JP7333626B2 - Composition for preventing and treating Alzheimer's dementia, composition for reducing amyloid β oligomer neurotoxicity - Google Patents

Description

Application of Patent Act Article 30, Paragraph 2 August 3, 2019 Website address https://content. iospress. com/articles/journal-of-alzheimers-disease/jad190098 JOURNAL OF ALZHEIMER'S DISEASE - VOLUME 70, ISSUE 3 935-952, 2019 1

TECHNICAL FIELD The present invention relates to a composition for preventing and treating Alzheimer's dementia and a composition for reducing amyloid β oligomer neurotoxicity.

Alzheimer's dementia is a type of dementia that causes cognitive impairment such as memory impairment and learning impairment, and is the most common disease affecting elderly people. In addition, before the onset of the disease, there is a prodromal period called mild cognitive impairment (MCI), so it is considered important to prevent the transition from MCI to dementia.

Although the cause of developing Alzheimer's dementia is partially unknown, in the human brain, amyloid beta (hereinafter referred to as "Aβ") forms soluble aggregates (hereinafter referred to as "Aβ oligomers", "AβO"). , further aggregate as fibrils and deposit as senile plaques. Accumulation of Aβ , especially Aβ oligomers, causes abnormalities in the microtubule-associated protein tau, neuropathies such as synaptic disorders, and inflammatory reactions, which are thought to cause dementia by causing neurodegeneration. In addition, it is known that Aβ is already accumulated in the stage of MCI against the background of Alzheimer's disease.

Therefore, various measures have been developed to prevent the onset of Alzheimer's dementia. and (3) removing and degrading accumulated Aβ.

Regarding the above technologies, for example, Patent Document 1 below discloses a method for inhibiting amyloid beta protein production, Patent Document 2 below discloses a technique related to a phenol derivative having an inhibitory effect on amyloid beta aggregation, and Patent Document 3 below discloses a method for inhibiting amyloid beta protein production. each disclose techniques relating to specific antibodies for promoting the elimination of amyloid beta.

Japanese translation of PCT publication No. 2007-533989 Japanese Patent Application Laid-Open No. 2008-231102 JP 2012-24098 A

By the way, AβO itself is known to cause neurotoxicity, and no report has yet been made on candidate compounds for preventive/therapeutic drugs based on reducing this. If the neurotoxicity of AβO itself can be reduced, it will be possible to realize more effective prophylaxis and treatment of Alzheimer's disease in combination with other therapeutic methods shown in the above literature.

Accordingly, in view of the above problems, an object of the present invention is to provide a composition having an AβO-induced neurotoxicity-reducing effect and a composition for the prevention and treatment of Alzheimer's disease .

The present inventors have searched a wide variety of compounds for compounds having the above effects, and discovered that a certain plant extract can reduce amyloid β oligomer neurotoxicity. The inventors have identified a specific compound that is the basis of the present invention and have completed the present invention.

That is, a composition for preventing/treating Alzheimer's disease according to one aspect of the present invention contains at least one of tyrosol, derivatives thereof, and salts thereof as active ingredients.

A composition for reducing amyloid β oligomer neurotoxicity according to another aspect of the present invention contains at least one of tyrosol, derivatives thereof, and salts thereof as active ingredients.

As described above, the present invention can provide a composition having an effect of reducing AβO- induced neurotoxicity and a composition for preventing and treating Alzheimer's dementia .

A brief description of the figures in the experimental examples is as follows.
FIG. 4 shows the results of Western blotting of plant extracts in Experimental Examples. FIG. 2 is a diagram showing the results of Western blotting on various compounds contained in the plant koukateen in Experimental Examples. FIG. 10 is a diagram showing the results of Western blotting when the amount of tyrosol was varied in Experimental Examples. FIG. 2 shows the results of confirming the influence of tyrosol on Aβ42 aggregation using ThT assay in experimental examples. FIG. 2 is a diagram for explaining treatment of AD mice and administration of tyrosol in experimental examples. FIG. 3 is a diagram showing the results of measuring body weight changes in each mouse after the start of tyrosol administration in Experimental Examples. It is a figure which shows the result of the Barnes maze test in an experimental example. The results of confirmation of Aβ deposition in the hippocampus (upper) and cerebral cortex (lower) of Tg mice (Tg-Veh) given only water for 20 weeks and Tg mice (Tg-Tyr) given water and tyrosol in the experimental example are shown. It is a photographic view showing. FIG. 10 is a diagram showing the results of quantitative determination of the area occupied by Aβ plaques at each position in the above diagram. FIG. 3 is a diagram showing the results of quantitative analysis of the amount of accumulation of Aβ40 and Aβ42 in Aβ plaques in Experimental Examples. FIG. 4 is a diagram showing regions of interest in the hippocampus of mice in experimental examples. FIG. 10 shows the results of immunostaining of the hippocampus of each mouse at 20 weeks with a spinophilin antibody in Experimental Examples. FIG. 10 shows quantitative data of spinophilin intensity at 12 weeks and 20 weeks in Experimental Examples. FIG. 10 shows the results of immunostaining with 4-HNE antibody in the hippocampal CA3 region at 20 weeks in Experimental Examples. FIG. 10 is a diagram showing quantitative results of 4-HNE intensity in the hippocampal CA3 region at 12 and 20 weeks in Experimental Examples. FIG. 10 shows immunostaining with hexanoyllysine adduct (HEL) antibody of neurons treated with Aβ oligomers or Aβ oligomers and tyrosol in Experimental Examples.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, the present invention can be embodied in many different forms, and is not limited to the specific exemplifications described in the embodiments and examples given below.

(Composition)
First, the composition for prevention and treatment of Alzheimer's disease dementia according to the present embodiment (hereinafter referred to as "the present composition") contains at least one of tyrosol, derivatives thereof, and salts thereof as active ingredients. Since the preventive/therapeutic composition has an effect of reducing amyloid β oligomer neurotoxicity, it is also a composition for reducing amyloid β oligomer neurotoxicity.

In the present composition, "tyrosol" refers to a compound represented by the following formula. This compound is an active ingredient that exerts the effects of the present composition, and has been confirmed by experimental examples described later. can be effective in both prevention and treatment of Alzheimer 's dementia .

In addition, the term "derivative" as used in the present composition refers to a compound in which a part of the structure of tyrosol is partially modified within a pharmaceutically acceptable range, and which is capable of obtaining an effect equivalent to that of tyrosol. However, an ester in which tyrosol and an acid are combined, a substituted compound in which hydrogen in tyrosol is replaced with another substituent, and the like can be exemplified. Examples of acids in esters include formic acid, acetic acid, propionic acid, and butyric acid, but are not limited thereto.

In addition, the "salt" in the present composition refers to a salt obtained by reacting tyrosol or a tyrosol derivative with a base, which is pharmaceutically acceptable and can provide effects equivalent to those of the present composition. Examples include sodium salts, potassium salts, calcium salts, magnesium salts and the like.

In addition, the present composition can be used for at least one of prevention and treatment as described above, and in addition to being in the form of a medicine (medicine), it can be in the form of a food (functional food). can.

When the present composition is a pharmaceutical, prevention or treatment can be achieved by administering it to patients with MCI already having Alzheimer's disease background or those who are at high risk of becoming patients in the future.

In addition, when the present composition is a pharmaceutical, the administration method is not limited as long as it can exhibit its effect, and it may be in the form of oral medicine such as tablets, capsules, granules, syrups, etc. It may be in any form, such as a drug, an external medicine such as an ointment, or an injection such as a liquid. That is, the present composition can be administered orally, injected, sublingually, cutaneously, and the like.

In addition, when the present composition is a pharmaceutical, from the viewpoint of facilitating the production of the composition in the above form, stabilizing the quality, or enhancing the effect, depending on the form of the pharmaceutical, excipients , stabilizers, preservatives, buffers, suspending agents, emulsifiers, coloring agents, flavoring agents, viscosity modifiers, and other ingredients other than the above active ingredients, so-called pharmaceutical additives. Of course, it is also possible to add a solvent such as water or physiological saline to the extent that the quality, effects, etc. of the present composition are not changed.

Also, when the present composition is a food, it can be used for the prevention and treatment (improvement) of Alzheimer 's dementia , as in the case of the pharmaceuticals. Here, "food" means beverages such as tea, coffee, lactic acid beverages, milk, etc., meats, seafood, eggs, grains, beans, potatoes, vegetables, seaweeds, fruits and other natural ingredients. Fermented foods such as yogurt and natto, processed foods made from natural ingredients, oils and fats including edible oils such as salad oil, olive oil, soybean oil and corn oil, seasonings such as soy sauce and miso, etc. It can be exemplified and is not limited as long as it can be ingested by humans. The food also naturally includes a so-called supplement form in which the above-mentioned active ingredients are mixed with at least one of the above-mentioned food powders, concentrates, excipients, and the like. Moreover, of course, when extracting from raw materials as an extract, the extract itself can also be said to be a food composition.

In addition, the dosage of the active ingredient of the present composition can vary depending on whether the composition is in the form of a drug or food, and can be adjusted as appropriate according to the symptoms, body weight, etc. of the subject of administration. Although not limited, it is preferably 0.5 mg or more, more preferably 1.0 mg or more per 1 kg of body weight per day. By setting it as this range, it becomes possible to exhibit the effect of this composition.

In addition, tyrosol, derivatives thereof and salts thereof (hereinafter referred to as "tyrosol etc.") in the present composition can be produced synthetically, but can also be obtained by extraction from plants containing them. In this case, the type of plant is not limited as long as it contains tyrosol or the like. , Sinopodophyllum emodi (WALL.) YING, Rhodiola crenulata (HOOK.f.et Thoms.) H.; OHBA, Rhodiola sachalinensis, Olea europaea, Engelhardia roxburghiana, Fraxinus americana and the like can be exemplified.

Moreover, when tyrosol or the like is extracted from plants, the extraction method is not particularly limited, and can be carried out according to a known method. As an extracting solvent that can be used for this extraction, water, an organic solvent such as alcohol, acetone, or hexane, or a mixture of at least two of these can be suitably used. The alcohol is not limited, but can be exemplified by, for example, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, etc. From the viewpoint of human consumption, ethanol is more preferable. . In the case of a mixture of water and ethanol, it is more preferable to use hydrous ethanol with an ethanol content of 5 wt % or more and 95 wt % or less, more preferably 10 wt % or more and 90 wt % or less.

As described above, the present composition can exhibit effects as a composition having an effect of reducing neurotoxicity due to AβO and as a composition for preventing and treating Alzheimer's dementia . In particular, since the present composition contains tyrosol and the like as active ingredients, it can be easily administered orally. In addition, as described above, the mechanism is different from that of known compositions for the prevention and treatment of Alzheimer's dementia , so that it can be used in combination with other compositions, and a synergistic effect can be expected. In addition, the present composition has a relatively simple chemical structure, is easy to produce, and is plant-derived, so it can be said to be highly safe. Furthermore, tyrosol has a relatively low molecular weight and is easily transferred to the hindbrain when it is taken into the body, and high effects can be expected.

Here, the effects of the compositions referred to in the above embodiments will be described based on experiments that were actually conducted.

(extract)
First, the rhizome of Rhodiola rosea was extracted with water at 80°C and centrifuged to obtain an extract.

(Identification of active ingredient)
Next, the primary cultured neurons on the ninth day of culture were allowed to stand for 3 days with nothing added, only 2.5 μM AβO added, or this AβO and the plant extract added, Evaluation was performed by Western blot using an activated caspase-3 antibody. In this experiment, AβO was prepared using synthetic Aβ42 by the previously reported method, diluted with culture medium, and then used. The results are shown in FIG. In the figure, C indicates the results when nothing was added, O indicates the results when only AβO was added, and E1 indicates the results when AβO and an extract (10 μg/ml) of koukeiten were added. Activated caspase-3 was used as an indicator of apoptosis activation.

According to this result, the expression level of activated caspase-3 could be significantly decreased in the state where the extract of koukeiten was added. In other words, it was confirmed that the extract of koukeiten may have the effect of reducing neurotoxicity.

Next, Tyrosol, Salidroside, Rosavin, Rosarin, Rosin, and Rosiridin, which are candidate compounds contained in the extract of koukeiten, were each set to 5 μM, and the above A similar experiment was conducted. The results are shown in FIG. In the figure, T is tyrosol, S is salidroside, R1 is rosavin, R2 is rosaline, R3 is rosin, and R4 is the case where rosylidine was added.

According to this result, it was confirmed that tyrosol significantly decreased the expression level of activated caspase-3, and it was considered that tyrosol in the Koukiten extract was mainly responsible for this effect.

Further, experiments similar to those described above were conducted with different concentrations of tyrosol. Specifically, FIG. 3 shows the results obtained when the concentration of tyrosol was 5 μM and 10 μM. In the figure, T5 indicates the results when tyrosol was added at 5 μM, and T10 indicates the results when tyrosol was added at 10 μM.

As a result, it was confirmed that the expression level of activated caspase-3 was lowered at any amount of addition. This effect can be confirmed even at 20 μM, and furthermore, it has been confirmed that no toxicity is exhibited up to at least 200 μM (not shown).

(Effect of compound on Aβ aggregation)
Thioflavin T (ThT) assay was then used to confirm the effect of tyrosol on Aβ42 aggregation. The results are shown in FIG.
In the figure, the horizontal axis indicates time and the vertical axis indicates luminescence intensity. 50 μM Aβ plus 10 μM tyrosol, Aβ+T25 is 50 μM Aβ plus 25 μM tyrosol, Aβ+T50 is 50 μM Aβ plus 50 μM tyrosol, and Aβ+T100 is 50 μM Aβ plus 100 μM tyrosol. there is

As a result, after 72 hours, it was confirmed that EGCG significantly inhibited aggregation, but it was confirmed that tyrosol added did not have a significant aggregation inhibitory effect. That is, it was confirmed that the above-mentioned effect of tyrosol is not mainly from the viewpoint of suppressing Aβ aggregation.

(AD model mouse)
Next, the efficacy of tyrosol was confirmed using AD (Alzheimer's Disease) model mice (5XFAD) transgenic (Tg) mice) and wild-type mice (Non mice) that actually developed Alzheimer's disease. Ta. 5XFAD mice, which overexpress mutated amyloid β precursor protein and mutated presenilin 1, were obtained from MMRRC in the United States and maintained by mating with C57BL/6 mice.

Therefore, first, to each of Tg mice and Non mice, a group that was administered only water (Tg-Veh mice, Non-Veh mice), a group that was given water and tyrosol (dose 12.5 mg / kg / day) (Tg -Tyr mice, Non-Tyr mice). For the time of giving water and tyrosol, preparations were made separately for two months to 20 weeks after birth (until 7 months after birth) and for 4 months to 12 weeks after birth (until 7 months after birth). It should be noted that around 3 months after birth is the time when Aβ plaques appear due to aggregation of Aβ. An image diagram in this case is shown in FIG. In this case, tyrosol was administered to mice by adding tyrosol-containing water (25 μM) to their water-fed stools and freely drinking the water.

(Safety confirmation of Tyrosol)
FIG. 6 shows the results of measuring body weight changes in each mouse after the start of administration of tyrosol. According to this result, almost no difference was observed between the administration of water alone and the administration of tyrosol. The values of albumin, urea nitrogen, and alanine aminotransferase (ALT) were also confirmed, but no difference was observed between the Tg mice and the Non mice. As a result, the toxicity of tyrosol itself could not be confirmed, and the safety could be confirmed within the range of administration.

(Barnes maze test)
Here, the Barnes maze test was performed to evaluate the spatial memory of the above mice. This result is shown in FIG. According to this test, withdrawal latencies of Tg mice fed water only or water and tyrosol for 12 weeks were longer than those of Non-Veh mice on days 2 and 3 of the 5 training days, but on day 5 It was confirmed that the escape latency of the Tg-Veh mice was significantly longer than that of the non-Veh mice, but there was no significant difference between the Tg-Tyr mice and the non-Veh mice. Further, when a paired 2-way ANOVA was performed on the Tg-Veh and Tg-Tyr mice, it was confirmed that the Tg-Tyr mice had significantly and slightly improved spatial cognition function. In Non mice, no difference was observed between the case where only water was given and the case where tyrosol was also given.

On the other hand, in Tg mice given water alone or water and tyrosol for 20 weeks, the escape latency of Tg-Veh mice was significantly longer than that of Non-Veh mice on the 4th and 5th days, while Tg-Tyr mice had non- It was confirmed that there was no significant difference from Veh. In addition, when a paired 2-way ANOVA was performed on the Tg-Veh and Tg-Tyr mice, there was a significant difference between the two groups, and the spatial cognitive function was significantly improved in the Tg-Tyr mice. confirmed that there is

In this case, a probe test was performed on the 6th day. Tg-Tyr mice spent more time near the hole in the quadrant with the target hole than in the other quadrants, whereas Tg-Veh mice did not. . (illustration omitted)

(accumulation of Aβ)
By the way, the accumulation of Aβ in AD mice was confirmed by immunohistochemical and biochemical analyses. FIG. 8 Immunization of Aβ accumulation in the hippocampus (upper) and cerebral cortex (lower) of Tg mice fed water only (Tg-Ver) for 20 weeks, Tg mice fed water and tyrosol (Tg-Tyr) for 20 weeks. A histochemical analysis is shown. In addition, FIG. 9 shows the quantitative determination of the amount of Aβ accumulation at each position. As a result, Aβ plaques were confirmed in all mice. Note that in this case, mice given tyrosol for 12 weeks had no significant difference in Aβ plaques compared to mice given water only, whereas mice given tyrosol for 20 weeks showed no significant difference compared to mice given water only. There weren't many differences. In addition, as shown in FIG. 10, Aβ plaques were further analyzed by ELISA for the amount of accumulation of Aβ40 and Aβ42. No significant difference was observed. This result also confirmed that tyrosol did not affect the accumulation of Aβ. Moreover, as a result of Western blotting of cerebral cortex extracts, the levels of amyloid β precursor protein were similar between the above two groups of mice. (illustration omitted)

(synaptic abnormality)
In addition, it has been reported that the main effect of AβO neurotoxicity is synaptic toxicity, and synaptic function and structure are disturbed in AD model mice. Therefore, to investigate whether tyrosol exerts a beneficial effect on synaptic abnormalities, we focused on spinophilin, a scaffolding protein for dendritic synapses. The results are shown in FIGS. 11-13. FIG. 11 shows the region of interest in the mouse hippocampus, and FIG. 12 shows the results of immunostaining of the hippocampus of each mouse at 20 weeks. FIG. 13 also shows quantitative data of spinophilin intensity at 12 weeks and 20 weeks respectively.

Hippocampal immunostaining with this anti-spinophilin confirmed positive signals in the CA3, CA1, and hilar regions of the dentate gyrus (DG). Spinophilin intensity in these regions was reduced in Tg-Veh mice compared to Non-Veh mice, but appeared to return to control levels in Tg-Tyr mice. Quantitative analysis also confirmed that the spinophylline intensity was reduced by about 30% in Tg-Veh mice, but the intensity was almost the same in Non-Veh mice and Tg-Tyr mice. This result was similar in both 12-week-old mice and 20-week-old mice. In addition, there was no difference in the above spinophilin immune reaction intensity between Non-Veh mice and Non-Tyr mice. (illustration omitted)

(Oxidative stress response)
AβO is also known to induce oxidative stress. Therefore, to gain insight into the mechanisms underlying the protective effects of tyrosol on synaptic damage, immunostaining with anti-4-HNE revealed that tyrosol administration on 4-HNE, a well-known marker of the oxidative stress response. analyzed the effects. The results are shown in FIGS. 14 and 15. FIG. FIG. 14 shows the results of 4-HNE immunostaining in the hippocampal CA3 region, and FIG. 15 shows the quantitative results.

As a result, relatively strong punctate immunoreactive signals could be confirmed in neurons in the hippocampal CA3 region. In particular, double staining with anti-4-HNE and DAPI shows that 4-HNE immunoreactive signals are present around neurons. Interestingly, the intensity of 4-HNE immunoreactivity was stronger in Tg-Veh mice than in Non-Veh mice.

Quantitative data also showed that the intensity of 4-HNE immunoreactivity in the CA3 region was significantly increased by 15-20% in Tg-Veh mice compared to Non-Veh mice, whereas Tg- Tyr mice recovered to the level of Non-Veh mice in both the 12-week and 20-week groups. In addition, there was no difference in the above 4-HNE immune reaction intensity between Non-Veh mice and Non-Tyr mice. (illustration omitted)

Here, in order to investigate whether tyrosol can prevent AβO-induced oxidative stress, an antibody against hexanoyllysine adduct (HEL), which is known to be a marker of oxidative damage due to lipid peroxidation, was used. Immunocytochemical analysis was performed. The results showed that HEL immunoreactivity was present in the soma and proximal part of the neurites and was stronger in AβO-treated neurons than in controls, reflecting the induction of oxidative stress by AβO, as shown in FIG. confirmed. In contrast, however, the intensity of neurons co-treated with AβO and tyrosol (5 μM) was similar to controls, so an antioxidant effect of tyrosol could be inferred.

(Other results)
At concentrations of 5 to 10 μM, tyrosol did not affect the amount of Aβ40 and Aβ42 secreted from primary cultured neurons (not shown), suggesting that tyrosol does not affect Aβ production in neurons. .

From the above results, it was confirmed that tyrosol, one of the main components of the plant Rhodiola rosea, can be a drug that protects neurons from AβO neurotoxicity. Long-term oral administration of tyrosol to AD model mice can moderate cognitive deficits and significantly reverse synaptic impairment and oxidative responses without affecting Aβ accumulation. These results collectively suggest that the natural agent tyrosol is a safe, effective and unique drug candidate for AD with a mechanism of action distinct from that of existing drugs of inhibiting Aβ accumulation. ing.

INDUSTRIAL APPLICABILITY The present invention has industrial applicability as a composition for preventing and treating Alzheimer's disease and a composition for reducing amyloid β oligomer neurotoxicity.

Claims (2)

An amyloid β oligomer neurotoxicity-reducing agent containing at least one of tyrosol and a salt thereof as an active ingredient. A composition for reducing amyloid β oligomer neurotoxicity, comprising a plant extract containing tyrosol as an active ingredient.