Application of hyperforin 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 hyperforin in preparation of a medicine for preventing or treating amyotrophic lateral sclerosis. The hyperforin can improve the survival rate of stable transfection cell lines of amyotrophic lateral sclerosis related cell models (SOD1G93A / TDP43M337V), and the hyperforin is prompted to play a protective role in the two cell models. After the hyperforin is given, the expression levels of HO-1 and Drp1 proteins in SOD1G93A cells are increased, which indicates that the hyperforin has certain antioxidant activity; and mitochondrial division in cells can be inhibited, so that cell apoptosis is inhibited and retarded, and a protection effect is achieved. Hypericin can help to improve the SOD1G93A cell morphology and promote synaptic generation, and it is prompted that hypericin has a positive effect on maintaining normal functions of cells. In conclusion, the hyperforin plays a protective role in an SOD1G93A cell model and a TDP43M337V cell model, and the mechanism of the hyperforin is possibly related to the antioxidant activity, the mitochondrial function improvement and the like of the hyperforin.

Description

Technical Field

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

[0002] Amyotrophic lateral sclerosis is a progressive degenerative disease of unknown etiology that selectively invades the anterior horn cells of the spinal cord, the motor nuclei of the brainstem, and the pyramidal tract. The onset of amyotrophic lateral sclerosis is insidious, and the clinical manifestations are characterized by combined damage to upper and lower motor neurons. Patients experience progressive muscle weakness, muscle atrophy, fasciculations, bulbar palsy, and pyramidal tract signs. Usually, death occurs 3 to 5 years after onset due to respiratory muscle paralysis and respiratory failure. According to different clinical manifestations, ALS is divided into classic ALS type, flail arm syndrome, flail leg syndrome, and ALS with frontotemporal dementia. Currently, the FDA has approved the marketing of only four drugs for the treatment of ALS: riluzole, edaravone, sodium phenylbutyrate / taurine diol, and Tofersen. Riluzole is beneficial for antagonizing glutamate excitotoxicity, edaravone is a free radical scavenger that can exert antioxidant effects, sodium phenylbutyrate / taurine diol can improve the state of the endoplasmic reticulum and mitochondria, and the antisense oligonucleotide drug Tofersen can target the removal of toxic proteins, but the four drugs have limited effects on prolonging patient survival. In addition, drugs including masitinib, ibudilast, and ION363 that improve neuroinflammation are currently in Phase III clinical development. The current status of ALS drug development is characterized by a high failure rate in research and development and an overall lack of effective therapeutic drugs. Therefore, ALS, as a disease with a high mortality rate, urgently needs more clear mechanism research and effective treatments to delay or stop the progression of the disease. Among ALS patients, 10% have a family history of familial ALS, and the remaining approximately 90% of patients have sporadic ALS. In familial ALS patients, hexanucleotide (GGGGCC) repeat expansion of C9orf72 gene accounts for about 30%, superoxide dismutase 1 (Cu / Zn superoxide dismutase 1, SOD1) gene mutation accounts for about 20%, TARDBP gene mutation encoding TAR DNA-Binding Protein 43 (TDP-43) and FUS gene mutation account for about 4% respectively. The above four genes are the most common and most studied related genes in ALS. In European and American populations, C9orf72 has the highest frequency, and SOD1 has a higher frequency in Asian and African populations. The above gene mutations have also been found in sporadic ALS, but the cause of most of them is still unknown. At present, the pathogenesis of ALS is still unclear. The mainstream theory believes that the combined effects of gene mutations, toxic protein aggregation, oxidative stress, mitochondrial dysfunction, glutamate excitotoxicity, neuroinflammation, axonal transport disorders and other mechanisms cause neuronal loss, thereby leading to ALS-related pathological changes.

[0003] Superoxide dismutase 1 (Cu / Zn superoxide dismutase 1, SOD1) is an important antioxidant enzyme in cells. It is mainly located in the cytoplasm and the inner membrane space of mitochondria. It can control the level of oxidative substances and mainly catalyze the conversion of superoxide anions into hydrogen peroxide and oxygen molecules. Human SOD1 exists in the form of homodimers. Each monomer can bind a copper ion and a zinc ion, and the dimers are connected by disulfide bonds. SOD1 mutation is the earliest ALS-related gene discovered and is also one of the most studied ALS-related genes in the field of ALS. At present, more than 180 gene mutations have been found in the SOD1 gene of ALS patients. Common mutations include D90A, A4V and G93A. SOD1 mutations cause changes in its protein conformation and form toxic aggregates, causing cellular oxidative stress damage. The accumulation of toxic aggregates in mitochondria will interfere with many biological processes in cells, including free radical scavenging, protein quality control, mitochondrial function, protein degradation and axonal transport.

[0004] TDP-43 can bind to DNA and RNA. Under normal circumstances, it is mainly located in the cell nucleus and plays an important role in the processing, stability, transport of mRNA and the biosynthesis of miRNA. The TDP-43 protein is highly conserved, and its structure includes an N-terminal nuclear localization signal (NLS) and a nuclear export signal (NES), two RNA recognition motifs (RRM), and a C-terminal glycine-rich region. TARDPB gene mutations account for approximately 0.05 of fALS and 0.01 of sALS. Common missense mutations include A382T, M337V, A315T, Q331K, D169G, G294A, G294V and Q343R. TARDPB mutations increase the aggregation tendency and cytoplasmic mislocalization of TDP-43 protein, cause changes in the stability of TDP-43 protein, and affect the interaction of TDP-43 protein with other proteins.

[0005] Hyperforin is a major component of Hypericum perforatum L. extract and is a polycyclic polyisopentenyl-substituted acylphloroglucinol (PPAPs) compound. Hyperforin was first discovered to have antidepressant pharmacological activities, mainly including neurotransmitter reuptake inhibition, the ability to increase intracellular sodium and calcium levels, typical transient receptor potential activation, N-methyl-D-aspartate receptor antagonism, antioxidant and anti-inflammatory neurobiological activities. In addition, hyperforin has been shown to have cognitive enhancement and memory-promoting properties, and has been shown to have neuroprotective effects on Alzheimer's disease neuropathology, including the ability to decompose amyloid aggregates in vitro, reduce the activation of astrocytes and microglia, and improve spatial memory in vivo. Hyperforin has also been reported to have antioxidant and antibacterial activities.

[0006] The main purpose of the present invention is to use two common cell models of amyotrophic lateral sclerosis (SOD1 G93A / TDP43M337V stable transfected cells) to study whether hyperforin can improve amyotrophic lateral sclerosis-related pathological damage and the specific mechanism, so as to provide effective drug targets and treatment strategies for the treatment intervention of amyotrophic lateral sclerosis. Summary of the invention:

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

[0008] In order to solve the technical problem of the present invention, the present invention provides the following technical solutions:

[0009] The invention uses an in vitro stably transfected SOD1 G93A or TDP43 M337V NSC-34 cell injury model to evaluate whether hyperforin can be used to prevent or treat amyotrophic lateral sclerosis. The invention also provides a method for extracting and preparing hyperforin.

[0010] The first aspect of the technical solution of the present invention is to provide the use of hyperforin and its pharmaceutically acceptable salts in the preparation of drugs for preventing or treating amyotrophic lateral sclerosis. Among them, the structural formula of hyperforin is

[0011]

[0012] As a preferred embodiment, the pharmaceutically acceptable salt includes: hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, cyclohexylammonium salt or dicyclohexylammonium salt.

[0013] 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 hyperforin 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.

[0014] The hyperforin 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, silica gel column chromatography separation, and liquid chromatography purification to prepare hyperforin.

[0015] Beneficial technical effects:

[0016] The present invention provides the application of hyperforin as a new treatment strategy for amyotrophic lateral sclerosis. Hyperforin plays a protective role in the cell model related to amyotrophic lateral sclerosis, which is specifically manifested in that hyperforin can increase the survival rate of SOD1G93A cells and TDP43 M337V cells, increase the expression levels of heme oxygenase-1 (HO-1) and mitochondrial dynamin-related protein 1 (Drp1) in SOD1 G93A cells, improve the morphology of SOD1 G93A cells, and promote synapse formation. As a potential drug for the treatment of amyotrophic lateral sclerosis, hyperforin has application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Hyperforin can improve the survival of SOD1 G93A stably transfected cells. A: Effects of different doses of hyperforin on the survival rate of SOD1 G93A cells after 4 hours of treatment. B: Effects of different doses of hyperforin on the survival rate of SOD1 G93A cells after 8 hours of treatment. C: Effects of different doses of hyperforin on the survival rate of SOD1 G93A cells after 24 hours of treatment.

[0018] Figure 2 Hyperforin can improve the survival of TDP43 M337V stably transfected cells. A: Effects of different doses of hyperforin on TDP43 M337V cells for 4 hours on cell survival. B: Effects of different doses of hyperforin on TDP43 M337V cells for 8 hours on cell survival. C: Effects of different doses of hyperforin on TDP43 M337V cells for 24 hours on cell survival.

[0019] Figure 3 Hyperforin increases the expression levels of HO-1 and Drp1 proteins in SOD1 G93A cells.

[0020] Figure 4 Hyperforin improves the morphology of SOD1 G93A cells. DETAILED DESCRIPTION

[0021] Example 1. Hyperforin can improve the survival of SOD1 G93A stably transfected cells

[0022] 1. Cells

[0023] Cells: The SOD1 G93A stably transfected NSC-34 cell line and the control pEGFP empty vector cell line were constructed by our laboratory. The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0024] 2. Methods

[0025] 2.1 MTT assay for cell viability

[0026] SOD1 G93A cells and pEGFP control group cells were cultured at 5*10 4 The cells were seeded at a density of 100 μL / mL in a 96-well plate and cultured for 24 h. Different concentrations of hyperforin were added and incubated for 4 h, 8 h, and 24 h. Then 10 μL of MTT solution (5 mg / mL) was added to the cultured cells in each well. After incubation for 4 h, 100 μL of the triplicate solution was added to each well, dissolved at 37 °C overnight, and the absorbance was measured at 570 nm.

[0027] 2.2 Data Statistical Analysis

[0028] Data are presented as mean ± SD, and statistical analysis was performed using Student's t-test. "#" indicates #P<0.05, ##P<0.01 compared with the pEGFP control group, and "*" indicates *P<0.05, **P<0.01, ***P<0.001 compared with the SOD1 G93A model group.

[0029] 3. Results

[0030] 3.1 Hyperforin can improve the survival of SOD1 G93A stably transfected cells

[0031] The results of this example show that compared with the pEGFP control group, the cell survival rate of the SOD1 G93A model group was significantly reduced. Compared with the SOD1 G93A model group, the cell survival rate was significantly increased after administration of hyperforin 10, 30 μM (incubation for 4 h), 3, 10, 30 μM (incubation for 8 h), and 30 μM (incubation for 24 h). Figure 1 ).

[0032] In summary, the results of this example show that hyperforin can increase the survival rate of SOD1G93A cells after being administered at appropriate concentrations and for a certain period of time, indicating that hyperforin can improve cell survival and has a protective effect in the SOD1 G93A cell model.

[0033] Example 2. Hyperforin can improve the survival of TDP43 M337V stably transfected cells

[0034] 1. Cells

[0035] Cells: The TDP43 M337V stably transfected NSC-34 cell line and the control pCI hygro empty vector cell line were constructed by our laboratory. The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0036] 2. Methods

[0037] 2.1 MTT assay

[0038] TDP43 M337V cells and pCI control group cells were cultured at 5*10 4 The cells were seeded at a density of 100 μL / mL in a 96-well plate and cultured for 24 h. Different concentrations of hyperforin were added and incubated for 4 h, 8 h, and 24 h. Then 10 μL of MTT solution (5 mg / mL) was added to the cultured cells in each well. After incubation for 4 h, 100 μL of the triplicate solution was added to each well, dissolved at 37 °C overnight, and the absorbance was measured at 570 nm.

[0039] 2.2 Data Statistical Analysis

[0040] Data were expressed as mean ± SD, and Student's t-test was used for statistical analysis. "#" indicates that compared with the pCI control group, ##P<0.01, and "*" indicates that compared with the TDP43 M337V model group, *P<0.05, **P<0.01, ***P<0.001.

[0041] 3. Results

[0042] 3.1 Hyperforin can improve the survival of TDP43 M337V stably transfected cells

[0043] The results of this example show that compared with the pCI control group, the cell survival rate of the TDP43 M337V model group was significantly reduced. Compared with the TDP43 M337V model group, the cell survival rate was significantly increased after administration of hyperforin at 3, 10, 30 μM (incubation for 4, 8 h), 1, 3, 10, 30 μM (incubation for 24 h). Figure 2 ).

[0044] In summary, the results of this example show that hyperforin can increase the survival rate of TDP43M337V cells after being administered at appropriate concentrations and for a period of time, indicating that hyperforin can improve cell survival and has a protective effect in the TDP43 M337V cell model.

[0045] Example 3 Hyperforin has an increasing effect on the expression level of HO-1 protein

[0046] In this example, Western Blot was used to detect the effect of hyperforin at different doses for 24 h on the expression level of HO-1 protein in SOD1G93A stably transfected cells.

[0047] 1. Cells

[0048] Cells: NSC-34 cell line stably transfected with SOD1 G93A and control empty vector cell line were the same as those in Example 1.

[0049] 2. Methods

[0050] 2.1 Cell protein extraction and sample preparation

[0051] Aspirate the supernatant in the cell culture dish, wash the cells twice with pre-cooled PBS, take a 6-well plate as an example, add 150 μL RIPA lysis buffer (supplemented with protease inhibitors) to each well, lyse on ice for 1 minute, collect the lysate into a 1.5 mL EP tube, and place it in an ice water bath for sonication (6% power, sonication for 3 seconds, pause for 3 seconds, cycle 3 times). Centrifuge at 4°C, 13000rpm for 10 minutes, collect the supernatant, and quantify the protein. Protein quantification is mainly based on Pierce TM The BCA protein quantification kit was operated according to the instructions. According to the protein quantification results, the samples were diluted with double distilled water to the same concentration, and after adding the loading buffer, the samples were boiled at 100℃ for 5 minutes, and then the samples were collected and transferred to -20℃ for use.

[0052] 2.2 Western Blot

[0053] (1) SDS-PAGE electrophoresis

[0054] Add each component in sequence according to the separation gel formula, add the separation gel to the gap between the thick and thin glass plates, and seal with anhydrous ethanol. Let stand at room temperature for about 40 minutes, discard the anhydrous ethanol after the separation gel solidifies, and use filter paper to absorb the residual anhydrous ethanol, add each component of 5% concentrated gel according to the formula, mix gently, add to the gap between the glass plates above the separation gel, and immediately insert a clean comb with an appropriate number of holes. The concentrated gel solidifies in about 15-20 minutes.

[0055]

[0056] Place the SDS-PAGE gel into a vertical electrophoresis tank and add an appropriate amount of electrophoresis buffer. After removing the comb, add protein samples and markers in order. When starting electrophoresis, adjust the voltage to 80V. After observing bromophenol blue electrophoresis to the separation gel, adjust the voltage to 120V. Stop the electrophoresis when the bromophenol blue is close to the bottom of the SDS-PAGE gel.

[0057] (2) Transfer

[0058] Prepare a PVDF membrane with a pore size of 0.22 μm, which is the same size as the SDS-PAGE gel, and soak it in anhydrous methanol for a few seconds to activate it. Soak the transfer filter paper and sponge pad with transfer buffer, and place them in the order of black transfer clamp-sponge pad-3 layers of filter paper-SDS-PAGE gel-activated PVDF membrane-2 layers of filter paper-sponge pad-transparent transfer clamp. Remove all bubbles between the SDS-PAGE gel and PVDF membrane, clamp them and place them in the transfer tank, and wet-transfer them at a constant current of 200 mA for 2 hours.

[0059] (3) Antigen-antibody reaction

[0060] After the transfer, the PDVF membrane was soaked in TBST containing 5% skim milk powder, placed on a shaker, and blocked at room temperature for 2 hours. The PVDF membrane was appropriately cut according to the molecular weight of the target protein and the internal reference protein, and then placed in an incubation box, added with the corresponding antibody and placed on a shaker at 4°C overnight. The next day, the primary antibody was removed, and the membrane was washed with TBST at room temperature for 10 minutes, a total of 3 times; horseradish peroxidase-labeled secondary antibody was added, incubated at room temperature for 2 hours, the secondary antibody was removed, and the membrane was washed with TBST at room temperature for 10 minutes, a total of 3 times.

[0061] (4) Color reaction

[0062] The PVDF membrane was placed on a color development tray, and Immobilon Western HRP substrate (solution A:solution B = 1:1 mixture) was added to the protein surface of the membrane for luminescence. The bands were imaged and analyzed using a GE ImageQuantLAS 500 biomolecular imager, and the grayscale analysis of the protein bands in the image was performed using Image J software to obtain the results.

[0063] 2.3 Data Statistical Analysis

[0064] Data are presented as mean ± SD, and Student's t-test was used for statistical analysis. "#" indicates that compared with the pEGFP control group, #P<0.05, and "*" indicates that compared with the SOD1 G93A model group, *P<0.05.

[0065] 3. Results

[0066] 3.1 Hyperforin has an enhancing effect on HO-1 protein expression level

[0067] The results of this example show that compared with the pEGFP control group, the expression level of HO-1 protein in the SOD1 G93A model group was significantly downregulated. Compared with the SOD1 G93A model group, 1, 10, and 30 μM hyperforin administration for 24 hours significantly increased the expression level of HO-1 protein in the cells. There was a certain trend of increasing the expression level of HO-1 protein in the cells after 3 μM hyperforin administration for 24 hours ( Figure 3 -AB).

[0068] In summary, the results of this example show that various doses of hyperforin can significantly increase the expression level of HO-1 protein in SOD1G93A cells 24 hours after administration, indicating that hyperforin has an improving effect on the expression level of HO-1, can improve the antioxidant capacity of cells, and play a protective role in SOD1 G93A cells.

[0069] Example 4 Hyperforin has an increasing effect on the expression level of Drp1 protein

[0070] In this example, Western Blot was used to detect the effect of hyperforin at different doses for 24 h on the expression level of Drp1 protein in SOD1G93A stably transfected cells.

[0071] 1. Cells

[0072] Cells: NSC-34 cell line stably transfected with SOD1 G93A and control empty vector cell line were the same as those in Example 1.

[0073] 2. Methods

[0074] The specific method is the same as that of Example 3.

[0075] 3. Results

[0076] 3.1 Hyperforin has an enhancing effect on the expression level of Drp1 protein

[0077] The results of this example show that compared with the pEGFP control group, the expression level of Drp1 protein in the SOD1 G93A model group was significantly downregulated. Compared with the SOD1 G93A cell model group, 10 μM hyperforin administration for 24 hours can significantly increase the expression level of Drp1 protein in the cells. 30 μM hyperforin administration for 24 hours has a certain trend of increasing the expression level of Drp1 protein in the cells ( Figure 3 -CD).

[0078] In summary, the results of this example show that various doses of hyperforin can increase the expression level of Drp1 protein in SOD1 G93A cells 24 hours after administration, indicating that hyperforin has an effect of increasing the expression level of Drp1, inhibiting mitochondrial fission, thereby inhibiting and slowing down cell apoptosis, and playing a protective role in SOD1 G93A cells.

[0079] Example 5 Hyperforin improves the morphology of SOD1 G93A cells

[0080] 1. Cells

[0081] Cells: NSC-34 cell line stably transfected with SOD1 G93A and control empty vector cell line were the same as those in Example 1.

[0082] 2. Methods

[0083] pEGFP control group cells and SOD1 G93A cells were seeded in 24-well plates, and different concentrations of hyperforin were added and incubated for 24 hours. Then, the cell morphology was recorded under an optical microscope.

[0084] 3. Results

[0085] 3.1 Hyperforin improves the morphology of SOD1 G93A cells

[0086] The results of this example show that compared with the pEGFP control group cells, the SOD1 G93A model group cells changed in morphology, showing a decrease in synapses and a rounded overall cell morphology. Compared with the SOD1 G93A model group, 3, 10, and 30 μM hyperforin administration for 24 hours significantly improved cell morphology and promoted cell synapse formation ( Figure 4 ).

[0087] In summary, the results of this example show that various doses of hyperforin can promote synapse formation in SOD1 G93A cells and improve cell morphology 24 hours after administration, indicating that hyperforin has a positive effect on cell synapse formation and can improve the morphology of SOD1G93A cells.

[0088] Example 6 Extraction and preparation process of hyperforin from Hypericum perforatum

[0089] 1. Plant Materials

[0090] The dried aerial parts of Hypericum perforatum.

[0091] 2. Extraction and preparation process

[0092] (1) The dried aerial part of Hypericum perforatum was crushed and added with anhydrous ethanol at a solid-liquid ratio of 1:4 and soaked for 36 h;

[0093] (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;

[0094] (3) The ethanol extract of Hypericum perforatum was dispersed in an appropriate amount of water, adsorbed by macroporous resin, and gradient eluted with 0%, 50%, 70%, and 100% ethanol; the 70% and 100% ethanol elution fractions were combined and evaporated to dryness under reduced pressure to obtain a fraction mainly containing hyperforin;

[0095] (4) using C18 column chromatography for separation, gradient elution, combining the fractions containing hyperforin, and recovering the solvent under reduced pressure to obtain a crude product of hyperforin;

[0096] (5) The crude product of hyperforin is then purified by preparative high performance liquid chromatography to obtain hyperforin.

[0097] 3. Results

[0098] The extraction and preparation process of hyperforin disclosed in this embodiment has a simple implementation process and a high extraction rate, and can prepare hyperforin simply and efficiently.

[0099] Chinese and English references in the drawings of the specification: Control: control; Model: model; Hyperforin: hyperforin.

[0100] 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 hyperforin and its pharmaceutically acceptable salts as shown in formula (I) in the preparation of drugs for preventing or treating amyotrophic lateral sclerosis, wherein the structural formula of hyperforin is:

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salts include hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, cyclohexylammonium salt or dicyclohexylammonium salt.

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 hyperforin and a pharmaceutically acceptable salt thereof according to 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 the group consisting of injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nanosuspensions.

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

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

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