out.

(6) Mouse stick-turning experiment

In the rotary bar test, a rotary bar having a diameter of 3 cm and a length of 50 cm was used. At the beginning of the experiment, mice were placed on a rotating rod and the rotational speed was gradually increased from 4 to 40 revolutions per minute for a total test period of 300 seconds. During the experiment, the Time the mouse dropped from the rotating rod was recorded and the holding Time (Time to fall) was calculated. Each mouse was tested 3 times and the mean value was taken for statistical analysis. After the end of the test, the rotating rod was cleaned with 75% ethanol, ensuring that the subsequent mice test was not affected.

In the bar rotation experiment, the drop time of mice in different treatment groups is compared, and the experiment result shows that the CRSE group can significantly improve the movement coordination of mice with the effect (figure 9). The drop time of a53T-asyn-Tg mice was significantly shortened (p < 0.001) compared to control (WT), showing poor motor coordination. The CRSE group had a significant improvement in the motor coordination of mice (p < 0.05) compared to the a53T-asyn-Tg model group.

(7) Mouse Y maze experiment

In the Y maze experiment, a maze consisting of three arms each having a length of 35 cm and an inter-arm angle of 120 degrees was used. The mice were placed in the central region of the Y maze and were free to explore for 5 minutes. The sequence and frequency of mice entering each arm was recorded by a behavioural video analysis system, and the percentage alternation (Alternation), the ratio of the number of mice entering different arms in succession to the total number, was calculated. After the end of each mouse experiment, the maze was cleaned with 75% ethanol to avoid residual odors affecting the behavior of the next mouse.

The Y maze experiment evaluates the memory capacity of mice from different treatment groups, indicating that treatment with CRSE6# helps to improve the working memory of mice (fig. 9). The alternation rate (alternation) is used as an index. The CRSE group had a significant increase (p < 0.01) compared to the a53T-asyn-Tg group.

(8) Influence of CRSE6# on brain tissue apoptosis of parkinsonism model mice and TUNEL detection experimental scheme

After 8 weeks, mice were anesthetized and brains were fixed with 4% by volume paraformaldehyde for 24 hours, followed by gradient dehydration of brain tissue and embedding in paraffin. After dewaxing and hydration of brain tissue sections, apoptosis was detected using TUNEL staining. Sections were simultaneously stained with DAPI to label nuclei. After staining, images were observed and captured using a fluorescence microscope, TUNEL positive cells were apoptotic cells, DAPI positive cells were all cells. The number of apoptotic cells per sample was counted by selecting 5 fields and calculating the percentage of apoptotic cells to total cell number. TUNEL staining refers to the terminal deoxynucleotidyl transferase mediated dUTP notch end labeling method, DAPI chinese name 4', 6-diamidino-2-phenylindole.

Experimental results show that CRSE can obviously inhibit apoptosis in brain tissues of A53T-asyn-Tg mice, and has neuroprotection. (FIG. 10) the number of apoptotic cells was significantly higher in the A53T-asyn-Tg group than in the WT group (p < 0.001), and CRSE6# significantly reduced the level of apoptosis (p < 0.001) compared to the A53T-asyn-Tg group.

(9) Inhibition study of alpha-syn protein expression in PD mouse model by CRSE6#

As above, after embedding brain tissue in paraffin, dewaxing brain tissue sections, hydrating, and detecting protein expression level by adopting alpha-syn protein immunofluorescence staining. Sections were stained for nuclei using DAPI staining, and fluorescent detection was performed in combination with an alpha-syn specific antibody staining. After staining, DAPI positive cells were all cells and α -syn staining positive cells were cells expressing α -syn protein were observed and imaged using a fluorescence microscope. Each sample was obtained from 5 fields of view randomly selected, and analyzed for fluorescence intensity of α -syn, and the expression of α -syn in the brains of mice in each group was compared to evaluate the effect of CRSE6# on the expression of α -syn protein in mice in parkinson's disease model.

CRSE was effective in reducing alpha-synuclein aggregation in brain tissue of A53T-asyn-Tg mice, suggesting a potential neuroprotective effect (FIG. 11). The alpha-Syn fluorescence intensity was significantly higher in the A53T-asyn-Tg group than in the WT group (p < 0.001), and the CRSE group significantly reduced the alpha-Syn fluorescence intensity (p < 0.001) compared to the A53T-asyn-Tg group.

(11) Transcriptomics-based mechanism validation

Transcriptome sequencing heatmap results of mouse striatal tissue demonstrated differential gene expression between the a 53T-asyn-Tg group and the CRSE treated group (fig. 12). The color gradient indicates the level of gene expression, red for high expression and blue for low expression. The results show that CRSE treatment significantly affects the expression of various genes, and relates to different types of protein coding genes, lncRNA, miRNA and the like. Further studies could explore the function and signaling pathways of these differential genes through GO and KEGG enrichment assays.

FIG. 13 shows the results of GO enrichment analysis of the Differentially Expressed Genes (DEGs) between the CRSE treated group and the A53T- αsyn-Tg group. The horizontal axis represents the significantly enriched GO term (GOterms) and the vertical axis represents the number of genes enriched to that GO term. GO terms are classified by biological process (BP, blue), cellular component (CC, orange), molecular function (MF, green). The results show that CRSE treatment affects a number of biological processes, such as ion transport, neuronal signaling, transmembrane channel activity, etc., suggesting that CRSE may exert its neuroprotective effects by modulating these key biological functions.

The KEGG heat map (fig. 14) shows the results of KEGG signaling pathway enrichment analysis of the Differentially Expressed Gene (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 KEGG signaling pathway that is significantly enriched. The color gradient represents the p-value, red represents a more significant enrichment pathway, and blue represents a relatively lower significance. Analysis results indicate that CRSE treatment affects primarily neuroactive ligand-receptor interactions, calcium signaling pathways, cAMP signaling pathways, and multiple neurorelated pathways such as glutamatergic, cholinergic, gabaergic synapses. In addition, circadian rhythm regulation, addictive pathways (morphine, nicotine) and immune-related signaling pathways (chemokines, gnRH secretion) are also significantly enriched. These findings suggest that CRSE may play an important role in neuroprotection and amelioration of neurodegenerative diseases by modulating neurotransmitter signaling and cellular signaling pathways. Among them, calcium signaling pathways have attracted our attention and interest.

The GSEA graph (fig. 15) shows the results of enrichment analysis of the calcium signaling pathway, using the Gene Set Enrichment Analysis (GSEA) method. The green curve in the figure represents the enrichment fraction of the calcium signaling pathway in the overall gene order data, indicating that the pathway exhibits a strong enrichment in a specific gene region. The lower ranked list of indicators shows genes associated with this pathway, with the red region representing those genes closely associated with the calcium signaling pathway. Analysis showed that the pathway had a significant degree of enrichment, 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.

The tangerine core active ingredient can prevent PD through an IP3Rs-MCU calcium ion axis, and p is less than 0.05. The present invention then validated the relevant genes of mouse brain tissue by RT-Qpcr technique (fig. 16). The experimental results show that the mRNA expression levels (Foldchange) of the WT group, the A53T- αsyn-Tg group, the CRSE treated group and the L-DOPA treated group vary among different genes (Cav 1, cav2, cav3, TRPC4, adcy, pde1a, grm2, camk2d, canna 1g, and Canng 6). The results show that in the a53T- αsyn-Tg group, expression of multiple genes (e.g., cav1, cav3, TRPC4, etc.) is significantly upregulated, whereas CRSE treatment can modulate its expression levels to varying degrees, bringing it closer to the WT group, suggesting a potential role in regulating calcium signal-related genes. These results indicate that CRSE may improve neuropathological characteristics in a53T- αsyn-Tg mouse model by modulating calcium signaling pathway genes.

The tangerine core was then further verified on cells to prevent PD by modulating the IP3Rs-MCU calcium ion axis (fig. 17). CRSE6# regulates Rotenone-induced protection in cell injury via the IP3Rs-MCU calcium ion axis. The expression of GRP78 was detected by immunofluorescent staining, endoplasmic reticulum stress response was assessed, rotenone treated groups showed higher GRP78 expression, while different concentrations of CRSE6# helped reduce GRP78 expression, indicating that CRSE6# was able to relieve endoplasmic reticulum stress. The result of western blot analysis of GRP78 protein expression verifies the result of immunofluorescent staining. Subsequent western blot analysis of the expression results of IP3R, GRP and VDAC1 (these proteins are associated with intracellular calcium homeostasis and mitochondrial function) proteins showed that CRSE6# was able to significantly regulate the levels of these proteins. Subsequent Rhod-2 and Mito-Tracker immunofluorescence results showed that CRSE6# was able to reduce Rotenone-induced calcium overload and mitochondrial damage. Overall, CRSE6# has protective effects at certain concentrations and endoplasmic reticulum stress, calcium imbalance and mitochondrial function impairment caused by IP3Rs-MCU calcium axis modulation Rotenone.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (10)

1. Use of a tangerine seed 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 a tangerine seed material. 2. The use according to claim 1, characterized in that the citrus aurantium extract contains Zapoterin. 3. The use according to claim 1, characterized in that the drug contains the citrus aurantium extract as the only active ingredient. 4. The use according to claim 1, characterized in that the drug alleviates neuronal dysfunction. 5. The use according to claim 1, characterized in that the drug improves motor ability. 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 core tangerine extract is: soaking the core tangerine 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%. 8. The use according to claim 7, characterized in that the orange seed medicinal material is soaked for 10 h to 20 h. 9. The use according to claim 7, characterized in that the recovered eluent is ethanol with a volume fraction of 90%. 10. The use according to claim 7, characterized in that the temperature of the heat reflux is 65°C to 75°C.