Application of polygonatum cyrtonema polysaccharide in preparation of anti-depression drug

Abstract

The invention discloses an application of polygonatum cyrtonema polysaccharide (PCP1) in preparation of an anti-depression drug. The polysaccharide is extracted from rhizomes of polygonatum cyrtonema and is proved to be used for preparing antidepressant drugs and drugs for improving expression of hippocampal brain-derived neurotrophic factors (BDNF) for the first time. Experiments show that the PCP1 can significantly improve depression-like behaviors, inhibit pathological changes such as hippocampal neuronal vacuole and immobilization, effectively up-regulate BDNF protein expression and reverse abnormal biochemical indexes of related serum. The compound has the characteristic of multiple target points, and shows a good patent medicine prospect.

Description

Technical Field

[0001] This invention relates to an antidepressant drug, and more particularly to the application of a Polygonatum odoratum polysaccharide in the preparation of an antidepressant drug. Background Technology

[0002] Polygonatum is an important medicinal and edible plant in traditional Chinese medicine. There are many varieties. The Chinese Pharmacopoeia includes three main types of Polygonatum, namely Yunnan Polygonatum, Polygonatum (also known as Chicken Head Polygonatum) and Polygonatum multiflorum. They differ greatly in appearance, production area and efficacy.

[0003] Polysaccharides are the most abundant active ingredient in Polygonatum sibiricum, and have been extensively studied for their hypoglycemic and lipid-lowering effects. However, the variety of Polygonatum sibiricum, its growing environment, harvesting season, and storage methods all significantly influence the structure and activity of polysaccharides. While there are precedents for using oligosaccharides or stem and leaf polysaccharides extracted from Polygonatum sibiricum to treat depression and improve sleep, there are few reports on the therapeutic efficacy of polysaccharides from Polygonatum multiflorum for nervous system disorders, especially for depression. Summary of the Invention

[0004] Objective of this invention: The objective of this invention is to provide an application of Polygonatum multiflorum polysaccharide in the preparation of antidepressant drugs, thereby solving the problem of how to prepare antidepressant drugs. Another objective of this invention is to propose the application of Polygonatum multiflorum polysaccharide in the preparation of drugs that increase BDNF protein expression, thereby solving the problem of how to prepare drugs that increase BDNF protein expression.

[0005] Technical solution: The application of the Polygonatum multiflorum polysaccharide containing the following repeating sugar chain structure described in this invention in the preparation of antidepressant drugs:

[0006] The values ​​of n1, n2, n3, n4, and n5 range from 5 to 15.

[0007] n1, n2, n3, n4, and n5 represent the number of repeated segments, preferably 11. Preferably, the Polygonatum multiflorum polysaccharide is prepared by the following method: The aqueous extract was obtained by mixing the powdered rhizome of Polygonatum multiflorum with water and then heating it. The extract was concentrated and then ethanol was added to precipitate the precipitate. The precipitate was collected and freeze-dried to obtain crude polysaccharide of Polygonatum odoratum. The crude polysaccharide of Polygonatum odoratum was redissolved in water to obtain a crude polysaccharide solution. The crude polysaccharide solution was mixed with Sevage reagent to remove proteins. After removing Sevage reagent, the polysaccharide solution was obtained. The polysaccharide solution was decolorized, sterilized, dialyzed, and freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was redissolved in water to obtain a preliminarily purified polysaccharide solution. The preliminarily purified polysaccharide solution was loaded onto a cellulose anion exchange chromatography column, eluted with water, and the eluent was collected. The eluent was concentrated and dialyzed to obtain the water-washed fraction. The water-washed components were loaded onto a gel chromatography column, eluted with water, and the eluent was collected. The eluent was then concentrated, dialyzed, and lyophilized to obtain Polygonatum polysaccharide.

[0008] Preferably, the Polygonatum polysaccharide contains fructose, glucose, mannose and galactose residues in a molar ratio of 88.1 : 9.7 : 1.6 : 0.3.

[0009] Preferably, the antidepressant drug further comprises pharmaceutically acceptable excipients.

[0010] Preferably, the dosage form of the antidepressant includes one of the following: injection, powder for injection, aerosol, eye drops, nasal drops, capsule, pill, and suspension.

[0011] Preferably, the Polygonatum multiflorum polysaccharide targets and regulates at least one of Loxl2, Tshb, Dusp27, and BDNF proteins to exert an antidepressant effect.

[0012] Preferably, the polysaccharide from Polygonatum cyrtonema targets the neuroactive ligand-receptor interaction pathway and the cytokine ligand-receptor interaction pathway to exert its antidepressant effect.

[0013] In another aspect, this invention discloses the application of the above-mentioned Polygonatum multiflorum polysaccharide in the preparation of drugs that enhance BDNF protein expression.

[0014] Preferably, the BDNF protein is the BDNF protein found in hippocampal tissue.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention obtains a polysaccharide of Polygonatum multiflorum through screening. This polysaccharide can effectively improve depressive-like behavior, inhibit pathological changes such as vacuolation and shrinkage in the hippocampus of depressed mice, and effectively increase the expression level of BDNF protein in the hippocampus of mice, reverse the abnormality of related serum biochemical indicators. It also acts on multiple targets and signaling pathways, and has good prospects for drug development. Attached Figure Description

[0016] Figure 1 Results of mouse hippocampal transcriptome sequencing and analysis; Figure 2 For differential gene enrichment analysis; Figure 3 The results of Nielsen staining in the mouse hippocampus; Figure 4 The results of BDNF immunohistochemical staining in the mouse hippocampus; Figure 5 To detect biochemical indicators in mouse serum using the ELISA method. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] Example 1: Polysaccharide of Polygonatum multiflorum was prepared according to the method described in the literature. The method is as follows: (1) Fresh Polygonatum cyrtonema rhizomes were washed and de-rooted, then cut into 6-8 mm thick slices and dried in a constant temperature oven at 60℃ for 96 h. After being pulverized, the powder was passed through a 60-mesh sieve to obtain uniform Polygonatum cyrtonema rhizomes powder. An appropriate amount of Polygonatum cyrtonema rhizomes powder was added to deionized water at a material-to-liquid ratio of 1:20 (m:v), and extracted in a constant temperature water bath (75℃) for 2 h. After standing at room temperature, the powder was centrifuged at 8000 r / min for 15 min, and the supernatant was retained. The precipitate was then extracted again with the above-mentioned proportion of deionized water for 2 h. The supernatants were combined and concentrated under reduced pressure at 60℃ to 1 / 10 of the original volume. Ethanol was added until the final volume fraction of ethanol was 75%, and the powder was precipitated at 4℃ for more than 12 h. After centrifugation at 8000 r / min for 20 min, the precipitate was collected and freeze-dried at -80℃ for 72 h to obtain crude Polygonatum cyrtonema polysaccharide powder.

[0019] (2) Prepare Sevage reagent in advance according to the ratio of n-butanol:trichloromethane = 1:4 (v:v), and seal and store it in the dark. Add an appropriate amount of deionized water to the crude polysaccharide powder of Polygonatum odoratum obtained in step (1) until the solution is clear. Centrifuge at 12000r / min for 15min to remove impurities, collect the supernatant, and obtain the polysaccharide solution.

[0020] Sevage reagent was added to the polysaccharide solution at a ratio of 3:1 (v:v). The mixture was stirred at low temperature on a magnetic stirrer for 2 hours, then centrifuged at 8000 rpm for 10 minutes to separate the layers. The layers were transferred to a separatory funnel and allowed to stand for 10 minutes. The protein and organic layers were discarded, and the polysaccharide solution was collected. This process was repeated until the protein layer disappeared. The protein concentration was measured using a Bradford protein assay kit (<1%). The solution was then collected and rotary evaporated at 60°C to remove the organic reagent, yielding a preliminarily purified polysaccharide solution. The preliminarily purified polysaccharide solution was decolorized using D101 macroporous resin. The decolorized solution was concentrated to 1 / 10 of the original volume and filtered through a 0.22 μm microporous membrane for sterilization. The solution was then dialyzed at 4°C for at least 72 hours using a dialysis bag with a molecular weight cutoff of 1000 Da to remove small molecules. Finally, the dialyzed polysaccharide solution was freeze-dried at -80°C for 72 hours to obtain preliminarily purified Polygonatum odoratum polysaccharide powder.

[0021] (3) Weigh 200 mg of preliminarily purified Polygonatum polysaccharide powder, add 40 mL of deionized water to dissolve, filter through a 0.22 μm microporous membrane, and load the sample onto a DEAE-52 cellulose anion exchange chromatography column (2.5 × 40 cm). First, elute with deionized water at a flow rate of 0.4 mL / min, then elute with 0.1-0.5 mol / L NaCl solution. Collect one tube every 10 min, and use anthrone-sulfuric acid reagent to check whether the polysaccharide has been completely eluted. After the polysaccharide has been completely eluted, concentrate the eluent under reduced pressure to 1 / 5 of the original volume, then dialyze using a dialysis bag with a molecular weight cutoff of 1000 Da at 4 °C for 72 h, and then freeze-dry at -80 °C to obtain the water-washed (neutral) fraction and the NaCl-eluted (acidic) fraction of Polygonatum polysaccharide.

[0022] (4) Weigh 30 mg of the washed fraction powder of Polygonatum odoratum polysaccharide and the NaCl elution fraction, respectively, add 6 mL of deionized water to dissolve them, filter through a microporous membrane (0.22 μm), and load them onto a Sephadex G-75 gel chromatography column (1.6 × 60 cm). Elute with deionized water at a flow rate of 0.38 mL / min, collecting one tube every 10 min. After concentrating the eluent to 1 / 5 of its original volume, dialyze it using a 1000 Da dialysis bag in a refrigerator at 4℃ for 72 h. Finally, freeze-dry the dialysate at -80℃ for more than 72 h to obtain Polygonatum odoratum polysaccharide PCP1 and Polygonatum odoratum polysaccharide PCP2, respectively. For the identification methods and results of PCP1 and PCP2, please refer to the reference ([1] Liu Wei. Structural analysis of Polygonatum odoratum polysaccharide and its protective effect on non-alcoholic fatty liver disease [D]. Wannan Medical College, 2023. DOI:10.27374 / d.cnki.gwnyy.2023.000144.).

[0023] Comparative Example 1: Everything else is the same as in Example 1, except that: Replace the rhizome of Polygonatum multiflorum with the rhizome of Polygonatum esculentum.

[0024] The PCP1 and PCP2 prepared in Example 1 and the Polygonatum sibiricum polysaccharide prepared in Comparative Example 1 were used to treat depression. The experimental methods are as follows: (1) Animal grouping (7-week-old male C57 mice): Normal group: No CUMS modeling was performed, and no drug treatment was administered; CUMS model group: CUMS continuous stimulation for 7 weeks; PCP1 group: CUMS continuous stimulation for 7 weeks + 400mg / kg / day of PCP1 was administered by gavage to the Polygonatum group from week 5 to week 7 after modeling. PCP2 group: CUMS continuous stimulation for 7 weeks + PCP2 400mg / kg / day was administered by gavage to the Polygonatum group from week 5 to week 7 after modeling. Chicken head Polygonatum polysaccharide group: CUMS continuous stimulation for 7 weeks + 5-7 weeks after modeling, the Polygonatum group was given 400mg / kg / day of chicken head Polygonatum polysaccharide by gavage; Fluoxetine group: The Huangjing group was given 10 mg / kg / day of fluoxetine by gavage during weeks 5-7 after CUMS continuous stimulation for 7 weeks.

[0025] CUMS stimulation methods include: water and food restriction (24 h), swimming in 10 ℃ ice water (5 min), tail pricking (3 times / 3-5 s), day and night reversal, tail suspension (3 times / 1 min), damp bedding for 24 h, and 45° tilted cage (24 h).

[0026] The above seven stimuli were randomly arranged over a seven-week period, with one stimulus per day. The same stimulus was not to be presented consecutively, so that the animals could not predict the occurrence of the stimulus.

[0027] Behavioral experiments (tips ended in week 4 and week 7, respectively): (1) Sucrose preference experiment For 48 consecutive hours, two water bottles were placed in the mouse cages. One bottle contained a 1% sucrose solution, and the other contained plain water for sugar water training. After the training period, water was withheld (but food was not restricted) for 12 hours, and the amount of water consumed by the mice from both bottles was measured over 8 hours. The water bottle positions were changed every 2 hours. Sucrose preference (%) = (Sucrose water consumption / (Sucrose water consumption + Plain water consumption)) × 100%.

[0028] (2) Forced swimming test The water temperature in the test chamber was adjusted to 23-25℃ and the water depth to 18cm. The mice swam for 6 minutes, and the swimming time and immobility time were recorded for the next 4 minutes.

[0029] (3) Open field test A mouse was placed in the center of a 50x50 cm square apparatus made of polypropylene. The apparatus was divided into 16 equal areas using software. During the experiment, a camera recorded the mouse's movement trajectory, total distance traveled, and time spent in the central area. The experiment was preset to 30 seconds, with a total duration of 5.5 minutes. After the experiment, mouse feces were cleaned up, and alcohol was sprayed to prevent the previous mouse's scent from affecting the experimental results. The results are as follows: Table 1. Effects of different Polygonatum polysaccharides on depressive-like behavior in mice.

[0030] Note:** p <0.01 indicates a comparison with the normal control group; ## p <0.01 indicates a comparison with the model group; ns indicates a comparison with the model group with no statistical difference.

[0031] As shown in Table 1, at the end of the 7-week experiment, the normal control group mice maintained strong exploratory behavior, with longer and more evenly distributed movement trajectories; while the depression model group mice showed reduced exploratory desire and significantly shorter exploration distances. The fluoxetine group (FLU) and the Polygonatum multiflorum polysaccharide group (PCP1) mice exhibited significantly improved depressive-like behavior, specifically: compared to the model group mice, the fluoxetine group and the Polygonatum multiflorum polysaccharide group mice had increased movement distances and more evenly distributed movements. In forced swimming, the immobility time of the model group mice was significantly longer than that of the normal group mice, while the immobility time of the fluoxetine group (FLU) and the Polygonatum multiflorum polysaccharide group (PCP1) mice was significantly shorter than that of the depression group (CUMS). In the sucrose preference test, the sucrose preference of the depression group mice was lower than that of the normal group mice, while the sucrose preference of the fluoxetine group (FLU) and the Polygonatum multiflorum polysaccharide group (PCP1) mice was significantly higher than that of the model group mice. In these test results, the Polygonatum sibiricum group and the PCP2 group showed no difference from the model group. It is evident that only PCP1 has a therapeutic effect on depression, while neither Polygonatum sibiricum polysaccharide nor PCP2 has a therapeutic effect on depression.

[0032] After anesthetizing the mice, the brain regions were dissected, and the hippocampus was extracted. Transcriptome sequencing of the hippocampal tissues from each group of mice was performed. Results of hippocampal transcriptome sequencing and analysis are as follows: Transcriptome results ( Figure 1 The sequencing results showed that the constructed cDNA library had high sequencing quality and could be used for subsequent bioinformatics analysis. Correlation analysis of gene expression levels among samples showed that the correlation coefficients (R) among biological replicates were all greater than 0.8 (e.g., ...). Figure 1 (As shown in Figure A); differential gene expression analysis was performed using DESeq. The criteria for screening differentially expressed genes were: fold change |log2FC|>1, and statistical significance. P <0.05, the results showed that there were 58 differentially expressed genes (DEGs) in the normal control group and the model group (CUMS group), of which 40 were upregulated and 18 were downregulated. There were 24 DEGs in the PCP1 group and the model group, of which 12 were upregulated and 12 were downregulated. There were 9 DEGs in the fluoxetine and CUMS groups, of which 8 were upregulated and 1 was downregulated (e.g., ...). Figure 1 (As shown in the BE diagram); the ensemble diagram shows the common DEGs between the normal control group and the model group, and between the model group and the PCP1 group. The results show that three genes were differentially expressed between the two comparison groups: Loxl2, Tshb, and Dusp27 (as shown in the BE diagram). Figure 1 (As shown in Figure F); Differential gene enrichment analysis of the sequencing results of each group revealed that GO function was co-enriched in the intercellular matrix between the normal control group and the model group, and between the model group and the PCP1 group (e.g., Figure 2As shown in Figure AB), further KEGG analysis revealed that the signaling pathways with common enrichment of differentially expressed genes between the two comparison groups were neuroactive ligand-receptor interactions and cytokine ligand-receptor interactions. Previous results showed that BDNF expression was low in the model group, while Polygonatum polysaccharide could increase BDNF protein expression, further supporting the conclusion that the occurrence of CUMS may be due to weakened binding of brain-derived nerve growth factor to its responsive ligands, exacerbating damage to the interstitial space of nerve cells (e.g., Figure 2 (As shown in the CD diagram).

[0033] The method for detecting Nissl staining in the mouse hippocampus is as follows: (1) Dewaxing and hydration: The paraffin sections of brain tissue were successively immersed in xylene for 10 min*2 → 50% xylene for 5 min → 100% ethanol for 5 min*2 → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min for dewaxing and hydration. Then rinsed with tap water for about 5 min. Note that you should not rinse the sections directly to avoid tissue damage.

[0034] (2) Immerse the slides in toluidine blue staining solution for 30 minutes. This process is carried out in a constant temperature incubator (temperature 50~60℃). Observe under a microscope during the process and stop staining when clear Nissl bodies are seen. (3) Rinse the slices with tap water, and then soak them in 70% ethanol for 1-3 min, 95% ethanol for 3-5 min, and 100% ethanol for 3 min in sequence. (4) Use xylene to clear sections twice, 5 min each time, and then seal with neutral resin. (5) Take pictures of the slides using a microscope.

[0035] like Figure 3 As shown, Nissl staining results indicated that in the normal group mice, the morphological structure of neurons in the CA1 region of the hippocampus was intact and orderly arranged, with virtually no pathological changes such as vacuolation and condensation. Nucleoli were clearly visible, and Nissl bodies appeared blue-purple, evenly distributed in the cytoplasm, some exhibiting a tiger-stripe pattern. Compared to the normal control group, in the model group (CUMS group), neurons in the CA1 region of the hippocampus were atrophied, smaller, looser, and more disordered, with less prominent Nissl bodies. Quantitative analysis showed a significant reduction in the number of neurons. In contrast, interventions in the *Polygonatum sibiricum* polysaccharide group and the PCP2 group did not show significant improvement, and their Nissl staining results showed no statistically significant difference compared to the model group. P >0.05). After intervention with fluoxetine and Polygonatum polysaccharide, the pathological changes such as vacuolated cells and condensation in the CA1 region of the mouse hippocampus were significantly reduced, the cells tended to be arranged more regularly, and the number of Nissl bodies and normal nerve cells increased significantly.

[0036] The immunohistochemical assay for BDNF was performed as follows: (1) Dewaxing and hydration: The paraffin sections of brain tissue were successively immersed in xylene for 10 min*2 → 50% xylene for 5 min → 100% ethanol for 5 min*2 → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min for dewaxing and hydration. Then rinsed with tap water for about 5 min. Note that you should not rinse the sections directly to avoid tissue damage.

[0037] (2) Staining: Wipe the tissue section dry and add an appropriate amount of freshly prepared DAB staining solution. Observe under a microscope at the same time. When the staining is observed to be brown (3~5 min), stop the staining under tap water.

[0038] (3) Counterstaining: Place the sections in hematoxylin for 1 min and rinse the sections with running water for 3 min. Then differentiate with 1% hydrochloric acid ethanol for 1 s and rinse the sections with running water 3 times, 2 min each time.

[0039] (4) Dehydration and clearing mounting: Dehydrate the sections with alcohol (80% ethanol 2s → 95% ethanol 3min → anhydrous ethanol I 5min → anhydrous ethanol II 5min). Clear the sections with xylene (xylene I 5min → xylene II 5min). Then mount the sections with neutral resin.

[0040] The experimental results are as follows: like Figure 4 As shown in the immunohistochemical results of BDNF, BDNF was mainly expressed in the cytoplasm of nerve cells in the mouse hippocampus. Compared with the normal control group, the expression level of BDNF in the model group was significantly reduced after CUMS modeling, indicating that hippocampal nerve cells in depressed mice were damaged. However, there was no statistically significant difference in BDNF expression levels between the chicken head polygonatum polysaccharide group and the PCP2 group and the model group. P >0.05). In contrast, fluoxetine and PCP1 intervention significantly upregulated BDNF expression.

[0041] ELISA was used to detect serum biochemical parameters in mice.

[0042] The method is as follows: After the animal behavioral test was completed, the animals were quickly decapitated and blood was collected after anesthesia. The blood was placed in centrifuge tubes and left at room temperature for 2 hours, then centrifuged at 3000 rpm / min for 15 minutes at 4°C. The supernatant was slowly collected and aliquoted (50 μL / aliquot) into clean centrifuge tubes. Further, the levels of serum CORT (serum cortisol), IL-1β (interleukin-1β), 5-HT (serotonin), and IL-10 (interleukin-10) in each group of mice were detected using ELISA. The specific detection steps for each indicator were performed according to the ELISA kit instructions.

[0043] The results are as follows Figure 5 As shown, Polygonatum multiflorum polysaccharides improved the detection of biochemical indicators in CUMS mice. Figure 5 As shown in the ELISA results, compared with the normal control group, CUMS mice had significantly increased serum CORT, IL-1β, and TNF-α levels, and significantly decreased 5-HT levels. Administration of Polygonatum cyrtonema polysaccharide and fluoxetine reversed these biochemical indicators to varying degrees. Polygonatum cyrtonema polysaccharide and fluoxetine significantly reduced serum CORT, TNF-α, and IL-1β levels in CUMS mice, while increasing serum 5-HT levels. However, there were no statistically significant differences in these indicators between the Polygonatum cyrtonema polysaccharide group and the PCP2 group compared with the model group. P >0.05). The above results suggest that Polygonatum multiflorum polysaccharide and fluoxetine can reverse changes in depression-related biochemical indicators in CUMS mice.

Claims

1. Application of Polygonatum multiflorum polysaccharides containing the following repeating sugar chain structure in the preparation of antidepressant drugs: ,in, The values ​​of n1, n2, n3, n4, and n5 range from 5 to 15.

2. The application according to claim 1, characterized in that, The polysaccharide from Polygonatum odoratum was prepared by the following method: The aqueous extract was obtained by mixing the powdered rhizome of Polygonatum multiflorum with water and then heating it. The extract was concentrated and then ethanol was added to precipitate the precipitate. The precipitate was collected and freeze-dried to obtain crude polysaccharide of Polygonatum odoratum. The crude polysaccharide of Polygonatum odoratum was redissolved in water to obtain a crude polysaccharide solution. The crude polysaccharide solution was mixed with Sevage reagent to remove proteins. After removing Sevage reagent, the polysaccharide solution was obtained. The polysaccharide solution was decolorized, sterilized, dialyzed, and freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was redissolved in water to obtain a preliminarily purified polysaccharide solution. The preliminarily purified polysaccharide solution was loaded onto a cellulose anion exchange chromatography column, eluted with water, and the eluent was collected. The eluent was concentrated and dialyzed to obtain the water-washed fraction. The water-washed components were loaded onto a gel chromatography column, eluted with water, and the eluent was collected. The eluent was then concentrated, dialyzed, and lyophilized to obtain Polygonatum polysaccharide.

3. The application according to claim 1, characterized in that, The Polygonatum polysaccharide contains fructose, glucose, mannose and galactose residues in a molar ratio of 88.1:9.7:1.6:0.

3.

4. The application according to claim 1, characterized in that, The antidepressant also contains pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The dosage forms of the antidepressant include one of the following: injection, powder for injection, aerosol, eye drops, nasal drops, capsule, pill, and suspension.

6. The application according to claim 1, characterized in that, The polysaccharide from Polygonatum multiflorum targets and regulates at least one of the proteins Loxl2, Tshb, Dusp27, and BDNF to exert an antidepressant effect.

7. The application according to claim 1, characterized in that, The polysaccharide from Polygonatum odoratum exerts its antidepressant effect by targeting the neuroactive ligand-receptor interaction pathway and the cytokine ligand-receptor interaction pathway.

8. The application of the Polygonatum polysaccharide according to claim 1 in the preparation of drugs that increase the expression level of BDNF protein.

9. The application according to claim 8, characterized in that, The BDNF protein mentioned is the BDNF protein found in hippocampal tissue.

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