Application of hypericin in preparation of medicine for preventing or treating amyotrophic lateral sclerosis

Abstract

The invention belongs to the technical field of medicines, and discloses application of hypericin in preparation of a medicine for preventing or treating amyotrophic lateral sclerosis. Compared with an empty vector control cell group, the survival rates of SOD1G93A cells and TDP43M337V cells are remarkably reduced, and the survival rates of the SOD1G93A cells and the TDP43M337V cells can be remarkably improved after 1-30 [mu] M of hypericin is given for 4 hours and 24 hours. Meanwhile, after 30 [mu] M of hypericin is given for 4 h, the SOD1G93A protein level can be significantly reduced through an autophagy pathway, but the endogenous SOD1 protein level exerting the effects of oxidation resistance and the like in vivo is not affected.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of hypericin in preparing a drug for preventing or treating amyotrophic lateral sclerosis. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology that mainly affects the cerebral cortex, brainstem and spinal motor neurons. The clinical manifestations are mainly progressive skeletal muscle atrophy, weakness, fasciculations, bulbar palsy and pyramidal tract signs. The incidence of ALS in China is about 0.6 / 100,000 people, and the prevalence is about 3.1 / 100,000 people. The disease has an insidious onset, and patients often have difficulty breathing in the later stages of the disease, with an average survival of 3-5 years. Therefore, ALS is a disease with a short survival period and poor prognosis. On May 11, 2018, the "First List of Rare Diseases" was formulated, and ALS was included.

[0003] At present, the U.S. Food and Drug Administration (FDA) has approved a total of four drugs for the treatment of ALS, namely riluzole, edaravone, relyvrio and toferson. Among them, riluzole and edaravone were launched on the FDA in 1996 and 2017, respectively. Riluzole is a glutamate antagonist that can extend the survival of ALS patients by about 3 months. Edaravone is a free radical / reactive oxygen scavenger that can slightly improve patient symptoms. Relyvrio and toferson are drugs approved by the FDA for marketing in the past two years. Relyvrio works by improving the health of the endoplasmic reticulum and delaying the death of nerve cells. According to the results of its Phase II clinical trial, relyvrio can delay the survival of ALS patients by 6.5 months, while toferson is only suitable for ALS patients with SOD1 mutations, but the efficacy of both still needs to be further proven by Phase III clinical trials. Therefore, there is an urgent need for effective drugs to treat ALS.

[0004] ALS is divided into two types: familial ALS (fALS) and sporadic. Among them, fALS accounts for 5-10% of all ALS patients, and the rest are sporadic. However, studies have confirmed that gene mutations are actually related to the onset of both familial and sporadic ALS, and superoxide dismutase 1 (SOD1) is the first known mutant gene. In fALS and sporadic ALS, approximately 20% and 2% of patients have mutations related to their illness. In recent years, more studies have shown that gene mutations such as 43kDa TAR DNA-binding protein (TAR DNA-Binding Protein 43, TDP43) are also closely related to the onset of ALS.

[0005] Hypericin is an extract of Hypericum perforatum. It was first isolated from the plant of the family Garcinia in 1957. It belongs to the dianthrone class of compounds and is the most biologically active substance in Hypericum perforatum. It is a grayish yellow to brown flowing powder and has multiple functions such as antidepressant and antiviral. It is speculated that it may also play a certain preventive or therapeutic role in ALS.

[0006] Therefore, the main purpose of the present invention is to use SOD1 G93A stable transfected cells and TDP43 M337V stable transfected cells as models to study whether hypericin can improve the related pathological damage caused by SOD1 G93A or TDP43 M337V and the specific mechanism, so as to provide effective data support for the treatment of amyotrophic lateral sclerosis with hypericin. Summary of the invention:

[0007] The technical problem solved by the invention is application of hypericin in preparing medicine for preventing or treating amyotrophic lateral sclerosis.

[0008] To solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the technical solution of the present invention is to provide the use of hypericin and its pharmaceutically acceptable salts in the preparation of drugs for preventing or treating amyotrophic lateral sclerosis. As a preferred embodiment, the pharmaceutically acceptable salt includes: hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, cyclohexylammonium salt or dicyclohexylammonium salt.

[0010] The second aspect of the technical solution of the present invention is to provide a use of a pharmaceutical composition in the preparation of a drug for preventing or treating amyotrophic lateral sclerosis. The pharmaceutical composition comprises the hypericin and its pharmaceutically acceptable salts and a pharmaceutically acceptable carrier as described in the first aspect. The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes. The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nanosuspensions.

[0011] The hypericin described in the first aspect to the second aspect is extracted from plant materials. The preparation method is: ultrasonic extraction with organic solvent, adsorption of the extract with macroporous resin, separation with silica gel column chromatography, and purification with liquid chromatography to prepare hypericin.

[0012] Beneficial technical effects:

[0013] Compared with the respective empty vector control cell groups (pEGFP and pCI), the survival of SOD1 G93A cells and TDP43 M337V cells was significantly decreased, and the survival of SOD1 G93A cells and TDP43 M337V cells was significantly improved after administration of 1-30 μM hypericin. 10 and 30 μM hypericin can promote the clearance of SOD1 G93A protein in SOD1 G93A cells through the autophagy pathway, but have no effect on the endogenous SOD1 protein level in SOD1 G93A cells. Hypericin has good application prospects as a therapeutic drug for amyotrophic lateral sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 1-30μM hypericin improves the survival of SOD1 G93A stable cells and TDP43 M337V stable cells. A: MTT assay was used to detect the difference in proliferation between empty vector control pEGFP stable cells and SOD1 G93A stable cells, as well as the effect of 0.1-30μM hypericin on the survival of SOD1 G93A stable cells after 4 hours of administration of SOD1G93A stable cells. B: MTT assay was used to detect the difference in proliferation between empty vector control pEGFP stable cells and SOD1 G93A stable cells, as well as the effect of 0.1-30μM hypericin on the survival of SOD1 G93A stable cells after 24 hours of administration of SOD1 G93A stable cells. C: MTT assay was used to detect the difference in proliferation between empty vector control pCI stable cells and TDP43 M337V stable cells, and the effect of 0.1-30 μM hypericin on the survival of TDP43 M337V stable cells after 4 hours of administration of TDP43 M337V stable cells. D: MTT assay was used to detect the difference in proliferation between empty vector control pCI stable cells and TDP43 M337V stable cells, and the effect of 0.1-30 μM hypericin on the survival of TDP43 M337V stable cells after 24 hours of administration of TDP43 M337V stable cells. “#” indicates that compared with the empty vector group, #P<0.05, ##P<0.01, and “*” indicates that compared with the model group, *P<0.05, **P<0.001, ***P<0.001.

[0015] Figure 2 30μM hypericin significantly downregulated the SOD1 G93A protein level in SOD1 G93A stably transfected cells.

[0016] A: Western Blot results of the effect of 1-30μM hypericin on SOD1 G93A protein level in SOD1 G93A stably transfected cells for 4 hours, GAPDH as internal reference. B: Western Blot statistical results of SOD1G93A in SOD1 G93A stably transfected cells after 1-30μM hypericin for 4 hours. C: Western Blot results of the effect of 1-30μM hypericin on SOD1 G93A protein level in SOD1 G93A stably transfected cells for 24 hours, GAPDH as internal reference. D: Western Blot statistical results of SOD1 G93A in SOD1 G93A stably transfected cells after 1-30μM hypericin for 24 hours.

[0017] Figure 3 30μM hypericin had no effect on the endogenous SOD1 protein level in SOD1 G93A stably transfected cells.

[0018] A: Western Blot results of the effect of 3-30 μM hypericin on endogenous SOD1 protein levels in SOD1 G93A stably transfected cells for 4 hours, GAPDH was used as an internal reference. B: Western Blot statistical results of endogenous SOD1. *P<0.05. Figure 4 30μM hypericin significantly downregulated the SOD1 G93A protein level in SOD1 G93A stably transfected cells through the autophagy pathway. A: Western Blot results of the effect of 3-30μM hypericin on p62 protein level in SOD1 G93A stably transfected cells for 4 hours, GAPDH as internal reference. B: Western Blot statistical results of p62. *P<0.05. DETAILED DESCRIPTION

[0019] Example 1. Hypericin has a protective effect on SOD1 G93A and TDP43 M337V stably transfected cells

[0020] This example found that compared with the corresponding empty vector control group cells, SOD1 G93A and TDP43 M337V stably transfected cells can significantly reduce cell survival, and hypericin has a significant protective effect in SOD1 G93A and TDP43 M337V stably transfected cells after 4 hours and 24 hours, specifically, 1-30 μM hypericin has a significant improvement in the survival rate of the cells.

[0021] 1. Cells and Plasmids

[0022] Cell lines:

[0023] NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0024] The SOD1 G93A stable transfection cell line and the pEGFP empty vector control cell line were constructed by our laboratory.

[0025] The TDP43 M337V stable transfection cell line and the pCI empty vector control cell line were constructed by our laboratory.

[0026] 2. Methods

[0027] 2.1 MTT assay to determine cell viability

[0028] pEGFP empty vector control cells and SOD1 G93A stably transfected cells were cultured at 5*10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and 100 μL was added to each well of the plate and placed in an incubator for culture. After 24 hours of culture, the medium was changed and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pEGFP empty vector control cells and the SOD1 G93A model group cells; 10 μL of medium containing hypericin was added to the wells of each SOD1G93A drug-treated group cells, so that the final concentrations in the wells of each drug-treated group were 0.1, 0.3, 1, 3 and 10 μM. After incubation in the incubator for 4 hours and 24 hours, 10 μL of MTT was added to each well. After incubation in the incubator for another 4 hours, 100 μL of the triplicate solution was added to each well, dissolved at 37°C overnight, and the absorbance was measured at 570 nm.

[0029] pCI empty vector control cells and TDP43 M337V stably transfected cells were cultured at 5*10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and 100 μL per well were placed in an incubator for culture. After 24 hours of culture, the medium was changed and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pCI empty vector control cells and the TDP43 M337V model group cells; 10 μL of medium containing hypericin was added to the wells of each TDP43M337V drug-treated group cells, so that the final concentrations in the wells of each drug-treated group were 0.1, 0.3, 1, 3 and 10 μM. After incubation in the incubator for 4 hours and 24 hours, 10 μL of MTT was added to each well. After incubation in the incubator for another 4 hours, 100 μL of the triplicate solution was added to each well, dissolved at 37°C overnight, and the absorbance value was measured at 570 nm.

[0030] 2.2 Data Statistical Analysis

[0031] Data are presented as mean ± SD, and statistical analysis was performed using Student's t-test. "#" indicates that the model group cells were compared with the empty vector control group cells (pEGFP and pCI). #P<0.05, ## P<0.005, "*" indicates that the cells in each drug-treated group were compared with the cells in the model group, where *P<0.05, **P<0.01, ***P<0.001.

[0032] 3. Results

[0033] 3.1 Hypericin improves the survival of SOD1 G93A cells and TDP43 M337V cells

[0034] The results of this example show that compared with the empty vector control cell group, the survival rate of SOD1 G93A cells and TDP43 M337V cells was significantly reduced ( Figure 1 -A and Figure 1 -B), while administration of 1-30 μM hypericin significantly increased the survival rate of SOD1 G93A cells and TDP43M337V cells ( Figure 1 -C and Figure 1 -D).

[0035] Example 2. Hypericin promotes the clearance of SOD1 G93A protein in SOD1 G93A cells via the autophagy pathway

[0036] 1. Cells

[0037] Cells: SOD1 G93A stable transfection cell line and pEGFP empty vector control cells were constructed by our laboratory.

[0038] 2. Western Blot

[0039] (1) SDS-PAGE electrophoresis

[0040] Take 10% separation gel as an example, add each component according to the following formula, add the separation gel to the gap between the thick glass plate and the thin glass plate, and seal it with anhydrous ethanol. After the separation gel solidifies at room temperature for 0.5-1 hour, discard the anhydrous ethanol, and use filter paper to absorb the residual anhydrous ethanol. Add each component of 5% concentrated gel according to the formula and add it on top of the separation gel, immediately insert a clean comb, and the concentrated gel solidifies in about 15-20 minutes.

[0041]

[0042]

[0043] Place the gel in a vertical electrophoresis tank and add an appropriate amount of electrophoresis buffer. After pulling out the comb, start electrophoresis at a constant voltage of 80V according to the order of sample loading and protein markers. When bromophenol blue enters the separation gel, increase the voltage to 120V and stop electrophoresis when bromophenol blue approaches the bottom.

[0044] (2) Transfer

[0045] Prepare a 0.22μm PVDF membrane of the same size as the gel block, immerse it in anhydrous methanol to activate it, soak the filter paper and sponge pad in transfer buffer, and place the gel and membrane in the order of black transfer clamp-sponge pad-3 filter papers-gel-membrane-3 filter papers-sponge pad-white transfer clamp. Expel bubbles, clamp and place in the transfer tank, and transfer at a constant current of 200mA for 1.5 hours.

[0046] (3) Antigen-antibody reaction

[0047] After the transfer, the PVDF membrane was blocked with TBST containing 5% skimmed milk powder and incubated on a shaker at room temperature for 2 hours. The PVDF membrane was cut according to the molecular weight of the target protein and placed in an incubation box. After pouring the corresponding antibody, it was shaken at 4°C overnight. On the next day, the primary antibody was aspirated and the membrane was washed with TBST at room temperature for 10 minutes, for a total of 3 times; horseradish peroxidase-labeled secondary antibody was added, incubated on a shaker at room temperature for hours, the secondary antibody was aspirated and the membrane was washed with TBST at room temperature for 10 minutes, for a total of 3 times.

[0048] 2.5 Data Statistical Analysis

[0049] The data were expressed as mean ± SD, and the Student's t-test was used for statistical analysis. "*" indicates that each drug-treated cell group was compared with the SOD1 G93A model cell group, where *P<0.05.

[0050] 3. Results

[0051] 3.1 Hypericin promotes the clearance of SOD1 G93A protein in SOD1 G93A cells

[0052] Administration of 10 and 30 μM hypericin significantly reduced SOD1 G93A protein levels after 4 and 24 hours ( Figure 2 -A, Figure 2 -B, Figure 2 -C, Figure 2 -D).

[0053] 3.2 Hypericin has no effect on endogenous SOD1 protein levels in SOD1 G93A cells

[0054] Administration of 1-30 μM hypericin had no effect on endogenous SOD1 protein levels 4 hours later ( Figure 3 -A, Figure 3 -B), indicating that hypericin has no effect on the level of endogenous SOD1 protein that plays an antioxidant role in the body.

[0055] 3.2 Hypericin promotes the clearance of SOD1 G93A protein through the autophagy pathway

[0056] 30 μM hypericin significantly reduced p62 protein content 4 hours after administration ( Figure 4 -A, Figure 4 -B), indicating that hypericin promoted the clearance of SOD1 G93A protein in SOD1 G93A cells through the autophagy pathway.

[0057] In summary, hypericin can promote the clearance of SOD1 G93A protein and maintain the level of endogenous SOD1 protein through the autophagy pathway, which is the basis for the protective effect of hypericin.

[0058] Example 3. Extraction and preparation process of hypericin from Hypericum perforatum

[0059] 1. Plant Materials

[0060] The dried aerial parts of Hypericum perforatum.

[0061] 2. Extraction and preparation process

[0062] (1) After the dried aerial part of Hypericum perforatum was crushed, anhydrous ethanol was added in a solid-liquid ratio of 1:4 and soaked for 36 h;

[0063] (2) ultrasonic extraction was performed at room temperature for 3 times, each time for 0.5 h, and the solvent was recovered under reduced pressure to obtain the ethanol extract of Hypericum perforatum;

[0064] (3) adding an appropriate amount of water to disperse the ethanol extract of Hypericum perforatum, using a macroporous resin for adsorption, and eluting with 0%, 50%, 70%, and 100% ethanol in a gradient manner; combining the 70% and 100% ethanol elution fractions and evaporating under reduced pressure to dryness to obtain a fraction containing hypericin;

[0065] (4) separating by C18 column chromatography, gradient elution, combining the fractions containing hypericin, and recovering the solvent under reduced pressure to obtain a crude product of hypericin;

[0066] (5) The crude hypericin is then purified by preparative high performance liquid chromatography to obtain hypericin.

[0067] 3. Results

[0068] The hypericin extraction and preparation process disclosed in this embodiment has a simple implementation process and a high extraction rate, and can prepare hypericin simply and efficiently.

[0069] The above embodiments are only for illustrating the technical features of the present invention, and their purpose is to enable people familiar with this field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of hypericin represented by formula (I) and its pharmaceutically acceptable salt in the preparation of a drug for preventing or treating amyotrophic lateral sclerosis, 2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from organic acid salts or inorganic acid salts, including hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate or methanesulfonate.

3. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating amyotrophic lateral sclerosis, characterized in that: The pharmaceutical composition comprises the hypericin and a pharmaceutically acceptable salt thereof as claimed in claim 1 and a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

5. The use according to claim 4, characterized in that The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral solutions, suspensions, sustained-release preparations, subcutaneous implants or nanosuspensions.

6. The use according to claim 1, characterized in that The hypericin is extracted and prepared from plant materials.

7. The use according to claim 6, characterized in that The preparation method of hypericin comprises the following steps: taking Hypericum perforatum as plant material, extracting by ultrasonic method using organic solvent, adsorbing the extract by macroporous resin, separating by C18 column chromatography, and purifying by liquid chromatography to prepare hypericin.

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