Use of Dihuangoside D in the Preparation of a Drug for Preventing and / or Treating Parkinson's Disease

Abstract

The application discloses application of Dendrofin D in preparation of a medicine for preventing and / or treating Parkinson's disease, wherein the Parkinson's disease is caused by deficiency of a trace element, and the trace element is selenium; Dendrofin D can reduce MPP+-induced cell damage and damage of TH+ neurons in the substantia nigra, and can reduce motor disorders and non-motor disorders caused by Parkinson's disease; meanwhile, Dendrofin D and catalpol are combined to be applied in preparation of the medicine for preventing and / or treating Parkinson's disease, and the combination can effectively improve the protection on Parkinson's disease and related symptoms caused by the Parkinson's disease, has the effects of preventing and treating Parkinson's disease, and can improve motor disorders and non-motor disorders related to Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The application relates to application of Densiflorin D in preparation of a medicine for preventing and / or treating Parkinson's disease. BACKGROUND

[0002] Parkinson's disease (PD) is the second largest neurodegenerative disease after Alzheimer's disease, and PD patients are often accompanied by non-motor disorders such as constipation and loss of smell, and 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 deficiency of liver and kidney, and on this basis, phlegm and blood stasis are generated, which blocks the brain collaterals and aggravates the internal wind.

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

[0004] Radix Rehmanniae Preparata is prepared from the rhizome of Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. and has the effects of tonifying blood and yin, nourishing essence and filling marrow, and can be used to treat liver-kidney yin deficiency and the like, and is commonly used to treat PD in clinic. The main chemical components of Radix Rehmanniae Preparata include iridoid glycosides, amino acids, phenylethanoid glycosides, trace elements, polysaccharides and ionones, which have the effects of antioxidant, anti-inflammatory and inhibition of cell apoptosis (Chinese Journal of Experimental Traditional Medical Formulae, 2022, 28(23):228-236.). The pharmacological effects of Radix Rehmanniae Preparata in treating PD have been verified in animals and humans, but the main effective components of Radix Rehmanniae Preparata in treating PD are still unknown, in addition, the complex chemical components of Radix Rehmanniae Preparata are not conducive to quality control and the development of related medicines, and the complex chemical components are prone to cause adverse reactions such as abnormal cholesterol metabolism. SUMMARY

[0005] The application aims to provide application of Densiflorin D in preparation of a medicine for preventing and / or treating Parkinson's disease, and also provides application of Densiflorin D and catalpol in combination in preparation of a medicine for preventing and / or treating Parkinson's disease.

[0006] Technical solution: The application discloses application of Dihuangoside D in preparation of a medicine for preventing and / or treating Parkinson's disease.

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

[0008] Among them, the trace element is selenium.

[0009] Among them, Dihuangoside D reduces MPP+-induced cell damage and damage of TH+ neurons in the substantia nigra, and reduces movement disorders and non-movement disorders caused by PD.

[0010] Among them, the non-movement disorder is olfactory dysfunction or gastrointestinal dysfunction.

[0011] Among them, the administration dose of the Dihuangoside D is 5-40 muM / day.

[0012] The application also discloses that Dihuangoside D is combined with catalpol to be applied in preparation of a medicine for preventing and / or treating PD.

[0013] Among them, the mass ratio of the catalpol and the Dihuangoside D is 10-2:1.

[0014] Among them, the medicine is in the form of nasal drops, eye drops, capsules, tablets, films, suppositories or injections.

[0015] Invention principle: The application discloses that Dihuangoside D can improve a PD related model, including an in-vitro cell model and an animal model. When the administration dose is 5 muM-40 muM, the Dihuangoside D shows obvious improvement on a MPP+-induced SH-SY5Y cell model; when treating PD and related movement disorders and non-movement disorders, the Dihuangoside D shows good improvement on PD in-vivo and in-vitro. After the selenium deficiency PD model mice are given the Dihuangoside D (10 mg / kg) for one month, the performance of the mice in the buried ball experiment and the habituation / dishabituation experiment is better than that of the model group mice, indicating that the olfactory function of the mice is obviously improved; the water content and the number of feces of the mice are detected, and it is found that the Dihuangoside D can reduce constipation of the selenium deficiency PD mice; the rotating rod experiment and the pole climbing experiment are used to detect the movement ability of the mice, and it is found that the Dihuangoside D can obviously improve the movement ability of the selenium deficiency PD mice. The TH+ neuron content in the midbrain of the mice is detected, and it is found that the TH+ neuron damage of the mice is obviously reduced after the Dihuangoside D is given. Therefore, the Dihuangoside D has the effects of preventing and treating PD and can improve the related movement disorders and non-movement disorders of PD.

[0016] The application also discloses that when the catalpol and the rehderin D are used in combination, the improvement effect is increased by about 10% to 30% compared with the rehderin D alone and is increased by about 3% to 14% compared with the catalpol alone. The catalpol has good anti-neuroinflammatory activity and can also improve the related symptoms of the MPTP-induced PD model mice by reducing oxidative stress; and the rehderin D in the present application can reduce the aggregation of alpha-synuclein in the brain of the PD model mice, so that the catalpol and the rehderin D used in combination act on different targets, reduce the administration concentration of a single drug, improve the efficacy of the drug and have good synergistic effect. Moreover, the catalpol and the rehderin D have different drug targets, and the combination of the drugs can reduce the drug resistance of a single drug.

[0017] Beneficial effects: Compared with the prior art, the application has the following remarkable advantages: (1) the rehderin D can reduce the MPP+-induced cell damage and reduce the related symptoms of the PD model mice, including olfactory dysfunction and gastrointestinal dysfunction, and in addition, the rehderin D also has a protective effect on the damage of TH+ neurons in the midbrain of the PD mice; (2) the catalpol and the rehderin D used in combination have good protective effect on PD and the related symptoms caused by PD. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 2 is a diagram of the effects of different doses of MPP+ and the rehderin D on the viability of SH-SY5Y cells, wherein A is the toxic effect of different doses of the rehderin 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 Fig. 4 is a diagram of the protective effect of the rehderin D on the MPP+-induced SH-SY5Y cell model;

[0020] Figure 3 Fig. 6 is a diagram of the protective effect of the catalpol on the MPP+-induced SH-SY5Y cell model;

[0021] Figure 4 Fig. 8 is a diagram of the protective effect of the combination of the catalpol and the rehderin D on the MPP+-induced SH-SY5Y cell model;

[0022] Figure 5 Fig. 10 is a diagram of the improvement of the rehderin D on the constipation symptoms of the PD selenium-deficient mice (n=10); wherein A is the fecal water content of the mice in each group, and B is the fecal output of the mice in each group;

[0023] Figure 6 Fig. 12 is a diagram of the improvement of the rehderin D on the olfactory dysfunction of the PD selenium-deficient mice (n=10); wherein A is the burying ball experiment result of the mice in each group, and B is the habituation / dishabituation experiment result of the mice in each group.

[0024] Figure 7Rehymarin D improves the motor ability of PD selenium deficiency mice (n=10); wherein, A is the results of the rotarod test of mice in each group; B is the results of the pole climbing test of mice in each group;

[0025] Figure 8 Rehymarin D improves the spontaneous activity of PD selenium deficiency mice (n=10); wherein, A is a representative trajectory chart of the open field test of mice in each group, and B is a comparison of the grid crossing times of mice in each group;

[0026] Figure 9 Rehymarin D improves the loss of TH+ neurons in the substantia nigra of PD selenium deficiency mice (n=3); wherein, A is a representative picture of immunohistochemistry of TH+ neurons in the mouse midbrain, and B is a statistical chart of immunohistochemistry of TH+ neurons in the mouse midbrain. DETAILED DESCRIPTION

[0027] The technical solutions of the present application are further described below in combination with examples. The test materials used in the examples can be purchased through conventional channels.

[0028] Example 1

[0029] Verification of the inhibition of MPP+-induced SH-SY5Y cell death by Rehymarin D or Catalpol in vitro:

[0030] SH-SY5Y cells were obtained from the laboratory of Professor Deng Xueyang, School of Chinese Medicine, China Pharmaceutical University, Rehymarin D (CAS: 81720-08-3) was purchased from Nanjing Dilige Pharmaceutical Technology Co., Ltd., Catalpol (CAS: 2415-24-9) was purchased from Baoji Chen Guang 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-antibiotic solution. The cells were cultured to about 4 passages before the experiment.

[0031] SH-SH5Y cells in the logarithmic growth phase were taken and treated with Rehymarin D at a certain concentration gradient (0 μM-80 μM) for 24 h. The CCK8 kit was used to detect cell viability and the cytotoxicity of Rehymarin D on SH-SY5Y. MPP+ was added at a certain concentration gradient (1 mM-4 mM) for 24 h. The CCK8 kit was used to detect cell viability and the cytotoxicity of MPP+ on SH-SY5Y.

[0032] As Figure 1The figure shows the cytotoxic effects of rehmannia glycoside D and MPP+ on SH-SY5Y cells. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results show that rehmannia glycoside D at 0 μM–40 μM had no significant cytotoxic effect, while MPP+ at 3 mM caused 50% cell viability loss.

[0033] SH-SH5Y cells in the logarithmic growth phase were pretreated with rehmannia glutinosa D at a certain concentration gradient (0 μM to 40 μM) for 4 h, and then co-incubated with 3 mM MPP+ for 24 h. Cell viability was detected using a CCK8 assay kit.

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

[0035] SH-SH5Y cells in the logarithmic growth phase were pretreated with catalpol at a certain concentration gradient (1 μM to 100 μM) for 4 h, and then co-incubated with 3 mM MPP+ for 24 h. Cell viability was detected using the CCK8 assay kit.

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

[0037] Example 2

[0038] Validation of the combined use of rehmannia glutinosa D and catalpol in in vitro inhibition of MPP+-induced SH-SY5Y cell death:

[0039] Rehmannia glutinosa D and catalpol were administered to cells in combination. SH-SY5Y cells in logarithmic growth phase were used. The concentrations of rehmannia glutinosa D were set at 0 μM, 5 μM, 10 μM, 20 μM, and 40 μM, and the concentrations of catalpol were set at 0 μM, 10 μM, 20 μM, 40 μM, and 100 μM, for a total of 25 administration concentrations. After treatment with each drug concentration in pairs for 4 h, the cells were incubated with 3 mM MPP+ for 24 h. Cell viability was assessed using a CCK8 assay kit.

[0040] As shown in Figure 4 Figure 9, the protective effect of combination of catalpol and rehderin D on MPP+-induced SH-SY5Y cell model. Data are expressed as Mean ± S.E.M. **p<0.01, ****p<0.0001. The experimental results show that when catalpol is 40 μM and rehderin D is 10 μM, the cell viability is the strongest, indicating the best efficacy, and the combination is significantly better than 10 μM rehderin D alone.

[0041] Example 3

[0042] Verification of rehderin D for improving the related symptoms of selenium-deficient PD mice:

[0043] Mouse modeling and drug administration: The test animals were C57BL / 6 mice, 4 weeks old, weighing 18-22 g, purchased from Nanjing Annuo Biological Technology Co., Ltd. MPTP (CNS: 28289-54-5) was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., rehderin D (CNS: 81720-08-3) was purchased from Nanjing Dilige Pharmaceutical Technology Co., Ltd., and mouse feed was ordered from Jiangsu Nantong Trolife Feed Technology Co., Ltd.

[0044] The laboratory room temperature was 20-22°C, the relative humidity was 40%-60%, the ventilation fan was ventilated, the natural light source was 12h / day, and the mice were caged, and the cage was cleaned every three days. The mice were divided into blank group, PD selenium-deficient group, PD selenium-deficient + rehderin D group and PD normal selenium group. After feeding the mice with different selenium contents for 5 weeks, the mice in the drug administration group were given rehderin D (10 mg / kg) by gavage every day for 3 weeks. After 3 weeks of drug administration, modeling was started, and the mice in the model group and the drug administration group were injected intraperitoneally with MPTP (30 mg / kg) every day for 7 days, and the mice in the blank group were given an equal amount of normal saline, and the mice in the drug administration group were given rehderin D by gavage half an hour before modeling.

[0045] Improvement of rehderin D on constipation of selenium-deficient PD mice:

[0046] After modeling, the mouse feces within half an hour were collected, placed in a clean ep tube and counted. The wet weight of the mouse feces with the ep tube was weighed and recorded as the wet weight. The feces were placed in an oven at 37°C and dried for 24 hours, and the dry weight of the mouse feces with the ep tube was weighed and recorded as the dry weight. The weight of the ep tube after drying the feces was discarded and recorded as the tube weight. The formula for calculating the water content of the mouse feces was: ((wet weight-tube weight)-(dry weight-tube weight)) / wet weight-tube weight x 100%

[0047] Results are shown in Figure 5The results showed that the PD selenium deficiency group of mice and the PD normal selenium group of mice spent significantly longer time to find the cheese ball buried in the litter compared with the blank group of mice.

[0048] Rehymoside D improves the olfactory function of selenium deficiency PD mice:

[0049] The olfactory function of mice was detected by the buried ball experiment. The mice were restricted in diet 1 day before the test and during the experiment to keep the body weight of the mice at 90% of the original body weight. Before the test, all mice were placed in the test room for 1 h to adapt to the environment, and 5 min 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 litter, and the food was buried 1 cm under the litter. The food used for the test was familiar to the mice before dietary restriction. The mice were placed in the test cage and kept a constant distance from the hidden food. The latency time of the mice to find the food was recorded. If the mice failed to find the buried food within 2 min, the latency score was recorded as 120 s.

[0050] The results showed that in the buried ball experiment, the PD selenium deficiency group of mice and the PD normal selenium group of mice spent significantly longer time to find the cheese ball buried in the litter compared with the blank group of mice.

[0051] The olfactory sensitivity of mice was detected by habituation / dishabituation experiment. 3 days before the test, the mice were placed alone in the cage and a cotton swab with no odor was placed in the cage for the mice to pre-adapt. 1 h before the formal experiment, the mice were moved to the experimental operation room for pre-adaptation, and the mice were placed in the cage. Two kinds of odorants (lemon essence, 1:100 dilution; vanilla essence, 1:100 dilution) were prepared, and the odorants were dipped with cotton swabs. The mice were exposed to one kind of odorant for 120 s for 3 times in succession, with an interval of 2 min each time, and the mice were exposed to a new odorant at the 4th experiment. The sniffing time of the mice to the new odorant within 120 s was recorded.

[0052] The results are shown in Figure 6 Rehymoside D improves the olfactory dysfunction of PD selenium deficiency mice (n=10). (A) Buried ball experiment results of mice in each group; (B) habituation / dishabituation experiment results of mice in each group. Data are expressed as Mean±S.E.M. **p<0.01, ****p<0.0001, #### p<0.0001 VS Control,ΦΦΦΦ p < 0.0001 (Trial 4 vs Trial 3). Experimental results show that, Figure 6 As shown in Figure A, in the cheese ball burial experiment, compared with the control group, the time taken for PD selenium-deficient mice and PD normal selenium group mice to find the cheese ball buried under the bedding was significantly longer. After administration of rehmannia glycoside D, the time required for PD selenium-deficient + rehmannia glycoside D group mice to find the cheese was significantly shortened.

[0053] like Figure 6 As shown in Figure B, in the habituation / dehabituation experiment, the olfaction time of mice in the PD selenium-deficient group and the PD normal selenium group was significantly reduced compared to the control group in the first and fourth experiments, while the olfaction time of mice in the PD selenium-deficient + rehmannia glutinosa D group was significantly increased compared to the PD selenium-deficient group in the first and fourth experiments. Compared to the third experiment, the olfaction time of mice in the control group and the PD selenium-deficient + rehmannia glutinosa D group was significantly increased in the fourth experiment, while the olfaction time of mice in the PD selenium-deficient group and the PD normal selenium group was not significantly prolonged.

[0054] Rehmannia glutinosa D improves motor function in selenium-deficient PD mice:

[0055] The rotarod test was used to assess the motor balance ability of mice. Mice were tested after 3 days of continuous training, using a rotarod speed of 20 rpm / min to measure the time spent on the rotarod within 2 minutes. The pole climbing time was used to assess the mice's motor ability. The pole was 1 cm in diameter and 50 cm long, with a 5 cm diameter ball fixed to the top. Gauze was wrapped around the pole to increase friction for climbing, and the pole was placed vertically. Mice had been trained for 3 days before the formal test. The mice were placed head-up at the top of the pole, and the total time taken for them to turn around and climb down was recorded.

[0056] like Figure 7 As shown, rehmannia glycoside D improved the motor function of selenium-deficient PD mice (n=10). (A) Results of rotarod test in each group of mice; (B) Results of pole climbing test in each group of mice. Data are expressed as mean ± SEM. **p<0.01, ****p<0.0001. The experimental results showed that the time spent on the rotarod was significantly reduced and the time required to climb the pole was significantly increased in the PD selenium-deficient group and the PD normal selenium group compared with the control group. However, the time spent on the rotarod was significantly prolonged and the time required to climb the pole was significantly reduced in the PD selenium-deficient group compared with the PD selenium-deficient group.

[0057] Rehmannia glutinosa 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 an open field device of 50 cm x 50 cm, and the ANY-MAZE behavior software was used to automatically record the action trajectory of the mice within 5 min. The number of grid crossings of the mice was calculated.

[0059] As shown in Figure 8 Figure 6, rehynboside D improved the spontaneous activity of PD selenium-deficient mice (n = 10). The data are presented as Mean ± S.E.M. **p < 0.01, ***p < 0.001, ****p < 0.0001. The experimental results showed that the number of grid crossings of the PD selenium-deficient group mice and the PD normal selenium group mice was significantly reduced; compared with the PD selenium-deficient group mice, the number of grid crossings of the PD normal selenium group mice and the PD selenium-deficient + rehynboside D group mice was significantly increased.

[0060] Rehynboside D improved the loss of TH+ neurons in the substantia nigra of selenium-deficient PD mice:

[0061] After the behavioral experiment, the mice were sacrificed, and the whole brain of the mice was fixed with 4% paraformaldehyde, paraffin-embedded, coronally sectioned, gradient alcohol dehydrated, and microwave oven antigen repaired for 10 min. After inactivation of endogenous hydrogen peroxidase with 3% hydrogen peroxide solution, the slices were placed in 4% BSA solution for blocking for 2 h. Then the blocking solution was discarded, and the anti-TH (1:1000, Aif Bioscience) primary antibody was added, and incubated at 4°C for 12 h. After incubation of the primary antibody, the slices were washed with PBS three times, 5 min each time, and incubated with the secondary antibody for 1 h. After PBS washing, 3, 3'-diaminobenzidine hydrochloride (DAB) staining, and hematoxylin re-staining, the slices were washed with PBS three times for 10 min each time after each step. Finally, the slices were mounted, dehydrated, xylene transparent, and neutral resin sealed. The positive proteins in the substantia nigra were observed under a slice scanner and photographed, and the optical density of the positive proteins was analyzed by Image Pro Plus 6.0 software.

[0062] As shown in Figure 9 Figure 7, rehynboside D improved the loss of TH+ neurons in the substantia nigra of PD selenium-deficient mice (n = 3). The data are presented as Mean ± S.E.M. **p < 0.01, ***p < 0.001. The experimental results showed that the content of TH+ neurons in the substantia nigra of the PD selenium-deficient group and the PD normal selenium group mice was significantly reduced, and the content of TH+ neurons in the PD selenium-deficient + rehynboside D group mice was significantly increased compared with the PD selenium-deficient group mice.

Claims

1. Application of Rehmannia glutinosa D as the sole active ingredient in the preparation of drugs for the prevention and / or treatment of Parkinson's disease caused by selenium deficiency.

2. The application according to claim 1, characterized in that, Rehmannia glutinosa D reduces MPP+-induced cell damage and TH+ neuronal damage in the substantia nigra, and alleviates motor and non-motor impairments associated with Parkinson's disease.

3. The application according to claim 2, characterized in that, The non-motor disorders are olfactory dysfunction or gastrointestinal dysfunction.

4. The application according to claim 1, characterized in that, The dosage of rehmannia glycoside D is 5 ~ 40 μM / day.

5. The application of rehmannia glycoside D in combination with catalpol in the preparation of drugs for the prevention and / or treatment of Parkinson's disease caused by selenium deficiency, characterized in that, The mass ratio of catalpol to rehmannia glycoside D is 10~2:

1.

6. The application according to claim 1, characterized in that, The drug is available in the form of nasal drops, eye drops, capsules, tablets, films, suppositories, or injections.

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