Application of rehmannia D in preparation of medicine for preventing and / or treating Parkinson's disease

Abstract

The invention discloses an application of rehmannia glutinosa libosch D in preparation of a medicine for preventing and / or treating Parkinson's disease, the Parkinson's disease is caused by deficiency of trace elements, and the trace elements are selenium; the rehmannia glutinosa libosch D can relieve MPP + induced cell injury and nigra TH + neuron injury, and can relieve dyskinesia and non-dyskinesia caused by Parkinson's disease; meanwhile, the rehmannia glutinosa libosch D and catalpol are jointly applied to preparation of the medicine for preventing and / or treating the Parkinson's disease, the protection effect on the Parkinson's disease and related symptoms caused by the Parkinson's disease can be effectively improved, the effect of preventing and treating the Parkinson's disease is achieved, and the effect of improving dyskinesia and non-dyskinesia related to the Parkinson's disease can be achieved.

Description

Technical Field

[0001] The present invention relates to the application of rehmannia glutinosine D in preparing medicine for preventing and / or treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. PD patients often have non-motor disorders such as constipation and loss of smell, as well as motor disorders such as tremor, bradykinesia, and postural instability. The typical pathological feature of PD is the loss of dopaminergic neurons in the substantia nigra of the midbrain, which is manifested as a decrease in tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. Traditional Chinese medicine believes that PD is caused by liver and kidney deficiency, and on this basis, phlegm and blood stasis are generated internally, blocking the brain meridians and exacerbating the dark movement of internal wind.

[0003] Selenium is an essential trace element for the human body. It participates in the synthesis of selenoproteins in the body and plays an antioxidant and anti-inflammatory role. The risk of PD is related to blood selenium levels (Environ Sci Pollut Res Int. 2023; 30(55): 117349-117359.). MPTP is a neurotoxic compound that can cause oxidative stress after systemic administration and produce stable and reliable damage to dopaminergic neurons in the substantia nigra and striatum. It is often used to establish PD model mice. Studies have shown that mice with a selenium-deficient diet are more susceptible to the effects of MPTP (Neurochem Res. 1991; 16(12): 1257-1263.), which aggravates the symptoms of PD.

[0004] Rehmannia glutinosa (Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey.) is a plant of the Scrophulariaceae family. It has the effects of nourishing blood and yin, replenishing essence and filling marrow, and can treat liver and kidney yin deficiency. It is often used to treat PD in clinical practice. The main chemical components of Rehmannia glutinosa include iridoid glycosides, amino acids, phenylethanol glycosides, trace elements, polysaccharides, and ionones, which have antioxidant, anti-inflammatory and anti-apoptotic effects (Chinese Journal of Experimental Traditional Chinese Medicine, 2022, 28(23): 228-236.). The pharmacological effects of Rehmannia glutinosa in the treatment of PD have been verified in both animals and humans, but the chemical composition of Rehmannia glutinosa is complex, and its main active ingredients in the treatment of PD are still unknown. In addition, the complex chemical composition of Rehmannia glutinosa is not conducive to quality control and the development of related drugs, and the complex chemical composition is prone to cause adverse reactions such as abnormal cholesterol metabolism. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide the use of rehmannia glycoside D in the preparation of a drug for preventing and / or treating Parkinson's disease, and also to provide the use of rehmannia glycoside D and catalpol in the preparation of a drug for preventing and / or treating Parkinson's disease.

[0006] Technical solution: The present invention discloses the use of rehmannia glutinosa D in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0007] Wherein, the Parkinson's disease is Parkinson's disease caused by trace element deficiency.

[0008] Wherein, the trace element is selenium.

[0009] Among them, rehmannia glutinosa D reduces MPP+-induced cell damage and substantia nigra TH+ neuronal damage, and alleviates movement disorders and non-motor disorders caused by PD.

[0010] Wherein, the non-motor disorder is olfactory dysfunction or gastrointestinal dysfunction.

[0011] Wherein, the dosage of the rehmannia glutinosa glycoside D is 5 to 40 μM / day.

[0012] The invention also discloses the combined use of rehmannia glutinosa D and catalpol in the preparation of a medicine for preventing and / or treating PD.

[0013] Wherein, the mass ratio of catalpol to rehmannia glutinosa D is 10-2:1.

[0014] Wherein, the drug is in the form of nasal drops, eye drops, capsules, tablets, films, suppositories or injections.

[0015] Principle of the invention: The present invention discloses related models in which rehmannia glutinosa D can improve PD, including in vitro cell models and animal models. At a dosage of 5μM to 40μM, rehmannia glutinosa D showed a significant improvement effect on the MPP+-induced SH-SY5Y cell model; when treating PD and its related movement disorders and non-movement disorders, it showed a good improvement effect on PD both in vivo and in vitro. After one month of administration of rehmannia glutinosa D (10 mg / kg), the performance of selenium-deficient PD model mice in the ball burying experiment and habituation / dehabituation experiment was better than that of the model group mice, indicating that the olfactory function of the mice was significantly improved; the fecal moisture content and fecal discharge amount of the mice were detected, and it was found that rehmannia glutinosa D can alleviate the constipation of selenium-deficient PD mice; the motor ability of the mice was tested by the rotating rod test and the climbing pole test, and it was found that rehmannia glutinosa D can significantly improve the motor ability of selenium-deficient PD mice. By detecting the content of TH+ neurons in the substantia nigra of mice in the midbrain, it was found that the damage to TH+ neurons in the substantia nigra of mice was significantly reduced after the administration of rehmannia glutinosa D. Therefore, rehmannia glutinosa D has the effect of preventing and treating PD and can improve PD-related movement disorders and non-movement disorders.

[0016] The present invention also discloses that when catalpol and rehmannia glycoside D are used in combination, the improvement effect thereof is increased by about 10% to 30% compared with the administration of rehmannia glycoside D alone, and the improvement effect thereof is increased by about 3% to 14% compared with the administration of catalpol alone. Catalpol has good anti-neuroinflammatory activity, and can also improve the relevant symptoms of MPTP-induced PD model mice by reducing oxidative stress; and rehmannia glycoside D in this study can reduce the aggregation of α-synuclein in the brain of PD model mice, so the combined administration of catalpol and rehmannia glycoside D acts on different targets, reduces the administration concentration of a single drug, and improves the efficacy of the drug, and has a good synergistic effect. Moreover, catalpol and rehmannia glycoside D have different drug targets, and the combined use of drugs can reduce the drug resistance of a single drug.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Rehmannia glutinosa D can alleviate MPP+-induced cell damage and alleviate related symptoms of PD model mice, including olfactory dysfunction and gastrointestinal dysfunction. In addition, Rehmannia glutinosa D also has a protective effect on the damage of TH+ neurons in the substantia nigra of PD mice; (2) When catalpol and Rehmannia glutinosa D are used in combination, they have a good protective effect on PD and the related symptoms caused by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of different doses of MPP+ and rehmannia glutinosa D on the viability of SH-SY5Y cells, where A is the toxic effect of different doses of rehmannia glutinosa D on SH-SY5Y cells, and B is the effect of different doses of MPP+ on the viability of SH-SY5Y cells;

[0019] Figure 2 The protective effect of rehmannia glutinosin D on the MPP+-induced SH-SY5Y cell model;

[0020] Figure 3 The protective effect of catalpol on the MPP+-induced SH-SY5Y cell model;

[0021] Figure 4 The protective effect of catalpol and rehmannia glutinosa D combination on MPP+-induced SH-SY5Y cell model;

[0022] Figure 5 The results show that rehmannia glutinosin D improves the constipation symptoms of PD selenium-deficient mice (n=10); A is the water content of feces of mice in each group; B is the amount of feces discharged by mice in each group;

[0023] Figure 6 The results show that rehmannia glutinosa D improves the olfactory dysfunction of PD selenium-deficient mice (n=10); A is the result of the ball burying experiment of each group of mice; B is the result of the habituation / dehabituation experiment of each group of mice.

[0024] Figure 7The results showed that rehmannia glutinosin D improved the motor ability of PD selenium-deficient mice (n=10); A was the results of the rotarod test of each group of mice; B was the results of the climbing pole test of each group of mice;

[0025] Figure 8 Rehmannia glutinosin D improves the spontaneous activity of PD selenium-deficient mice (n=10); A is a representative trajectory diagram of the open field test of each group of mice, and B is a comparison of the number of grid crossings of each group of mice in the open field test;

[0026] Fig. 9 Rehmannia glutinosin D improves the loss of TH+ neurons in the substantia nigra of PD selenium-deficient mice (n=3); wherein A is a representative immunohistochemical image of TH+ neurons in the midbrain of mice, and B is a statistical graph of the results of immunohistochemistry of TH+ neurons in the midbrain of mice. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0028] Example 1

[0029] Verification of the ability of rehmannia glutinosa or catalpol to inhibit MPP+-induced SH-SY5Y cell death in vitro:

[0030] SH-SY5Y cells were obtained from the laboratory of Associate Professor Deng Xueyang of the School of Traditional Chinese Medicine, China Pharmaceutical University. Rehmannia glutinosin D (CAS: 81720-08-3) was purchased from Nanjing Dierge Pharmaceutical Technology Co., Ltd., catalpol (CAS: 2415-24-9) was purchased from Baoji Chenguang Technology Co., Ltd., and MPP+ (CAS: 48134-75-4) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The cells were cultured in a 37°C constant temperature incubator (humidity 95%, CO2 concentration 5%), and the culture medium was DMEM / F12 containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution. The experiment was started when the cells were cultured to about the fourth generation.

[0031] SH-SH5Y cells in the logarithmic growth phase were treated with rehmannia glutinosine D for 24 hours according to a certain concentration gradient (0μM~80μM), and the cell viability was detected by CCK8 kit to detect the cytotoxic effect of rehmannia glutinosine D on SH-SY5Y. MPP+ was treated with MPP+ for 24 hours according to a certain concentration gradient (1mM~4mM), and the cell viability was detected by CCK8 kit to detect the cytotoxic effect of MPP+ on SH-SY5Y.

[0032] like Figure 1The figure shows the toxic effects of rehmannia glutinosa D and MPP+ on SH-SY5Y cells. The data are presented as Mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The experimental results show that when rehmannia glutinosa D is administered at 0μM to 40μM, it has no obvious cytotoxic effect, and MPP+ will cause 50% cell viability damage when administered at 3mM.

[0033] SH-SH5Y cells in the logarithmic growth phase were taken, and rehmannia glutinosin D was added for pretreatment for 4 hours according to a certain concentration gradient (0μM~40μM), and then 3mM MPP+ was added for co-incubation for 24 hours, and the cell viability was detected using the CCK8 kit.

[0034] like Figure 2 The figure shows the protective effect of rehmannia glutinosa D on the MPP+-induced SH-SY5Y cell model. The data are presented as Mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; the experimental results show that rehmannia glutinosa D exhibits significant improvement effects at a dosage of 5μM to 40μM.

[0035] SH-SH5Y cells in the logarithmic growth phase were taken, and catalpol was added for pretreatment for 4 hours according to a certain concentration gradient (1μM~100μM), and then 3mM MPP+ was added for co-incubation for 24 hours, and the cell viability was detected using the CCK8 kit.

[0036] like Figure 3 The figure shows the protective effect of catalpol on the MPP+-induced SH-SY5Y cell model. The data are presented as Mean±SEM. **p<0.01, ***p<0.001, ****p<0.0001. The experimental results show that catalpol exhibits a significant improvement effect at a dosage of 10μM to 100μM.

[0037] Example 2

[0038] Verification of the combined use of rehmannia glutinosin D and catalpol to inhibit MPP+-induced SH-SY5Y cell death in vitro:

[0039] Rehmannia glutinosin D and catalpol were co-administered to cells. SH-SY5Y cells in the logarithmic growth phase were taken. The concentrations of rehmannia glutinosin D were set to 0μM, 5μM, 10μM, 20μM, and 40μM, and the concentrations of catalpol were set to 0μM, 10μM, 20μM, 40μM, and 100μM, with a total of 25 administration concentrations. The above drug concentrations were combined in pairs and acted on the cells for 4 hours, and then 3mM MPP+ was added for co-incubation for 24 hours, and the cell viability was detected using the CCK8 kit.

[0040] like Figure 4 The figure shows the protective effect of catalpol and rehmannia glutinosa D combination on MPP+-induced SH-SY5Y cell model. Data are presented as Mean±SEM. **p<0.01, ****p<0.0001. The experimental results showed that when catalpol was 40μM and rehmannia glutinosa D was 10μM, the cell viability was the strongest, indicating the best efficacy, and the combination was significantly better than 10μM rehmannia glutinosa D alone.

[0041] Example 3

[0042] Verification of the effect of rehmannia glutinosin D on improving the symptoms of selenium-deficient PD mice:

[0043] Mouse modeling and drug administration: The experimental animals were C57BL / 6 mice, 4 weeks old, weighing 18 g to 22 g, purchased from Nanjing Annuokang Biotechnology Co., Ltd. MPTP (CNS: 28289-54-5) was purchased from Shanghai McLean Biochemical Technology Co., Ltd., rehmannia glutinosin D (CNS: 81720-08-3) was purchased from Nanjing Dierge Pharmaceutical Technology Co., Ltd., and mouse feed was ordered from Jiangsu Nantong Trofi Feed Technology Co., Ltd.

[0044] The room temperature in the laboratory was 20-22°C, the relative humidity was 40%-60%, the ventilation fan was used, the natural light source was 12h / day, the mice were kept in cages, and the cages were cleaned once every three days. The mice were divided into a blank group, a PD selenium-deficient group, a PD selenium-deficient + rehmannia glycoside D group, and a PD normal selenium group. After the mice were fed with different selenium contents for 5 weeks, the mice in the drug-treated group were gavaged with rehmannia glycoside D (10 mg / kg) every day for 3 consecutive weeks. Modeling began 3 weeks after administration. The mice in the model group and the drug-treated group were intraperitoneally injected with MPTP (30 mg / kg) every day. The mice in the blank group were given an equal amount of normal saline for 7 consecutive days. The mice in the drug-treated group were gavaged with rehmannia glycoside D half an hour before modeling.

[0045] Rehmannia glutinosin D improves constipation in selenium-deficient PD mice:

[0046] After the modeling is completed, collect the feces of the mice within half an hour, place them in a clean ep tube and count them. Weigh the wet weight of the mouse feces with the ep tube and record it as the wet weight. Place the feces in an oven at 37°C and dry it for 24 hours, weigh the dry weight of the mouse feces with the ep tube and record it as the dry weight. Discard the dried feces and weigh the weight of the ep tube, and record it as the tube weight. The calculation formula for the water content of mouse feces is: ((wet weight-tube weight)-(dry weight-tube weight)) / wet weight-tube weight×100%

[0047] The results are as follows Figure 5The figure shows that rehmannia glutinosa D improves the constipation symptoms of PD selenium-deficient mice (n=10). (A) Fecal water content of mice in each group; (B) Fecal excretion quantity of mice in each group. Data are presented as Mean±SEM. *p<0.05, **p<0.01, ****p<0.0001. The experimental results showed that the fecal water content of mice in the PD selenium-deficient group was significantly reduced, and the number of feces excreted within 30 minutes was significantly reduced. After administration of rehmannia glutinosa D, the fecal water content and fecal excretion quantity of mice were significantly increased compared with those of mice in the PD selenium-deficient group.

[0048] Rehmannia glutinosin D improves the olfactory function of selenium-deficient PD mice:

[0049] The buried ball test was used to detect the olfactory function of mice. The mice were restricted in diet 1 day before the test and during the experiment to keep the weight of the mice at 90% of their original body weight. Before the test, all mice were placed in the test room for 1 hour to adapt to the environment, and 5 minutes before the test, the mice were placed in the cage to adapt to the cage. A small piece of cheese ball was randomly placed in a corner of a clean test cage with 3 cm thick bedding, and the food was buried 1 cm under the bedding. The food used in the test was familiar to the mice before the dietary restriction. The mice were placed in the test cage at a constant distance from the hidden food. The latency time of the mice to find food was recorded. If the mouse failed to find the buried food within 2 minutes, the latency score was recorded as 120s.

[0050] The experimental results showed that in the ball burying experiment, compared with the blank group mice, the PD selenium-deficient group mice and the PD normal selenium group mice took significantly longer to find the cheese balls buried under the bedding.

[0051] The habituation / dehabituation experiment was used to detect the olfactory sensitivity of mice. Three days before the test, the mice were placed in cages alone, and odorless cotton swabs were placed in the cages to allow the mice to pre-adapt. One hour before the formal experiment, the mice were moved to the experimental operation room for pre-adaptation. The mice were placed in cages and two odorants (lemon essence, diluted 1:100; vanilla essence, diluted 1:100) were prepared. The odorants were dipped with cotton swabs. The mice were first exposed to one of the odorants for 120 seconds for three consecutive times, with an interval of 2 minutes each time. In the fourth experiment, the mice were exposed to a new odorant, and the sniffing time of the mice to the new odorant was recorded within 120 seconds.

[0052] The results are as follows Figure 6 The figure shows that rehmannia glutinosin D improves olfactory dysfunction in PD selenium-deficient mice (n=10). (A) Results of the ball burying experiment of mice in each group; (B) Results of the habituation / dehabituation experiment of mice in each group. Data are presented as Mean±SEM. **p<0.01, ****p<0.0001, #### p<0.0001VS Control,ΦΦΦΦ p<0.0001(Trial 4VS Trial 3). Figure 6 As shown in A, in the ball-burying experiment, the time taken by the PD selenium-deficient mice and the PD normal selenium-deficient mice to find the cheese ball buried under the bedding was significantly longer than that of the blank mice. After the administration of rehmannia glutinosine D, the time taken by the PD selenium-deficient + rehmannia glutinosine D-deficient mice to find the cheese was significantly shortened.

[0053] like Figure 6 As shown in B, in the habituation / dehabituation experiment, the sniffing time of the PD selenium-deficient mice and the PD normal selenium group in the first and fourth experiments was significantly shorter than that of the blank group mice, while the sniffing time of the PD selenium-deficient + rehmannia glycoside D group in the first and fourth experiments was significantly longer than that of the PD selenium-deficient mice. Compared with the third experiment, the sniffing time of the blank group mice and the PD selenium-deficient + rehmannia glycoside D group in the fourth experiment was significantly increased, while the sniffing time of the PD selenium-deficient mice and the PD normal selenium group mice was not significantly prolonged.

[0054] Rehmannia glutinosin D improves motor ability of selenium-deficient PD mice:

[0055] The rotarod test was used to detect the movement balance ability of mice. The test began after the mice were trained continuously for 3 days. The speed was 20rpm / min, and the time the mice spent on the rod within 2 minutes was detected. The climbing pole time was used to detect the movement ability of mice. The climbing pole device used was a rod with a diameter of 1cm, a length of 50cm, and a small ball with a diameter of 5cm fixed on the top. Gauze was wrapped around the rod to increase the friction of the mouse climbing, and the rod was placed vertically. Before the formal test, the mice had been trained for 3 days. The mouse was placed head up on the top of the climbing pole device, and the total time for the mouse to turn around and climb down the pole was recorded.

[0056] like Figure 7 As shown, rehmannia glutinosine D improves the motor ability of PD selenium-deficient mice (n=10). (A) Results of the rotating rod test of mice in each group; (B) Results of the climbing pole test of mice in each group. Data are presented as Mean±SEM. **p<0.01, ****p<0.0001. The experimental results showed that the time on the rod of mice in the PD selenium-deficient group and the PD normal selenium group was significantly reduced compared with the blank group mice, and the time required to climb the pole was significantly increased, while the time on the rod of mice in the PD selenium-deficient + rehmannia glutinosine D group was significantly longer than that of mice in the PD selenium-deficient group, and the time to climb the pole was significantly reduced.

[0057] Rehmannia glutinosin D improves spontaneous activity in selenium-deficient PD mice:

[0058] The open field test was used to detect the motor ability of mice. The mice were placed in a 50 cm × 50 cm open field apparatus. The ANY-MAZE behavioral software was used to automatically record the movement trajectory of the mice within 5 minutes and calculate the number of times the mice crossed the grid.

[0059] like Figure 8 As shown, rehmannia glutinosin D improved the spontaneous activity of PD selenium-deficient mice (n=10). Data are presented as Mean±SEM. **p<0.01, ***p<0.001, ****p<0.0001. The experimental results showed that the number of grid-piercing in PD selenium-deficient mice and PD normal selenium group mice was significantly reduced; compared with PD selenium-deficient mice, the number of grid-piercing in PD normal selenium group mice and PD selenium-deficient + rehmannia glutinosin D group mice was significantly increased.

[0060] Rehmannia glutinosin D improves the loss of TH+ neurons in the substantia nigra of selenium-deficient PD mice:

[0061] After the behavioral experiment, the mice were killed, and the whole brain of the mice was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned in the coronal plane, dehydrated with gradient alcohol, and microwaved for antigen repair for 10 minutes. After inactivating endogenous catalase with 3% hydrogen peroxide solution, the cells were placed in 4% BSA solution for blocking for 2 hours. The blocking solution was then discarded, and anti-TH (1:1000, Aifang Biology) primary antibody was added and incubated at 4°C for 12 hours. After the primary antibody incubation, the cells were washed three times with PBS, 5 minutes each time, and incubated with the secondary antibody for detecting the corresponding primary antibody for 1 hour. PBS washing, 3,3'-diaminobenzidine hydrochloride (DAB) staining, and hematoxylin counterstaining. After each step was completed, the sections were washed with PBS 3 times, 10 minutes each time. Finally, the sections were mounted, dehydrated, xylene transparent, and neutral gum sealed. The positive proteins in the substantia nigra were observed and photographed under a slice scanner, and the positive proteins were analyzed by optical density using Image Pro Plus 6.0 software.

[0062] like Fig. 9 As shown, rehmannia glutinosin D improved the loss of TH+ neurons in the substantia nigra of PD selenium-deficient mice (n=3). Data are presented as Mean±SEM. **p<0.01, ***p<0.001. The experimental results showed that the content of TH+ neurons in the substantia nigra of mice in the PD selenium-deficient group and the PD normal selenium group was significantly reduced, while the content of TH+ neurons in the PD selenium-deficient + rehmannia glutinosin D group was significantly increased compared with the PD selenium-deficient group.

Claims

1. Use of rehmannia glycoside D in the preparation of drugs for preventing and / or treating Parkinson's disease.

2. The use according to claim 1, characterized in that: The Parkinson's disease is Parkinson's disease caused by trace element deficiency.

3. The use according to claim 2, characterized in that: The trace element is selenium.

4. The use according to claim 1, characterized in that: Rehmannia glutinosin D reduces MPP+-induced cell damage and substantia nigra TH+ neuronal damage, and alleviates movement disorders and non-movement disorders caused by Parkinson's disease.

5. The use according to claim 1, characterized in that: The non-motor disorder is olfactory dysfunction or gastrointestinal dysfunction.

6. The use according to claim 1, characterized in that: The dosage of the rehmannia glutinosa glycoside D is 5 to 40 μM / day.

7. The use according to claim 1, characterized in that: Rehmannia glutinosa D and catalpol are used in combination in the preparation of medicines for preventing and / or treating Parkinson's disease.

8. The use according to claim 7, characterized in that: The mass ratio of catalpol to rehmannia glycoside D is 10-2:

1.

9. The use according to claim 1, characterized in that: The medicine is in the form of nasal drops, eye drops, capsules, tablets, films, suppositories or injections.

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