Application of ginsenoside Rg2 in the preparation of drugs for the treatment of multiple sclerosis

Abstract

This invention relates to the field of biomedical technology and provides the application of ginsenoside Rg2 in the preparation of drugs for treating multiple sclerosis (MS). Ginsenoside Rg2 exerts its therapeutic effect on MS through the following mechanisms: reducing the expression levels of pro-inflammatory factors, promoting regulatory T cell differentiation, and inhibiting the infiltration of immune cells into the central nervous system; inhibiting NLRP3 signaling pathway activation and reducing neuronal pyroptosis; preventing the immune system from destroying myelin and reducing the ratio of demyelinated area to white matter area; upregulating oligodendrocyte transcription factor expression, promoting oligodendrocyte precursor cell proliferation and differentiation, and promoting myelin regeneration; and inhibiting microglia activation. This invention solves the problems of limited efficacy, significant side effects, only delaying progression, and limited administration methods of existing drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of ginsenoside Rg2 in preparation of a multiple sclerosis treatment drug. BACKGROUND

[0002] Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), and its pathogenesis is closely related to autoimmune abnormalities, nerve inflammation damage and myelin sheath regeneration disorders. The characteristic is that the immune system abnormally attacks the nerve myelin sheath (demyelination), leading to nerve signal transmission disorders, and further causing various neurological dysfunctions. Its main pathological feature is to induce inflammatory reactions and promote microglial cell activation and T cell infiltration. The main clinical manifestations are limb weakness, sensory abnormalities, cognitive impairment and motor function decline, which seriously affect the quality of life of patients. At present, the incidence of MS is increasing year by year, and it has become one of the main nervous system diseases causing disability in young adults.

[0003] Existing studies have shown that the pathological core of MS is that: (1) immune cells (such as T cells, B cells and macrophages) are abnormally activated and infiltrate the CNS, release pro-inflammatory factors (such as TNF-α, IL-17 and IFN-γ) to trigger inflammatory cascade reactions; (2) oligodendrocyte damage leads to demyelination and nerve signal transmission obstruction; (3) irreversible damage to nerve axons, which further leads to permanent neurological dysfunction. Therefore, an ideal MS treatment drug should have multiple effects of anti-inflammatory regulation, nerve protection and promotion of myelin sheath regeneration.

[0004] Ginsenoside Rg2 is a tetracyclic triterpenoid saponin (chemical formula: C 42 H 72 O 13 ) extracted from ginseng. Existing studies have only reported that it has anti-apoptotic effect, improves cognitive dysfunction (animal model) and anti-inflammatory effect. And it is safe for human body without toxicity, and it has great potential to be developed as a clinical drug.

[0005] Current MS clinical treatment drugs are mainly divided into disease modifying treatment drugs (DMTs), symptomatic treatment drugs and immunosuppressants, but they have the following key defects:

[0006] Single efficacy: existing drugs (such as fingolimod) mainly focus on immunosuppression, and cannot effectively solve the problem of nerve degeneration in the middle and late stages of the disease, and cannot promote the repair of damaged myelin sheath. About 30% of patients have no response to existing DMTs, and long-term use can easily lead to decreased efficacy.

[0007] Significant side effects and poor safety: Immunosuppressants (such as cyclophosphamide, methotrexate) can easily cause systemic immunosuppression, leading to infection, liver and kidney function damage, and blood system abnormalities, and some DMTs (such as natalizumab) can induce fatal complications such as progressive multifocal leukoencephalopathy (PML).

[0008] Inability to achieve nerve repair: Existing drugs do not address the core pathological link of MS - nerve axon protection and myelin regeneration, and can only slow down disease progression, but cannot reverse the already formed nerve function defects.

[0009] Limited administration methods: Most drugs require subcutaneous injection or intravenous infusion, with poor patient compliance; oral drugs (such as fingolimod) have risks such as heart rate slowing and lung infection, and the applicable population is narrow.

[0010] In addition, there are few studies on the application of natural compounds in MS treatment, and existing natural drugs (such as curcumin, resveratrol) have problems such as low bioavailability and unclear mechanism of action, making it difficult to be converted into a clinical treatment method. SUMMARY

[0011] The purpose of the present application is to provide the use of ginsenoside Rg2 in the preparation of a multiple sclerosis treatment drug, aiming to solve the problems raised in the background art.

[0012] To solve the above problems, the present application provides the use of ginsenoside Rg2 in the preparation of a multiple sclerosis treatment or prevention drug.

[0013] Another purpose of the present application is to provide the use of ginsenoside Rg2 in the preparation of a drug for inhibiting inflammation in the central nervous system, wherein the ginsenoside Rg2 is used to reduce the expression level of pro-inflammatory factors, promote the differentiation of regulatory T cells, and inhibit the infiltration of immune cells into the central nervous system.

[0014] Another purpose of the present application is to provide the use of ginsenoside Rg2 in the preparation of a neuroprotective drug, wherein the ginsenoside Rg2 is used to inhibit the activation of the NLRP3 signaling pathway and reduce pyroptosis of nerve cells.

[0015] Another purpose of the present application is to provide the use of ginsenoside Rg2 in the preparation of a drug for promoting myelin regeneration.

[0016] Further, the ginsenoside Rg2 is used to prevent the destruction of the myelin sheath by the immune system and reduce the ratio of the area of myelin loss to the area of white matter.

[0017] Further, the ginsenoside Rg2 is used to up-regulate the expression of oligodendrocyte transcription factors, promote the proliferation and differentiation of oligodendrocyte precursor cells, and promote myelin regeneration.

[0018] Another object of the present application is to provide an application of ginsenoside Rg2 in preparing a medicine for inhibiting microglial cell activation.

[0019] Another object of the present application is to provide a medicine for treating or preventing multiple sclerosis, comprising a pharmaceutically acceptable carrier and ginsenoside Rg2.

[0020] Further, the concentration of the ginsenoside Rg2 is 20-80 μmol / L.

[0021] Further, the extraction method of the ginsenoside Rg2 comprises the following steps:

[0022] One or more of the roots, stems and leaves of Panax genus plants of Araliaceae are crushed, and 70%-80% ethanol aqueous solution is used for 80℃ reflux extraction to obtain an extraction liquid;

[0023] The above extraction liquid is reduced pressure concentrated to be alcohol-free, and then loaded onto a D101 macroporous resin column, and sequentially eluted with 30%, 50%, and 70% ethanol aqueous solution by volume, and the 70% ethanol aqueous solution elution component is collected to obtain an elution liquid;

[0024] The above elution liquid is purified by high performance liquid chromatography, the chromatographic column is a C18 column, the mobile phase is acetonitrile-water=25:75, the flow rate is 1.0 mL / min, the detection wavelength is 203 nm, the chromatographic peak with a retention time of 12.5-13.5 min is collected, and then freeze-dried to obtain ginsenoside Rg2.

[0025] The present application aims at the technical bottleneck and clinical needs in the current multiple sclerosis (MS) treatment field, solves the problems of limited efficacy, significant side effects, only delaying progression, and limited administration mode of the existing medicines through the novel application of natural compound ginsenoside Rg2. The ginsenoside Rg2 mainly plays the MS treatment role through the following mechanisms:

[0026] 1. Improving mouse behavior: reducing mouse clinical symptoms, significantly reducing symptoms, and basically eliminating the symptoms of bilateral hind limb paralysis accompanied by forelimb paralysis.

[0027] 2. Improving motor function: the mice treated with ginsenoside Rg2 stay on the rod for a longer time in the rotarod test, showing better motor ability.

[0028] 3. Anti-inflammatory regulation: reducing the expression level of pro-inflammatory factors (IL-1β, IL-18), promoting the differentiation of regulatory T cells (Treg), and inhibiting the infiltration of immune cells into the CNS.

[0029] 4. Neuroprotection: inhibiting the activation of the NLRP3 signaling pathway and reducing pyroptosis of nerve cells.

[0030] 5. Reducing spinal cord white matter demyelination: Ginsenoside Rg2 prevents the immune system from destroying myelin, reducing the ratio of demyelination area to white matter area.

[0031] 6. Promoting myelin regeneration: Myelin regeneration refers to the process of new oligodendrocytes wrapping around exposed axons to form new myelin, partially or completely restoring nerve signal transmission speed and protecting axons in multiple sclerosis (MS). Ginsenoside Rg2 can up-regulate oligodendrocyte transcription factor (Olig2, SOX10) expression, promote oligodendrocyte precursor cell (OPC) proliferation and differentiation, accelerate myelin regeneration, and repair demyelination lesions.

[0032] 7. Inhibiting microglial overactivation: In MS lesions, abnormal activation of microglia releases a large amount of inflammatory mediators, and ginsenoside Rg2 can reduce the number of activated microglia. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure for the effect of ginsenoside Rg2 on the clinical score of EAE mice.

[0034] Figure 2 Figure for the effect of ginsenoside Rg2 on the motor ability of EAE mice.

[0035] Figure 3 Figure for the effect of ginsenoside Rg2 on the content of inflammatory factor IL-1β in the spinal cord of EAE mice.

[0036] Figure 4 Figure for the effect of ginsenoside Rg2 on the activation of microglia in the spinal cord tissue of EAE mice.

[0037] Figure 5 Figure for the effect of ginsenoside Rg2 on the white matter demyelination in the spinal cord of EAE mice.

[0038] Figure 6 Figure for the effect of ginsenoside Rg2 on the expression of oligodendrocyte marker O4+.

[0039] Figure 7 Figure for the effect of ginsenoside Rg2 on the expression of myelin protein MBP. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0041] In one embodiment of the present application, it is first discovered that the natural compound Ginsenoside Rg2 has a brand-new use in the treatment of multiple sclerosis; wherein the chemical name of Ginsenoside Rg2 is 6-O-[alpha-L-rhamnopyranosyl-(1-2)-beta-D-glucopyranosyl]-dammar-24-ene-3beta, 6alpha, 12beta, 20S-tetrol, molecular formula C 42 H 72 O 13 Ginsenoside Rg2 is derived from the roots, stems or leaves of Panax ginseng C.A. Mey. or Panax quinquefolius L. and can be prepared by natural extraction or semi-synthetic method.

[0042] In one preferred embodiment of the present application, an extraction method of Ginsenoside Rg2 is also provided, which specifically comprises the following steps:

[0043] S1, crushing the roots, stems or leaves of Panax ginseng C.A. Mey. or Panax quinquefolius L., and extracting the crushed roots, stems or leaves with 70%-80% ethanol aqueous solution at 80°C for 3 times, each time for 2 hours, and then combining the extraction solutions;

[0044] S2, concentrating the above extraction solution to alcohol-free under reduced pressure, then loading the concentrated solution onto a D101 macroporous resin column, and eluting the column with 30%, 50% and 70% ethanol aqueous solution in sequence, collecting the elution fraction of 70% ethanol aqueous solution to obtain an elution solution;

[0045] S3, purifying the above elution solution by high performance liquid chromatography (HPLC) (chromatographic column: C18 column, mobile phase: acetonitrile-water=25:75, flow rate: 1.0 mL / min, detection wavelength: 203 nm), collecting the chromatographic peak with a retention time of 12.5-13.5 min, and then freeze-drying to obtain the monomer of Ginsenoside Rg2 with a purity of ≥98%.

[0046] In the embodiments of the present application, the ginsenoside Rg2 is first disclosed for treating multiple sclerosis, which is a novel medical application of the compound. The ginsenoside Rg2 can be used for preventing, treating and / or relieving multiple sclerosis and symptoms thereof, and fills the blank of natural compounds in the field of multiple sclerosis treatment. The ginsenoside Rg2 can achieve anti-inflammation, neuroprotection and myelin regeneration at the same time, solve the limitation of single target in the prior art, and has a significant effect: in the MS animal model (EAE), the ginsenoside Rg2 can not only significantly delay the onset and reduce the clinical score, but also prove its strong repair ability of reducing demyelination and promoting myelin regeneration in pathology, and the effect is equivalent to that of some positive control drugs (such as fingolimod), and has a unique advantage in promoting repair. The ginsenoside Rg2 has high safety: as a natural product, its safety is better than that of many synthetic immunosuppressants, and has less potential side effects. The embodiments of the present application provide a new potential treatment option for MS patients who are not responsive to existing immunotherapy or enter the progressive stage, especially for the unmet clinical need of nerve repair, and also provide a new lead compound and research idea for the development of natural product-based drugs for treating neurological and immunological diseases.

[0047] Example 1, Experimental Autoimmune Encephalomyelitis (EAE) Model and Ginsenoside Rg2 Treatment: 6-8-week-old female C57BL / 6 mice were used, and were randomly divided into: (1) model control group (Vehicle, normal saline), i.e. the Vehicle group in the accompanying drawings; (2) positive drug control group (fingolimod FTY720, 1 mg / kg / day), i.e. the FTY720 group in the accompanying drawings; (3) ginsenoside Rg2 treatment group (G-Rg2, 30 mg / kg / day), i.e. the G-Rg2 group in the accompanying drawings; 10 mice in each group. The mouse experimental autoimmune encephalomyelitis (EAE) model was established to simulate the signs of clinical MS, and the specific method was as follows: 200 μg myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) was used for immunization in Freund's adjuvant (CFA), which contained 4 mg / mL Mycobacterium tuberculosis and was subcutaneously injected. In addition, 200 ng pertussis toxin was injected intraperitoneally (ip) on the day of immunization and 48 hours after immunization.

[0048] From the 3rd day after immunization, the model control group of mice was orally administered with normal saline, the positive drug control group of mice was orally administered with fingolimod (at a dose of 1 mg / kg), and the ginsenoside Rg2 treatment group of mice was orally administered with ginsenoside Rg2 (at a dose of 30 mg / kg), for 27 days of continuous treatment. From the day of immunization, the clinical scores of the mice in each group were observed and recorded every day. The clinical scores were evaluated according to the internationally accepted 5-point scoring system: 0 points for no clinical symptoms; 1 point for tail tension loss and mild gait clumsiness; 2 points for one side of the hind limb weakness, which could be restored after passive turning over; 3 points for both hind limbs paralysis, which could be moved after stimulation; 4 points for both hind limbs paralysis accompanied by forelimb paralysis; and 5 points for moribund state or death.

[0049] On the 30th day after immunization, the mice were subjected to the rotarod test. Specifically, the mice were placed on an accelerating rotator, and the rotating rod was gradually accelerated from 5 rpm to 50 rpm within 5 minutes. The time of the mouse falling was recorded as the latency period. Each mouse was tested three times, and the average latency period was analyzed.

[0050] According to the above experimental method, the therapeutic effect of ginsenoside Rg2 on EAE mice was detected, and the detection indexes were clinical score and motor function. The detection results are shown in Figure 1 and Figure 2 .

[0051] From Figure 1 and Figure 2 , it can be seen that, compared with the model control group, the onset time of the ginsenoside Rg2 treatment group of mice was significantly delayed, and the highest clinical score was significantly reduced. The highest clinical score of the model control group was 3.8±0.5, and the highest score of the ginsenoside Rg2 treatment group decreased to 1.5±0.3 (P<0.01 vs. model control group). During the entire experimental period, the average clinical score was comparable to that of the positive drug control group, and was extremely significantly lower than that of the model control group. In the rotarod test, the rotarod fall latency of the model control group was 227.9±14.32 (s), and the rotarod fall latency of the ginsenoside Rg2 treatment group was 283.8±7.09 (s), which was comparable to that of the positive drug control group, and was extremely significantly lower than that of the model control group (P<0.01 vs. model control group).

[0052] Example 2, Inflammatory regulation effect of ginsenoside Rg2 on EAE mice: After the above rotarod test, the mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital solution. After anesthesia, the mice were perfused with normal saline, and the spinal cord was quickly dissected on ice after the liver turned white. The spinal cord tissue was homogenized and used for Elisa (inflammatory factor level) detection; the spinal cord protein was extracted and frozen at -80℃ for Western blot (protein expression level) detection.

[0053] Elisa (inflammatory factor level) detection is specifically using a commercial kit to detect the content of a certain substance in the body according to the instructions.

[0054] According to the above experimental method, the inflammatory regulation effect of ginsenoside Rg2 on EAE mice was detected, and the detection indexes were the content of inflammatory factor IL-1β in the spinal cord tissue of mice and the activation of microglia cells.

[0055] The detection results are shown in Figure 3 and Figure 4 The IL-1β content of the model control group mice was 8.1±0.44 (pg / mL pro), and the IL-1β content of the ginsenoside Rg2 treatment group mice was 5.6±0.2 (pg / mL pro) (P<0.01 vs model control group). During the entire experimental period, the IL-1β content was comparable to that of the positive drug control group, and was extremely significantly lower than that of the model control group. The number of activated microglia cells in the model control group was 29.7±2.2, and the number of activated microglia cells in the ginsenoside Rg2 treatment group was 12±1.1. During the entire experimental period, the number of activated cells was comparable to that of the positive drug control group, and was extremely significantly lower than that of the model control group (P<0.01 vs model control group).

[0056] According to the above experimental method, the reduction of spinal cord white matter demyelination by ginsenoside Rg2 was detected, and the detection index was myelin staining.

[0057] The detection results are shown in Figure 5 The model control group showed extensive demyelination in the spinal cord white matter, and the ratio of demyelination area to white matter area was 23.45±3.32, while the ginsenoside Rg2 group was 14.22±3.11, and the demyelination area was significantly reduced (P<0.01), and the effect was comparable to that of the positive drug control group.

[0058] Example 3, in vitro cell experiment to verify the effect of ginsenoside Rg2 on promoting myelin regeneration: OPC culture, OPCs in the cerebral cortex of newborn mice were isolated and cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin mixture, and the cells were maintained at 37°C, 5% CO2 relative humidity 98%. The OPCs were divided into model control group (no drug), ginsenoside Rg2 group (20 μM, 40 μM, 80 μM).

[0059] Each group was detected by immunofluorescence, and the cells were pretreated with ginsenoside Rg2 (20 μM, 40 μM, 80 μM) for 7 days, and the drug medium of the corresponding concentration was replaced every 2 days to ensure the stability of the drug concentration. After 7 days of culture, the culture medium was discarded, and the cells were gently washed with pre-cooled PBS buffer for 3 times (5 minutes each time); 1 mL of 4% paraformaldehyde was added to each well for fixation, and 1 mL of 5% BSA blocking solution was added to each well for blocking at 37°C for 1 hour, then the primary antibody (O4+ antibody and MBP antibody) was added, and incubated at 4°C overnight. The next day, after incubation with the secondary antibody, the cells were observed under a fluorescence microscope, and the statistical analysis was performed using ImageJ software.

[0060] Detection index: The expression of oligodendrocyte marker O4+ and myelin protein MBP was detected.

[0061] The expression detection results of oligodendrocyte marker O4+ are shown in Figure 6 The proportion of O4+ cells in the 40 μM and 80 μM ginsenoside Rg2 groups was 65.3±5.2, which was significantly higher than that in the model control group (32.5±4.8, P<0.01). Figure 7 The expression detection results of myelin protein MBP are shown in

[0062] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of ginsenoside Rg2 in the preparation of a drug for treating or preventing multiple sclerosis.

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