Natural compound composition as well as preparation method and application thereof

Abstract

The invention discloses a natural compound composition as well as a preparation method and application thereof, and relates to the technical field of medicines, effective components of the natural compound composition are ginsenoside Rb1 and periplocin, the compound composition has an obvious protection effect on mouse nerve microglial cells BV2 damaged by MPP +, zebrafish PD-like dyskinesia caused by MPTP can be obviously improved, and the effect of preventing and treating MPP + is achieved. The compound combination is expected to be used for preventing and treating Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a natural compound combination for preventing and treating Parkinson's disease, and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is the second largest neurodegenerative disease. Its main pathological feature is the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which causes a significant decrease in dopamine (DA) in the synaptic cleft of the substantia nigra and striatum. Clinically, it usually manifests as resting tremor, bradykinesia, movement disorders and muscle rigidity, accompanied by constipation, depression, memory loss and other symptoms. With the aging of my country, the number of Parkinson's patients over the age of 65 has exceeded 4 million, and the incidence rate is still rising, which not only seriously affects the quality of life of patients, but also brings a huge burden to families and society.

[0003] BV2 cells are a microglial cell line derived from mice and are widely used in neuroscience research. This immortalized cell line can be used as an in vitro model to study the pathological mechanisms of neurodegenerative diseases and to evaluate the efficacy and pharmacological analysis of potential anti-PD compounds. Studies have confirmed that inflammatory factors derived from microglia can promote the death of damaged neurons and the regeneration of new neurons, maintaining the normal function of the brain. When microglia die, the homeostasis of brain neurons will be broken and neuronal degeneration and death will be accelerated. Therefore, screening compounds that have a protective effect on BV2 cells can be used as alternative drugs for anti-PD.

[0004] MPTP is converted into a pyridinium salt intermediate (MPDP) by monoamine oxidase B (MAOB). + ), which is further oxidized to generate the toxic metabolite methyl-phenyltetrahydropyridinium salt (MPP + ), while MPP + In terms of structure-activity relationship, its structure is close to dopamine, so it will be selectively transported into the substantia nigra neurons through the midbrain dopamine transporter (DAT), causing oxidative stress and death of DA neurons in the substantia nigra compacta (SNc). + It exerts its neurotoxicity by specifically taking up dopaminergic nerve endings, inhibiting mitochondrial energy metabolism, causing neurons to degenerate due to lack of ATP necessary to maintain their functional integrity, thereby inducing Parkinson's disease. + As an inducer, it can induce typical symptoms or biochemical characteristics similar to those of Parkinson's patients at the cellular and animal levels.

[0005] At present, the drugs commonly used in clinical treatment of Parkinson's disease can be roughly divided into the following six categories: compound levodopa, dopamine receptor agonists, monoamine oxidase type B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, and amantadine. These drugs are mainly used to restore the balance of dopaminergic and acetylcholine nervous system functions, but the anti-PD drugs on the market have disadvantages such as limited efficacy, large toxic side effects, and short effective time. Traditional Chinese medicines such as ginseng and gastrodia contain a variety of natural compounds with neuroprotective effects, and their effectiveness and safety have been proven in hundreds of years of Chinese medicine practice. Selecting ingredients with good activity, low side effects, and low extraction cost from these natural compounds, and conducting scientific proportioning and verification, is expected to find new effective drugs or lead compounds for the prevention and treatment of PD, providing an important basis for analyzing the molecular mechanism of PD and the development of new anti-PD drugs. Summary of the invention

[0006] The present invention provides a natural compound combination with ginsenoside Rb1 (G-Rb1) and Periplocin as active ingredients, wherein under a specific concentration ratio and dissolution method, methyl-phenyl tetrahydropyridinium salt (MPP + )-induced BV2 cell damage, and has a significant therapeutic effect on MPTP-induced zebrafish Parkinson's disease motor symptoms. The natural compound combination of the present invention is expected to be used to prepare drugs for the prevention and treatment of Parkinson's disease (PD) patients, or as a lead compound for the development of new anti-PD drugs.

[0007] The technical solution of the present invention is as follows:

[0008] A combination of natural compounds, the main active ingredients of which include ginsenoside Rb1 (G-Rb1) and Periplocin.

[0009] The method for preparing the natural compound combination specifically comprises the following steps:

[0010] Step 1: Use sterile water to ultrasonically dissolve ginsenoside Rb1, add water to prepare a G-Rb1 stock solution with a concentration of 1-200 μM, use dimethyl sulfoxide to oscillate to dissolve dianthus glycoside, and add water to prepare a dianthus glycoside stock solution with a concentration of 14.4-70 μM;

[0011] Step 2: Mix the G-Rb1 stock solution and the dianthus glycoside stock solution in a volume ratio of 1:1 to obtain a natural compound combination.

[0012] The present invention also provides application of the natural compound combination in preparing drugs for preventing and treating Parkinson's disease.

[0013] The natural compound combination of the present invention does not require the addition of commonly used inert auxiliary materials in pharmacy, such as diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

[0014] The natural compound raw materials used in the present invention are easily available, highly safe, and have low extraction costs. The chemical structures and components of the compounds are clear, and the quality is controllable. Whether it is a compound monomer or a crude extract of a raw material drug, the efficacy is clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The MPP of each group of drugs + Resulting in BV2 cell viability detection diagram;

[0016] Figure 2 This is a diagram of zebrafish behavior trajectory;

[0017] Figure 3 This is a heat map of zebrafish behavior trajectories;

[0018] Figure 4 The results of zebrafish behavior analysis, (A) is the total movement distance of juveniles in different drug treatment groups; (B) is the scatter plot of the movement speed per minute of each juvenile in different drug treatment groups within 30 minutes. DETAILED DESCRIPTION

[0019] The invention will be further described below by way of examples and in conjunction with pharmacological tests.

[0020] The ginsenoside Rb1 (G-Rb1) and Periplocin used in the present invention are both commercially available and can also be extracted according to conventional methods disclosed herein.

[0021] The materials and instruments involved in the cell and animal experiments included in the present invention are as follows:

[0022] 1. Animals and cells

[0023] Zebrafish larvae: AB strain experimental zebrafish are spawned by natural mating of male and female in a ratio of 2:1. Fertilized eggs are collected 2 days later (2 dpf) and can be used for pharmacological and toxicological experiments.

[0024] BV2 cells: mouse microglial cells, purchased from Wuhan Pronocell Life Science Co., Ltd.;

[0025] 2. Drugs and main reagents

[0026] Fetal bovine serum and DMEM medium were purchased from Gibco;

[0027] MPP+ was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; MPTP was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ginsenoside Rb1 (G-Rb1) was from Yunnan Teanna Pharmaceutical Co., Ltd., and dianthus glycoside was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (purity ≥98%); 60×E3 water preparation method: weigh 696mg NaCl, 32mgKCl, 87.56mg CaCl2, and 195.6mg MgCl2.6H2O respectively, dissolve them in ultrapure water and make up to 40mL, and store at -20℃; 1×E3 water: dilute 60×E3 water with ultrapure water according to the required volume when using.

[0028] 3. Main test instruments

[0029] Zebrafish culture system: Beijing Aisheng Technology Development Co., Ltd.; HPG280BX light incubator: Harbin Donglian Electronic Technology Development Co., Ltd.; 6- and 96-well plates: NEST; ANY-maze animal behavior analysis software: Stoelting, USA; CCK-8 (cell proliferation-toxicity detection kit): Biosharp.

[0030] Example 1

[0031] Step 1: Use sterile water to ultrasonically dissolve ginsenoside Rb1, add water to prepare G-Rb1 stock solution A with a concentration of 1 μM, use dimethyl sulfoxide to oscillate to dissolve dianthus glycoside, and add water to prepare dianthus glycoside stock solution B with a concentration of 14.4 μM; Use sterile water to ultrasonically dissolve ginsenoside Rb1, add water to prepare G-Rb1 stock solution C with a concentration of 200 μM, use dimethyl sulfoxide to oscillate to dissolve dianthus glycoside, and add water to prepare dianthus glycoside stock solution D with a concentration of 70 μM;

[0032] Step 2: Mix the stock solution A and the stock solution B at a volume ratio of 1:1 to obtain the compound combination I treatment solution; mix the stock solution C and the stock solution D at a volume ratio of 1:1 to obtain the compound combination II treatment solution.

[0033] Example 2

[0034] Cell viability assay

[0035] 1. BV2 cell culture

[0036] BV2 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 100 g / mL in a 37°C, 5% CO2 incubator. The cells grew well and were cultured to the logarithmic growth phase. BV2 cells were seeded into 96-well plates at 3×10 3 cells / well, incubate with 5% CO2 for 24 hours, and perform drug administration after the cells grow to 80%-90%.

[0037] 2. Effect of G-Rb1 and G-Rb1 combined with Periploca dianthus glycosides (compound combination I treatment solution) on MPP + Resulting BV2 cell viability assay

[0038] The cultured BV2 cells were divided into blank control group (NC), MPP + The model group (1000 μM), the G-Rb1 administration group, the G-Rb1+periploca glycosides [0.5 μM+7.2 μM, 1 μM+7.2 μM, 2.5 μM+7.2 μM, 5 μM+7.2 μM, 10 μM+7.2 μM, 20 μM+7.2 μM, which is a compound combination obtained by mixing different concentrations of G-Rb1 stock solutions (1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM) and periploca glycosides stock solution (14.4 μM) in a volume ratio of 1:1] were used. Each group was set up with 4 replicate wells and 3×10 3 cells / well, incubated with 5% CO2 for 24 h, and then treated as follows: the G-Rb1 group was treated with G-Rb1, and the natural compound combination group was treated with the above-mentioned different concentrations of G-Rb1 and dianthus glycosides. After 6 h, MPP was added while keeping the concentrations of G-Rb1 and dianthus glycosides unchanged. + (1000 μM) continued to treat the cells for 24 h, while the model group only added 1000 μM MPP + Induce cell damage (same treatment for 24h), the blank group was not treated with any drugs, and after the culture was completed, 10μL CCK-8 reagent was added to each well, and cultured for 1.5h at 37℃ and 5% CO2 in the dark, and the absorbance A at 450nm and 630nm was measured on a multi-label enzyme-linked immunosorbent assay.

[0039] 3. Data processing and statistics

[0040] The results are as follows Figure 1 As shown, the effects of each group of drugs on MPP + The effect of loss of BV2 cell viability was expressed as the percentage of the absorbance values ​​of different groups to the absorbance value of the blank control group: (A450 administration-A630 administration) / (A450 blank-A630 blank)×100. The experiment was repeated 3 times and the results were statistically analyzed. Compared with the blank control group: *, P < 0.05, **, P < 0.01, and compared with MPP + Compared with the model group: #, P < 0.05, ##, P < 0.01, indicating that G-Rb1 + dianthus glycosides have an effect on MPP + The resulting loss of cell viability has a protective effect, and the use of 0.5μM G-Rb1+7.2μM Periplocin can enhance the viability of MPP+ damaged BV2 cells.

[0041] Example 3

[0042] Therapeutic effects of G-Rb1 and G-Rb1 combined with dianthus glycosides on MPTP-induced Parkinson's-like motor symptoms in zebrafish

[0043] 1. Animal grouping and treatment: Wild-type zebrafish embryos (2 dpf) with normal morphology hatched in the laboratory were selected. A 6-well plate was used with 25 embryos per well and a volume of 5-6 mL per well. The embryos were divided into a blank control group (NC), an MPTP (50 μM) model group, a G-Rb1 (200 μM) drug group, and a 100 μM G-Rb1+35 μM dianthus glycoside (natural compound combination II treatment solution) combination group. The blank control group was treated with 1×E3 water, and the model group and each drug group were treated with MPTP at a final concentration of 50 μM for 4 consecutive days. After the induction, each drug group was replaced with the corresponding final concentration of the drug and continued to be treated for 24 h. The blank control group and the MPTP model group remained unchanged and continued to be treated with the corresponding liquid. Each group was treated for a total of 5 days. After the treatment, each group of zebrafish fry was placed in the corresponding behavior trajectory shooting mold according to one per well, and video was shot. After the shooting, the video was imported into the behavior trajectory analysis software for behavior trajectory analysis of each group.

[0044] 2. Data processing and statistics

[0045] Analysis indicators: Behavior trajectory diagram and movement distance of zebrafish fry in each group. The experiment was repeated 3 times and the results were statistically analyzed. The results are as follows Figure 2-4 The following are the behavior trajectory of juveniles, heat map of juvenile residence time, and total movement distance of juveniles ( Figure 4 A) The speed of the young fish moving per minute ( Figure 4 B), each observation time was 30 minutes, 16 fry in each group, NC was the blank untreated group, MPTP group was the PD model group, the drug treatment time was 4 days, G-Rb1 treatment group was added with G-Rb1 for prevention and treatment at the same time as MPTP modeling. It can be seen from the figure that the combination of 100μM G-Rb1 and 35μM dianthus glycosides can improve the PD-like movement disorder induced by MPTP in zebrafish, as shown in the following: compared with the PD model group, the behavior trajectory and residence time of the fry in the G-Rb1 treatment group tended to be normal, the total moving distance recovered to the level equivalent to that of the normal group, and the distribution of the moving speed of each fish per minute within 30 minutes recovered to be similar to that of the normal group (normal distribution). Compared with the blank control group: *, P < 0.05, **, P < 0.01, compared with the MPTP model group: #, P < 0.05, ##, P < 0.01, indicating that compound combination III has a significant therapeutic effect on the MPTP-induced zebrafish fear model.

[0046] The results of animal experiments and cell experiments show that the drug combination of the present invention has an effect on MPP +The induced damaged BV2 cells had obvious protective effects and enhanced cell viability; it had obvious therapeutic effects on the MPTP-induced zebrafish Parkinson's model and improved the motor PD symptoms of zebrafish larvae. These results all indicate that the compound combination of the present invention is expected to be used for Parkinson's disease and is expected to serve as a lead compound for the development of new anti-PD drugs.

Claims

1. A natural compound combination, characterized in that: The main ingredients include ginsenoside Rb1 and ginsenoside.

2. The method for preparing the natural compound combination according to claim 1, characterized in that: The specific steps include: Step 1: Use sterile water to ultrasonically dissolve ginsenoside Rb1, add water to prepare a G-Rb1 stock solution with a concentration of 1-200 μM, use dimethyl sulfoxide to oscillate to dissolve dianthus glycoside, and add water to prepare a dianthus glycoside stock solution with a concentration of 14.4-70 μM; Step 2: Mix the G-Rb1 stock solution and the dianthus glycoside stock solution in a volume ratio of 1:1 to obtain a natural compound combination.

3. Use of the natural compound combination according to claim 1 in the preparation of drugs for preventing and treating Parkinson's disease.

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