CN111773254B

CN111773254B - Application of antrodia camphorata triterpene in preparation of medicines for treating neurodegenerative diseases

Info

Publication number: CN111773254B
Application number: CN202010705105.2A
Authority: CN
Inventors: 邹仙果; 杨开; 蔡铭; 能静; 王龑; 孙培龙
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2022-03-01
Anticipated expiration: 2040-07-21
Also published as: CN111773254A

Abstract

The invention discloses an application of an antrodia camphorata mycelium triterpene extract in preparing a medicament for treating neurodegenerative diseases, in particular Parkinson's disease. The invention provides the effect of the antrodia camphorata mycelium triterpene extract on the aspect of protecting cell nerves, provides a certain reference value for excavating the pharmacological action of the antrodia camphorata triterpene extract on the aspect of treating the Parkinson disease, and provides a basis for researching and developing natural novel Parkinson disease treatment medicines.

Description

Application of antrodia camphorata triterpene in preparation of medicines for treating neurodegenerative diseases

(I) technical field

The invention relates to an application of an antrodia cinnamomea mycelium triterpene extract in preparation of a medicament for treating neurodegenerative diseases.

(II) background of the invention

Parkinson's Disease (PD) is a common neurodegenerative disease in the middle-aged and elderly, caused by progressive degeneration and loss of Dopaminergic (DA) neurons in the substantia nigra compacta and aggregation of alpha-synuclein (alpha-syn) in the remaining DA-ergic neurons. The exact mechanism of PD occurrence is not clear at present, but oxidative damage caused by excess Reactive Oxygen Species (ROS), abnormal expression of key proteins regulating DA content, and apoptosis due to alpha-syn accumulation are considered as one of the key mechanisms.

ROS are important factors causing oxidative stress of cells, and play an important role in cell damage caused by many organ damages, controlling apoptosis.

DA is an important neurotransmitter involved in motor, cognitive, emotional, and endocrine regulation, is synthesized by DA-competent neurons and stored in vesicles, and is released by neurons by means of exocytosis. If the transmitter content is changed, the function of the whole dopaminergic nervous system is affected, and the corresponding diseases are caused. Tyrosine Hydroxylase (TH) is a rate-limiting enzyme responsible for catalyzing the conversion of the amino acid L-Tyrosine into dihydroxyphenylalanine (dopa), and it and dopamine transporter (DAT) are important proteins for the synthesis and transport of DA in DA-enabled nervous system pathways, which play a major role in maintaining the balance of DA content in synaptic cleft. If these proteins are dysfunctional, the brain DA levels become unbalanced, causing neurological diseases such as Parkinson's disease.

The structure of alpha-syn maintains a dynamic balance between normal, misfolded and oligomerized, and when this balance is broken, the alpha-syn fibrils rapidly aggregate into macromolecules, forming insoluble fibers, further forming lewy bodies to avoid greater damage to neurons, but pathological accumulation, precipitation of alpha-syn, ultimately leads to apoptosis or death. The clinical manifestations of PD are mainly bradykinesia, resting tremor, muscular rigidity and gait instability.

At present, the drugs clinically used for treating PD mainly include levodopa, amantadine, dopamine receptor agonists, anticholinergics, and the like. However, the drugs cannot slow down the development process of PD, bring many adverse reactions while treating, and limit the clinical application of the drugs. Therefore, it is of great practical significance to find more effective drugs or active ingredients for treating PD. In recent years, the search for active ingredients for delaying the onset of PD from traditional medicine-food dual-purpose resources has become a hot point of research.

Antrodia Cinnamomea (AC), also known as "Antrodia cinnamomea", "Antrodia camphorata", and the like belong to perennial fungi of Basidiomycota (Basidiomycota), Aphyllophorales (Aphyllophorales), Polyporaceae (Polyporaceae), and Antrodia, are unique and precious dual-purpose fungi for both medicine and food in Taiwan territory, and are called "ruby in forest". Antrodia camphorata has various physiological active ingredients including triterpenes, polysaccharides, adenosine, superoxide dismutase (SOD), etc. The triterpenes are one of the main effective components of antrodia camphorata. At present, researches on triterpenoids of antrodia camphorata mostly focus on optimization of an extraction preparation method and a process, and the researches on physiological activity are less.

In the prior art, the extraction and preparation method of antrodia camphorata triterpenes is mature, and the antrodia camphorata triterpenes has the physiological activity functions of resisting tumors and preventing alcoholic liver injury, and the neuroprotective pharmacological activity of the antrodia camphorata triterpenes is rarely reported. The prior application CN106692211B discloses a preparation method of an antrodia camphorata mycelium triterpenoid extract, which has the advantage that the antrodia camphorata triterpenoid extract with the purity of up to 70-90 percent is prepared by adopting ultrasonic-assisted extraction combined with solvent extraction and macroporous resin purification. However, the efficacy of the antrodia camphorata triterpene extract obtained by the method in the aspect of neuroprotection is not clear.

Disclosure of the invention

The invention aims to provide application of an antrodia cinnamomea mycelium triterpene extract in preparation of medicines for treating neurodegenerative diseases.

The technical scheme adopted by the invention is as follows:

application of Antrodia Camphorata mycelium triterpene extract in preparing medicine for treating neurodegenerative disease is provided.

Preferably, the neurodegenerative disease is parkinson's disease.

The invention provides the effect and the action mechanism of the antrodia camphorata mycelium triterpenoid extract in the aspect of protecting cell nerves, provides a certain reference value for exploring the pharmacological action of the antrodia camphorata triterpenoid extract in the aspect of treating the Parkinson disease, and provides a basis for researching and developing natural novel Parkinson disease treatment medicines.

Specifically, the antrodia camphorata mycelium triterpene extract is prepared by the following method: taking antrodia camphorata mycelium powder, carrying out ultrasonic extraction for 1-3 times by using 60-80% ethanol water solution, combining extracting solutions, concentrating, and carrying out freeze drying to obtain a crude extract; dispersing the crude extract in 10-20 times of water to obtain a dispersion liquid, adding petroleum ether with the same volume of 30-60 ℃ into the dispersion liquid for degreasing, adding ethyl acetate into the obtained degreasing liquid for extraction, collecting an ethyl acetate layer, and evaporating under reduced pressure to remove a solvent to obtain a concentrate; adding the concentrate into 20-30% ethanol water solution, adjusting the pH value to 2-5, purifying by macroporous adsorption resin, eluting by deionized water, 20-40% ethanol water solution and 60-90% ethanol water solution in sequence, collecting 60-90% ethanol eluent, evaporating under reduced pressure to remove the solvent, and freeze-drying to obtain the antrodia cinnamomea mycelium triterpene extract, wherein the triterpene purity of the antrodia cinnamomea mycelium triterpene extract is 70-90%.

The beneficial effects of the invention are mainly reflected in that: the invention provides the effect of the antrodia camphorata mycelium triterpene extract on the aspect of protecting cell nerves, provides a certain reference value for excavating the pharmacological action of the antrodia camphorata triterpene extract on the aspect of treating the Parkinson disease, and provides a basis for researching and developing natural novel Parkinson disease treatment medicines.

(IV) description of the drawings

Fig. 1 is a schematic diagram of the neuroprotective mechanism of the triterpene extract of antrodia camphorata on PC12 cells.

(V) detailed description of the preferred embodiments

For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

The materials and equipment used in the examples were as follows:

solid culture mycelium of antrodia cinnamomea: provided by research institute of medical fungi of Lishui Reynaudi;

and (3) vacuum drying oven: german Binder VD 23;

FS-1200 type ultrasonic cell disruptor: shanghai BioAnalyzer instruments Inc.;

a centrifuge: TD5A-WS, with NO.4 rotor, Hunan instrument centrifuge instruments GmbH;

rotating the evaporator: R2002B, circulating water pump, Shanghai Shensheng science and technology Limited;

AB-8 macroporous adsorption resin: sanxing resin science and technology, Inc., Anhui.

Example 1: obtaining triterpene extract from Antrodia Camphorata mycelium

(1) Drying Antrodia camphorata solid culture mycelium (provided by Lishui Recao medicinal fungus research institute) at 50 ℃ in vacuum, crushing and sieving to 80 meshes, weighing 50g of Antrodia camphorata mycelium powder, and mixing the powder according to the material liquid mass ratio of 1: 30, adding 1500g of 80 wt% ethanol water solution for ultrasonic-assisted extraction, wherein the extraction power is 100W, the extraction time is 40min, centrifuging at 5000r/min, collecting supernatant, repeatedly performing ultrasonic extraction on filter residues for 2 times, combining the obtained supernatants, performing rotary evaporation at 50 ℃ and the vacuum degree of 0.05Mpa, concentrating to 1/5 of the original volume, and then performing freeze drying to obtain the antrodia triterpenes crude extract;

(2) taking the antrodia camphorate triterpene crude extract obtained in the step (1), and ultrasonically dispersing the antrodia camphorate triterpene crude extract in 300mL of purified water to obtain a dispersion liquid; then adding 60 ℃ petroleum ether with the same volume as the obtained dispersion liquid for degreasing, and repeatedly degreasing for 3 times to obtain degreasing liquid; adding ethyl acetate with the same volume as the degreasing solution for extraction, repeatedly extracting for 3 times, collecting extract, and evaporating under reduced pressure to remove solvent to obtain concentrate;

(3) adding the concentrate obtained in step (2) into 400mL of 30 wt% ethanol aqueous solution with an original pH of 5.5, adjusting the pH to 2.5 with 0.1N hydrochloric acid aqueous solution, and then carrying out AB-8 macroporous adsorption resin column purification, glass chromatography column: 2.6 multiplied by 80cm, the sampling concentration is 20.4mg/mL, the sampling volume is 0.15 times of the column volume, the sampling flow rate is 1.0mL/min, deionized water and 30 wt% ethanol water solution which are 4 times of the column volume are respectively used for eluting and removing impurities, then 90 wt% ethanol water solution which is 6 times of the column volume is used for eluting, 90 wt% ethanol eluent is collected, the solvent is evaporated under reduced pressure, and the product, namely the antrodia camphorata mycelium triterpenes extract, is 3.9 g.

Analysis and detection prove that the extraction rate of the obtained product is 7.8 percent, and the content of the triterpene is 86.3 percent.

Example 2:

(1) culturing PC12 nerve cells in DMEM medium, inoculating the cells with the fusion degree of 80% to a 96-well plate for 24h, and treating the cells with 300 mu M6-OHDA for 4h to obtain an injured group; incubating 1000ppm triterpene purified Antrodia camphorata for 4h, and treating cells with 300 μ M6-OHDA for 4h to obtain medicinal group; cells cultured with DMEM were used as a negative control.

(2) After the treatment of the step (1), detecting the cell survival rate by adopting an MTT method, and determining the protective effect of the triterpenoid purified product of the antrodia camphorata on nerve cells.

The detection method comprises the following steps: after the completion of the treatment, the culture solution was removed, 100. mu.L of a culture solution containing 10% MTT was added, and after incubation at 37 ℃ for 4 hours, the culture solution was removed, 100. mu.L of DMSO was added to each well to dissolve MTT crystals, and the absorbance was measured at a detection wavelength of 570nm and a reference wavelength of 655nm and was designated as A. Cell viability was expressed as a percentage, and (%) -cell viability (experimental a-value-blank a-value)/(negative control a-value-blank a-value).

And (3) detection results: the cell survival rate detected by the MTT method is 39.40% in the injured group and 55.76% in the drug group compared with the negative control group.

(3) After the treatment of the step (1), detecting the ROS level by adopting a fluorescence spectrophotometry method, and determining the improvement effect of the triterpenoid purified product of the antrodia camphorata on the nerve cell damage.

The detection method comprises the following steps: after the treatment was completed, the culture medium was removed and a 300. mu.M 6-OHDA lesion was added for 4 hours. The mixture was then added to a culture medium containing DCFH-DA (20. mu.M) and the mixture was treated with light for 1 hour. The cells were washed 3 times with PBS and the fluorescence intensity was measured by a fluorescence spectrophotometer with an excitation wavelength of 485nm, an emission wavelength of 535nm and a fluorescence intensity B. ROS level (%) (experimental B-value-blank B-value)/(negative control B-value-blank B-value).

And (3) detection results: and (3) detecting the ROS level by a fluorescence spectrophotometry, wherein the ROS level of the damage group is 192.01%, and the ROS level of the medicine group is 144.73% compared with that of the negative control group.

(4) After the treatment of the step (1), protein immunoblotting is adopted to detect the expression levels of DAT, TH, alpha-syn and caspase-3 protein, and the mechanism of protecting nerve cells by the triterpenoid of antrodia camphorate is determined.

The detection method comprises the following steps: determining the protein concentration by using a BCA method, adding 6 × loading buffer to heat the protein sample at 100 ℃ for 5min for denaturation, and preparing SDS-PAGE loading protein; preparing 5% concentrated glue and 10% separation glue, adding 60 μ g of loading protein and Marker into each hole, performing electrophoretic separation (80V, 40min, 100V and 60min), taking out the glue after the electrophoresis separation is finished, preparing the glue into a filter paper, the glue, a PVDF membrane and a spongy cushion structure, and placing the filter paper, the glue, the PVDF membrane and the spongy cushion structure into a 200mA membrane transfer instrument to transfer a membrane for 12 h; blocking with 5% Milk/TBST for 1h, adding the corresponding primary antibody, incubating overnight at 4 deg.C, washing PVDF membrane with TBST (3 times for 5min each), then incubating with secondary antibody for 1h, washing with TBST; adding ECL chemiluminescence solution (A, B solution mixed at a ratio of 1: 1), and reacting for 30s in the absence of light. By ChemiDoc^TMThe XRS + chemiluminescent imager developed the band of interest.

And (3) detection results: compared with the negative control group, the DAT, TH, alpha-syn and caspase-3 protein expression levels of the injury group are 25.78%, 20.04%, 161.21% and 164.89% respectively, and the DAT, TH, alpha-syn and caspase-3 protein expression levels of the drug group are 50.24%, 60.21%, 134.72% and 141.31% respectively.

Example 3:

cell processing grouping: culturing PC12 nerve cells in DMEM medium, wherein the damage group is 300 mu M6-OHDA treated cells for 4 h; the drug group is prepared by adding 100ppm purified product of Antrodia camphorata triterpene, incubating for 4h, and treating cells with 300 μ M6-OHDA for 4 h; the negative control group was cells cultured with DMEM.

Index detection and results: the cell survival rate is detected by an MTT method, compared with a negative control group, the cell survival rate of the damaged group is 39.40%, and the cell survival rate of the medicine group is 64.05%; detecting ROS level by a fluorescence spectrophotometry, wherein compared with a negative control group, the ROS level of a damage group is 192.01%, and the ROS level of a medicine group is 121.31%; the protein immunoblotting method detects the expression levels of DAT, TH, alpha-syn and caspase-3, compared with the negative control group, the expression levels of DAT, TH, alpha-syn and caspase-3 in the damage group are respectively 25.78%, 20.04%, 161.21% and 164.89%, and the expression levels of DAT, TH, alpha-syn and caspase-3 in the drug group are respectively 55.21%, 68.28%, 125.06% and 135.10%.

Example 4:

cell processing grouping: culturing PC12 nerve cells in DMEM medium, wherein the damage group is 300 mu M6-OHDA treated cells for 4 h; the drug group is prepared by adding 10ppm of antrodia camphorata triterpene purified substance, incubating for 4h, and treating cells for 4h by 300 μ M6-OHDA; the negative control group was cells cultured with DMEM.

Index detection and results: the cell survival rate is detected by an MTT method, compared with a negative control group, the cell survival rate of the damaged group is 39.40%, and the cell survival rate of the medicine group is 69.23%; detecting ROS level by a fluorescence spectrophotometry, wherein compared with a negative control group, the ROS level of a damage group is 192.01%, and the ROS level of a drug group is 115.45%; the protein immunoblotting method detects the expression levels of DAT, TH, alpha-syn and caspase-3, compared with a negative control group, the expression levels of DAT, TH, alpha-syn and caspase-3 in a damage group are respectively 25.78%, 20.04%, 161.21% and 164.89%, and the expression levels of DAT, TH, alpha-syn and caspase-3 in a drug group are respectively 59.70%, 75.45%, 117.79% and 130.43%.

Example 5:

cell processing grouping: culturing PC12 nerve cells in DMEM medium, wherein the damage group is 300 mu M6-OHDA treated cells for 4 h; the drug group is prepared by adding 1ppm of antrodia camphorata triterpene purified substance, incubating for 4h, and treating cells for 4h by 300 μ M6-OHDA; the negative control group was cells cultured with DMEM.

Index detection and results: the cell survival rate is detected by an MTT method, compared with a negative control group, the cell survival rate of the damaged group is 39.40%, and the cell survival rate of the medicine group is 79.40%; detecting ROS level by a fluorescence spectrophotometry, wherein compared with a negative control group, the ROS level of a damage group is 192.01%, and the ROS level of a medicine group is 135.11%; the protein immunoblotting method detects the expression levels of DAT, TH, alpha-syn and caspase-3, compared with the negative control group, the expression levels of DAT, TH, alpha-syn and caspase-3 in the damage group are respectively 25.78%, 20.04%, 161.21% and 164.89%, and the expression levels of DAT, TH, alpha-syn and caspase-3 in the drug group are respectively 70.01%, 85.45%, 109.34% and 121.47%.

Example 6:

cell processing grouping: culturing PC12 nerve cells in DMEM medium, wherein the damage group is 300 mu M6-OHDA treated cells for 4 h; the drug group is prepared by adding 0.1ppm triterpenoid of Antrodia camphorata, incubating for 4h, and treating cells with 300 μ M6-OHDA for 4 h; the negative control group was cells cultured with DMEM.

Index detection and results: the cell survival rate is detected by an MTT method, compared with a negative control group, the cell survival rate of the damaged group is 39.40%, and the cell survival rate of the medicine group is 78.40%; detecting ROS level by a fluorescence spectrophotometry, wherein compared with a negative control group, the ROS level of a damage group is 192.01%, and the ROS level of a medicine group is 141.01%; the protein immunoblotting method detects the expression levels of DAT, TH, alpha-syn and caspase-3, compared with a negative control group, the expression levels of DAT, TH, alpha-syn and caspase-3 in a damage group are respectively 25.78%, 20.04%, 161.21% and 164.89%, and the expression levels of DAT, TH, alpha-syn and caspase-3 in a drug group are respectively 65.21%, 78.76%, 115.23% and 128.79%.

And (4) conclusion: as can be seen from the experiments, the antrodia camphorata mycelium triterpene extract can protect PC12 nerve cells by promoting cell survival, reducing cell oxidative damage caused by ROS, promoting and regulating DAT generated by DA and the expression of TH key protein and inhibiting alpha-syn accumulation and the expression of cell apoptosis protein caspase 3. Therefore, the antrodia camphorata mycelium triterpene extract can be used as a natural medicine for treating neurodegenerative diseases, particularly Parkinson's disease, has the advantages of good drug effect, low side effect and the like compared with a synthetic medicine, and has better application prospect.

Claims

1. The application of the antrodia camphorata mycelium triterpene extract in preparing the medicine for treating the Parkinson disease is characterized in that the antrodia camphorata mycelium triterpene extract is prepared by the following method: taking antrodia camphorata mycelium powder, performing ultrasonic extraction for 1-3 times by using 60-80% ethanol water solution, combining extracting solutions, concentrating, and performing freeze drying to obtain a crude extract; dispersing the crude extract in 10-20 times of water to obtain a dispersion liquid, adding petroleum ether with the same volume of 30-60 ℃ into the dispersion liquid for degreasing, adding ethyl acetate into the obtained degreasing liquid for extraction, collecting an ethyl acetate layer, and evaporating under reduced pressure to remove a solvent to obtain a concentrate; adding the concentrate into 20-30% ethanol water solution, adjusting the pH value to 2-5, purifying by macroporous adsorption resin, eluting by deionized water, 20-40% ethanol water solution and 60-90% ethanol water solution in sequence, collecting 60-90% ethanol eluent, evaporating under reduced pressure to remove the solvent, and freeze-drying to obtain the antrodia camphorata mycelium triterpene extract.