KR20200093976A - Pharmaceutical Composition for Inhibiting Amyloid Beta Deposition Comprising Uncaria Rhynchophylla Extract - Google Patents

Abstract

The present invention relates to a composition which can be usefully used for the prevention and treatment/symptom alleviation of diseases related to the amyloid beta deposition, by having an effect of suppressing the amyloid beta deposition, neurodegeneration, and neuroinflammation. More specifically, the present invention relates to a composition for inhibiting amyloid beta deposition using an Uncaria rhynchophylla extract as an active ingredient.

Description

Pharmaceutical Composition for Inhibiting Amyloid Beta Deposition Comprising Uncaria Rhynchophylla Extract}

The present invention relates to a composition that can be useful for the prevention and treatment/improvement of diseases related to the deposition of amyloid beta because it has the effect of inhibiting the deposition of amyloid beta and neurodegeneration and neuroinflammation.

Alzheimer's disease is a typical senile dementia and has a high prevalence of disease in one third of people over 85. The number of dementia patients in Korea is about 620,000, and it is expected to exceed 1 million in 2025. Recently, Alzheimer's disease is not limited to the individual patient or family, and has emerged as an important issue across society and the economy.

Alzheimer's disease causes a variety of symptoms, such as memory impairment, speech impairment, loss of cognitive function, neurodegeneration and nerve inflammation. As Alzheimer's disease progresses, more functional disorders appear, and neuropsychiatric symptoms also increase. Currently, as the level of amyloid beta (Aβ) increases in the brain, deposition occurs to form plaques, and accumulated Aβ plaques block signal transduction pathways between brain cells, causing brain cells to die and eventually causing dementia. The'amyloid beta hypothesis' is accepted as the established mechanism of dementia. Recently, a study in the United States dementia treatment research center reported that studies on the possibility of transition to dementia increase when the level of Aβ starts to increase even if there are no symptoms of dementia, such as decreased memory, which supports the amyloid beta hypothesis. . Because of this, more than half of the drugs in Phase III clinical trials as of 2018 target amyloid-related mechanisms, and clinical trials of many drugs, including BAN2401 and aducanumab (aducanumab), target direct reduction of amyloid beta deposition. It is ongoing. Previous studies suggest that Alzheimer's cognitive impairment is associated with neurodegeneration rather than the accumulation of amyloid beta itself. Neuronal degeneration in Alzheimer's can be characterized by synaptic and neuronal loss. In particular, recent studies have reported that neurodegeneration alone does not significantly affect cognitive decline without amyloid beta deposition. Therefore, a treatment strategy targeting to suppress aggregation and deposition of amyloid beta seems to be a possible strategy in itself, and treatment may be more effective when it includes inhibition of amyloid beta, neuroinflammatory and neurodegeneration.

Although some drugs targeting direct inhibition of amyloid beta deposition are in clinical trials, they are frustrated in clinical trials due to their low efficacy and side effects. As a typical example, Bapineuzumab received the most attention among new drug candidates targeting amyloid beta, but in Phase 3 of clinical trials, the effect compared to Placebo did not meet expectations, and development was discontinued. In 2012, Solanezumab, targeting Eli Lilly's amyloid beta, was also discontinued, but some patients have meaningful results and are currently re-promoting. However, there are still no treatments targeting amyloid beta. Therefore, current therapeutics for Alzheimer's are limited to disrupting the mechanism of neurotransmitters that regulate learning and memory, such as acetylcholinesterase inhibitors.

Diseases related to the deposition of amyloid beta increase the fragility of nerve cells due to the abnormality of amyloid production, resulting in neurodegeneration, and have a common characteristic that neuroinflammation occurs by activation of an inflammatory mechanism. In addition to Alzheimer's disease, diseases characterized by deposition of amyloid beta, Prion diseases such as Down's syndrome, Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, amyloid vasculosis, cerebral amyloid vasculosis, systemic amyloid disease, odor ( Dutch) type amyloidosis, inclusion body stomatitis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, hereditary cerebral hemorrhage related to amyloidosis and brain amyloid angiopathy. Therefore, medicines that inhibit the deposition of amyloid beta may be useful in the treatment of diseases associated with amyloid beta deposition in addition to Alzheimer's.

Cho Gu-deung (Uncaria rhynchophylla (Miq.) Jacks. (UR)) in Oriental Medicine has a dietary canon (熄風定痙: calming the wind and calming cramps) and blue-green interval (淸熱平肝: eliminating heat and lowering the liver of the liver) It has been used for symptoms such as competition, epilepsy, convulsions, headache, and dizziness (Shinbon elementary school, Hanol Publishing). In addition, Patent Publication No. 10-2001-0103341 reported that it has a neuronal protective activity against ischemic neuronal damage in the brain, and Patent Registration No. 10-1438415 inhibits acetylcholinesterase from trans-anetol extracted from jojo et al. It has been reported that it has the effect of preventing or recovering from memory impairment.

Patent Publication No. 10-2001-0103341 Registered Patent No. 10-1438415

An object of the present invention is to provide a composition useful for preventing and treating/improving symptoms of diseases associated with amyloid deposition by inhibiting deposition of amyloid beta.

The present invention further aims to provide a composition that inhibits neuroinflammation and neurodegeneration associated with amyloid deposition and promotes neurogenogenesis.

The present invention for achieving the above object relates to a pharmaceutical composition for inhibiting amyloid beta deposition containing an extract of coarse bulb as an active ingredient.

Jogudeung extract can be prepared by applying a conventional extraction method in the art. Specifically, the harvested raw, dried or frozen coarse can be extracted by cutting or grinding to an appropriate size to facilitate extraction, and as a solvent for extraction, lower alcohols of C1 to C4, water or Mixtures of these can be used. When considering the safety of the residual solvent, it is better to use an aqueous ethanol solution. As the extraction method, hot water extraction, cold needle extraction, reflux cooling extraction, supercritical extraction or ultrasonic extraction may be used.

Jogu-deung extract effectively inhibited the deposition of amyloid beta in in-vitro as well as in-vivo tests, and also inhibited neurodegeneration caused by amyloid beta deposition and neurodegeneration such as loss of synapses and nerve cells. Shown. Furthermore, it has an effect of promoting neurogenicity, and thus can be more effectively used for the prevention and treatment of diseases related to the deposition of amyloid beta. Diseases associated with the deposition of amyloid beta include Alzheimer's disease, Down's syndrome, Creutzfeldt-Jakob disease, Prion diseases such as Gerstmann-Straussler syndrome, amyloid vasculosis, cerebral amyloid vasculosis, systemic amyloid disease, Dutch ) Type amyloidosis, inclusion body stomatitis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, inherited cerebral hemorrhage related to amyloidosis and brain amyloid angiopathy, but are not limited thereto.

In particular, the composition of the present invention can be more usefully used for targeting at risk of amyloid beta deposition. The risk group for amyloid beta deposition is genetically vulnerable to diseases related to amyloid beta deposition (e.g., a patient with Alzheimer's disease or familial amyloidosis in the family) or an age group with a high incidence of amyloid beta deposition related diseases (e.g. For example, the elderly who have a high incidence of Alzheimer's disease), or a group in which amyloid beta deposition is not clearly observed but shows suspected symptoms of related diseases.

The composition of the present invention can be prepared by a method known in the pharmaceutical field for use as a drug for the inhibition of amyloid beta, itself or a pharmaceutically acceptable carrier, excipient (forming agent), It can be used in combination with diluents. The composition of the present invention can be formulated as a formulation for oral or parenteral administration and used as a preventive agent for diseases related to amyloid beta. The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount. In the present invention, the'pharmaceutically effective amount' means an amount sufficient to treat a disease at a ratio of rational benefit/risk applicable to medical treatment. Effective dose levels include the patient's disease type, severity, age, sex, drug activity, drug form, sensitivity to the drug, time of administration, route of administration and rate of discharge, duration of treatment, factors including drugs used concurrently, and other medicine. It can be determined by factors well known in the art. The typical dosage is 0.001 mg/kg·day to 10 g/kg·day. The composition of the present invention may be administered alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents.

In addition, the present invention provides a health functional food composition for preventing and improving symptoms of amyloid beta deposition-related diseases comprising an extract of jogu etc. as an active ingredient. The crude bulb extract may be added to the health functional food of the present invention, preferably 0.01 to 100% by weight. The health functional food of the present invention includes tablets, capsules, pills or liquids, etc. As foods to which the composition of the present invention can be added, for example, various foods, beverages, gums, teas, and vitamin complexes , Health functional foods, etc.

As described above, the composition containing the crude bulb extract of the present invention not only effectively suppresses amyloid beta deposition, but also inhibits neurodegeneration and neuroinflammation induced by amyloid deposition, and has an effect of promoting neurogenesis, and amyloid beta. It can be usefully used for prevention and treatment/improvement of diseases related to deposition.

In addition, the composition of the present invention is a long-term toxicity and safety for the human body has been verified as the main raw material for herbal medicines that have been used in herbal medicine for a long time, and can be used more safely.

1 is a graph showing the results of ThT assay showing the inhibitory effect of coarse bulb extract on amyloid beta aggregation in-vitro.
Figure 2 is a dyeing / immunofluorescence image and graph showing the inhibitory effect of amyloid beta aggregation of the bulbous extract in mice.
Figure 3 is an immunofluorescence image and graph showing the effect of suppressing neuroinflammation of jogudeung extract in the hippocampal part of the mouse.
Figure 4 is an immunofluorescence image and graph showing the efficacy of inhibiting synaptic and neuronal loss of the bulbous extract in the hippocampus of the mouse.
Figure 5 is an immunofluorescence image and graph showing the neuroinflammation and neurodegeneration inhibitory efficacy of the bulbous light extract in the cerebral cortex of the mouse.
Figure 6 is an immunofluorescence image and graph showing the efficacy of the bulbs in the mouse for neurogenic development.

Hereinafter, the present invention will be described in more detail with reference to the attached examples. However, these examples are merely examples for easily explaining the contents and scope of the technical spirit of the present invention, and the technical scope of the present invention is not limited or changed thereby. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical spirit of the present invention based on these examples.

[Example]

Example 1: Preparation of crude bulb extract

Dried jogu lamps were purchased from Sunil Herbal Medicine (Korea, Gunwi-gun) and extracted by immersion in 70% ethanol for 24 hours. The extract was filtered to remove the solvent, dried (yield 1%), and stored at -20°C for use.

Example 2: Evaluation of the efficacy of inhibiting amyloid beta aggregation in-vitro

It was confirmed that the crude bulb extract showed amyloid beta inhibitory activity in in-vitro by a ThT (Thioflavin T) assay using a Thioflavin T Aβ ₄₂ Aggregation Kit (ANASPEC, Fremont, CA, USA). The assay buffer was stored at 4°C to prevent amyloid beta aggregation, and the other solutions were stored at room temperature. The assay solution was prepared with an assay buffer containing 2 mM ThT. Aβ ₄₂ peptide solution was prepared by adding Aβ ₄₂ to a cold assay buffer at a concentration of 0.25 mg/ml. The peptide was allowed to stand at room temperature for 5 minutes to hydrate, and then completely solubilized by ultrasonic treatment for 5 minutes. Thereafter, the solution was centrifuged at 10,000 rpm and 4°C for 5 minutes to remove residual precipitate. Positive control phenol-red and morin (morin) solution, vehicle and crude bulb extract (UR) solution was also prepared using an assay buffer.

After adding 10 μl of assay solution and 85 μl of Aβ ₄₂ peptide solution to each well of a 96-well plate, 5 μl of positive control solution (final concentration = 100 μM), UR solution (final concentration = 100, 200, 300 , 400, and 500 μg/ml) or assay buffer (Vehicle). Then, the fluorescence intensity of ThT was measured (Ex/Em = 440 nm/484 nm) using an Infinite M200 Fluorescence Microplate Reader (Tecan, Mannedorf, Switzerland) to evaluate the degree of aggregation of amyloid beta.

Figure 1 shows the results, jogudeung extract significantly inhibited the aggregation of amyloid beta at a concentration of 200 ~ 500 μg / ml.

Example 3: Evaluation of the efficacy of inhibiting amyloid beta deposition in-vivo

1) Breeding of experimental animals and treatment of extracts such as coarse

The 5XFAD transgenic mouse, an Alzheimer's disease animal model, was purchased from The Jackson Laboratory (Bar Harbor, ME, USA; catalog number 006554). The experimental animals were used in the experiment after being purified in a laboratory environment with a 12:12 light and dark cycle while freely supplying a standard diet and water.

After acclimatization, female 5XFAD mice and wild-type (WT) mice with good health were classified to have 4 to 5 individuals per group. In the experimental group, 5XFAD mice (n=4) were diluted with saline solution immediately before administration, and orally administered daily for 4 weeks at a dose of 400 mg/kg. The control group and the comparison group were orally administered with the same amount of saline daily for 4 weeks to 5XFAD mice (n=5) and WT mice (n=5), respectively. All experiments were conducted in accordance with NIH's Guide to the Management and Use of Laboratory Animals (NIH publications No. 8023, 1978 amended), and were conducted under the supervision of the Steering Committee for Laboratory Animals, Konyang University.

2) Preparation of brain tissue samples

In Example 2, as it was confirmed that the crude bulb extract in-vitro inhibits aggregation of amyloid beta, whether it affects aggregation of amyloid beta in in-vivo, of the hippocampus (subiculum) and cerebral cortex Histological staining was performed.

When 4 weeks of oral administration was completed in 1), 0.05M PBS buffer containing 4% paraform aldehyde (PFA) was sacrificed by cardiac perfusion on the day following the last administration. Brains were removed and fixed at 4° C. for 20 hours using 4% PFA solution. For cryoprotection, the brain was immersed in 0.05M PBS containing 30% sucrose for 3 days. Samples are sliced into a series of coronal sections 30 μm thick in CM1850 cryostat (Leica Biosystems) and in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) until use for tissue analysis. It was stored at 4 ℃. For staining and analysis of brain tissue, three samples of brain sections of 210-240 µm were collected from the region between -2.6 and -4.3 mm for bregma in each mouse.

3) Evaluation of the effect of inhibiting amyloid aggregation by ThS staining

Brain tissue was stained with thioflavin S (ThS) to detect β-sheet amyloid plaques. Specifically, the ThS solution was prepared by dissolving ThS in 50% ethanol at a concentration of 5 mg/ml. The brain tissue sample prepared above was washed 3 times with PBS for 5 minutes and then incubated for 10 minutes at room temperature using an orbital shaker in the ThS solution. Thereafter, brain tissue was collected, washed twice with ethanol three times, and washed three times with PBS three times. ThS-stained brain tissue was mounted on a SuperFrost Plus ^TM Adhesion slide and covered with Fluoroshield ^TM . The prepared sample was photographed with Zeiss LSM 700 (Carl Zeiss AG, Oberkochen, German) and then analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the results are shown in FIG. 2.

2A is a ThS staining image for the hippocampal part (Sub) and the cerebral cortex (Ctx) of the experimental group (UR) and the control group (Vehicle), and B is a graph showing the quantified ThS area from the image. FIG. 2 shows that the ThS-positive region in the 5XFAD mice (experimental group) administered the bulbous extract was significantly reduced compared to the 5XFAD mice (control group) only administered saline in the hippocampus and the cerebral cortex.

4) Evaluation of the effect of suppressing amyloid aggregation, neurodegeneration and neuroinflammation by immunofluorescence analysis

Immunofluorescence assay

Amyloid aggregation, neurodegeneration and neuroinflammation were quantitatively analyzed by immunofluorescence analysis of brain tissue samples. Specifically, free-floating sections of brain tissue samples prepared in 2) to detect immunofluorescence of 4G8, NeuN, synaptophysin, GFAP, iba-1, DCX, and Ki67 are mouse anti-Aβ antibody 4G8(1:2000; Covance, Princeton, NJ), mouse anti-NeuN antibody (1:100; BioLegend, San Diego, CA), mouse anti-synaptophysin antibody (1:500; Sigma-Aldrich), rat anti-GFAP (1:200; Invitrogen), goat anti-iba-1 antibody (1:500; Abcam, Cambridge, UK), goat anti-DCX antibody (1:50; Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-Ki67 antibody (1:200; Abcam, Cambridge, UK). After incubation, washed 3 times with PBS for 5 minutes, and then the secondary antibody (1:500) goat Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA), donkey Alexa Fluor ^® 488-conjugated anti-rat antibody, ^{donkey Alexa Fluor ® 488-conjugated anti} -goat antibody, goat Alexa Fluor ® 488-conjugated was 1 hour incubation at room temperature with anti-rabbit antibody and ^{donkey Alexa Fluor ® 594-conjugated anti} -goat antibody. After washing with PBS three times, it was mounted on a SuperFrost Plus ^TM adhesion slide, covered with Fluoroshield ^TM with DAPI, and analyzed by the same method as 3).

Evaluation of the efficacy of inhibiting amyloid beta aggregation

C of FIG. 2 is an immunofluorescence image for 4G8 specific for Aβ _17-24 , and D and E are graphs of C images analyzed by Image J software. The experimental group showed a significant decrease in the number of 4G8-positive areas and 4G8-positive cells per unit area in both the hippocampus and the cerebral cortex compared to the control group, and showed that the coarse bulb extract significantly inhibited aggregation of amyloid beta, similar to the result of ThS staining.

Neuroinflammatory inhibition efficacy evaluation

Neuroinflammation is one of the characteristic conditions of Alzheimer's, and it is known that aggregation of amyloid beta activates glial cells to initiate proinflammatory and cytotoxic cascades to cause neurodegeneration. It was evaluated by immunofluorescence analysis of Iba-1, a marker of microglia, and GFAP, which is a marker of astrocytes, in which the bulb extracts affect neuroinflammation.

A and B in Figure 3 is a hippocampus, A in Figure 5 is a graph showing the immunofluorescence image of Iba-1 in the cerebral cortex and its analysis, Figure 5C is Iba-1 and NeuN in the entire cortical layer This is an image of double immunofluorescence analysis. The figure significantly increases the area of the Iba-1 positive region in the 5XFAD mouse compared to the wild-type mouse, and the crude bulb extract significantly reduced the area of the Iba-1 positive region in the 5XFAD mouse. This reduction of the Iba-1 positive region was more effective in the cerebral cortex than in the hippocampus.

C and D of Figure 3 is an image showing the immunofluorescence image of GFAP in the hippocampal region and its analysis results, and like Iba-1, the bulbous extract showed a significant reduction in the proportion of GFAP positive regions in 5XFAD mice.

This suggests that the crude bulb extract can effectively act on the suppression of neuroinflammation by aggregation of amyloid beta.

Evaluation of the efficacy of suppressing synaptic and neuronal cell loss

The loss of synapses and neurons is a major pathology of Alzheimer's. Aggregation of amyloid beta causes loss of synapses and neurons directly in the brain of Alzheimer's patients. It was evaluated by immunofluorescence analysis of NeuN, a marker for the nucleus of neurons, to determine whether the extract of Jogu-deung can reduce neurodegeneration with the effect of inhibiting amyloid beta aggregation in the brain of 5XFAD mice.

A and B in Figure 4 is a hippocampus, B in Figure 5 is a graph showing the immunofluorescence image of NeuN in the cerebral cortex and its analysis, Figure 5C is a double of Iba-1 and NeuN in the entire cortical layer Immunofluorescence analysis image. As expected, the number of NeuN-positive cells per unit area in the hippocampus and the cerebral cortex is significantly reduced in the control group compared to the control group, and the number of NeuN-positive cells in the hippocampal region is significantly higher than in the control group when administration of the bulbous extract is compared to the control group. It shows the increase. In the deep cerebral cortex of 5XFAD mice, there was no significant difference in the number of reduced NeuN positive cells, but it showed an increasing trend.

In order to quantify synaptic density in the hippocampus, immunofluorescence of synaptophysin (SYN), a marker of presynaptic vesicle protein, was analyzed. C and D of FIG. 4 are immunofluorescence analysis images and fluorescence intensity analysis graphs in the hippocampus, respectively, and the degree of immune response of SYN was also significantly decreased in 5XFAD mice compared to wild type mice. Jogu-deung extract significantly increased the immune response of SYN, suggesting that Jogu-deung extract has the effect of attenuating synaptic and neuronal degeneration caused by amyloid beta toxicity.

Evaluation of the improvement efficacy of damaged neurogenesis in the hippocampus

Neurogenesis of the hippocampus in the granulation zone (SGZ) of the adult hippocampus dentate gyrus (DG) plays an important role in the regulation of cognitive functions such as learning and memory. Alzheimer's animal models have been reported to lack the neurogenicity of the adult hippocampus, and restoring the neurogenicity of the damaged hippocampus has been suggested as a possible power of Alzheimer's treatment. In order to evaluate the effect of the bulbous extract on the proliferation of adult hippocampal neural stem cells in SGZ, immunofluorescence analysis of Ki67, a proliferation marker, was performed.

6 and A of FIG. 6 are the immunofluorescence images of Ki67 in SZG and the number of cells positive for Ki67 per unit length from the image, and the number of Ki67 positive cells was significantly increased compared to the control group by the bulbous extract. Show.

In addition, the effect of the bulbous extract on neuronal differentiation in adult hippocampal neurogenesis was confirmed by immunoassay for DCX, a marker of neuronal precursors in neurogenesis (C, D in FIG. 6). Compared to the wild type mouse (comparative group), the number of DCX positive cells in 5XFAD mice (control group) decreased to about 1/5 level, and the number of DCX positive cells in the experimental group increased by about 2 times compared to the control group by administration of the bulbous extract. Show.

This shows that Jogu et al extract has the effect of restoring the proliferation and differentiation of damaged neural stem cells of the adult hippocampus in the brain of Alzheimer's patients.

Claims (4)

A pharmaceutical composition for inhibiting amyloid beta deposition containing an extract of jogu etc. as an active ingredient.
According to claim 1,
Diseases associated with amyloid beta deposition: Alzheimer's disease, Down's syndrome, Creutzfeldt-Jakob disease, Prion diseases such as Gerstmann-Straussler syndrome, amyloid vasculosis, cerebral amyloid vasculosis, systemic amyloid disease, Dutch type Pharmaceutical composition for the prevention and treatment of diseases selected from the group consisting of amyloidosis, inclusion body stomatitis, reactive amyloidosis, idiopathic amyloidosis, familial amyloidosis, hereditary cerebral hemorrhage related to amyloidosis and brain amyloid angiopathy.
The method of claim 1 or 2,
Pharmaceutical composition characterized in that it further has the effect of preventing and treating neurodegeneration and neuroinflammation.
Health functional food composition for preventing and improving symptoms of amyloid beta deposition-related diseases containing crude bulb extract as an active ingredient.

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