JP7089755B2 - Acetylcholinesterase inhibitor - Google Patents

Description

The present invention relates to an acetylcholinesterase inhibitor useful for preventing dementia by inhibiting the activity of acetylcholinesterase.

According to the 2017 White Paper on Aging Society of the Cabinet Office, the number of elderly people with dementia aged 65 and over and the future estimation of the prevalence are estimated to be 4.62 million elderly people with dementia in 2012, 65 years old. The number of elderly people mentioned above was about 1 in 7 (prevalence rate 15.0%), but in 2025, the number of patients was about 7 million, which is about the number of elderly people aged 65 and over. It is said that one in five people will be.

There are types of dementia such as Alzheimer's type, vascular, Lewy body type, and frontotemporal type, but the number of patients with Alzheimer's type dementia is the largest, accounting for more than 60% of the total.
In Alzheimer-type dementia, since acetylcholine in the brain is reduced, an inhibitor of acetylcholinesterase (hereinafter, may be abbreviated as "AChE") involved in the reduction of acetylcholine is used as a therapeutic agent.

Currently, three agents, donepezil hydrochloride, galantamine, and rivastigmine, are clinically used as AChE inhibitors.
Although each of these drugs has its own characteristics, there are also development issues such as drugs with fewer side effects and drugs with different inhibition sites.

On the other hand, research and development aimed at preventing dementia from daily eating habits are also underway, and there are research cases of the preventive effect of habitual intake of green tea. The relationship between the intake of green tea and the prevention of dementia has been studied from various viewpoints, and in recent years, it has been reported that green tea has an AChE inhibitory effect, and tea varieties with a high AChE inhibitory effect have been identified. (See Non-Patent Document 1.). As described above, as one approach to the prevention of dementia, continuous intake of AChE-inhibiting foods in daily eating habits has been considered, and such foods are currently being searched for.

Nippon Paper Industries, Ltd., "Confirmed the" effect of suppressing deterioration of cognitive function due to aging "of high-performance tea" Saint Rouge "in animal tests" [online], November 18, 2016, [August 6, 2018 Search by day], Internet <URL: https://www.nipponpapergroup.com/news/year/2016/news161118003566.html>

An object of the present invention is to provide a novel AChE inhibitor useful for dementia prevention by inhibiting the activity of acetylcholinesterase.

The present inventors have found an AChE inhibitory effect on chalcones, which are the main components of yellow juice exuded from the stems and root cuts of Ashitaba (Angelica keiskei), which is an endemic species of the genus Umbelliferae in Japan. As a result of close examination in the in vitro enzyme inhibition experiment, the 2', 4-dihydrochalcone skeleton plays a large role in the AChE inhibitory action, and the geranyl group at the 3'position and the alkoxy group at the 4'position enhance the effect. I understood.

The present invention has been completed based on the above findings.
That is, the AChE inhibitor according to the present invention contains xanthangelol F as an active ingredient.
The AChE inhibitor according to the present invention includes components other than xanthangelol F (for example, various chalcones in Angelica keiskei described below) as long as xanthangelol F is an essential active ingredient. ) May be included as an active ingredient of the AChE inhibitor.

The AChE inhibitor of the present invention is useful for preventing dementia by inhibiting the activity of acetylcholinesterase. In particular, since the active ingredient can be obtained from Angelica keiskei, it can be used as an AChE inhibitory food that can be continuously ingested in daily eating habits.
Among them, xanthangelol F exhibits a high AChE inhibitory action comparable to that of galantamine hydrobromide which is clinically applied.

Hereinafter, the AChE inhibitor according to the present invention will be described in detail, but the scope of the present invention is not limited to these explanations, and other than the following examples, changes are appropriately made as long as the gist of the present invention is not impaired. Can be.

The chalcones which are the active ingredients of the AChE inhibitor according to the present invention have a 2', 4-dihydroxychalcone skeleton represented by the following formula (1) as a basic skeleton.

The AChE inhibitor according to the present invention contains chalcones having a geranyl group at the 3'position and / or a lower alkoxy group at the 4'position in the basic skeleton as an active ingredient.

According to the results examined by the present inventor, the 2', 4-dihydroxychalcone skeleton plays a large role in the AChE inhibitory action, and the AChE inhibitory activity is enhanced by the geranyl group at the 3'position and the lower alkoxy group at the 4'position. It turned out that. It was found that extremely high AChE inhibitory activity is exhibited when both of these, that is, having a geranyl group at the 3'position and a lower alkoxy group at the 4'position.
As long as the chalcones have these characteristics, hydrogen on the benzene ring in the basic skeleton may be further substituted as long as the effect of the present invention is not impaired.

In the above, the lower alkoxy group at the 4'position is, for example, a linear or branched alkoxy group having 1 to 6 carbon atoms, and a methoxy group is preferable.

As chalcones satisfying the above conditions, 4-hydroxydelicin, xanthangelol, and xanthangelol F are preferably exemplified.

4-Hydroxydericin is represented by the following formula (2) and has a methoxy group at the 4'position in the chalcone skeleton.

Xanthangelol is represented by the following formula (3) and has a geranyl group at the 3'position in the chalcone skeleton.

Xanthangelol F is represented by the following formula (4) and has a geranyl group at the 3'position and a methoxy group at the 4'position in the chalcone skeleton. As described above, since it has both a geranyl group and a methoxy group at a predetermined substitution position, the AChE inhibitory activity is particularly high.

The chalcones in the present invention may be obtained by chemical synthesis or may be obtained from a natural product.
For example, it is known that Ashitaba contains various chalcones, including chalcones satisfying the conditions of the present invention. In particular, it contains a large amount of 4-hydroxydelicin and xanthangelol, and also contains xanthangelol F.

Therefore, the present invention also includes an acetylcholinesterase inhibitor containing chalcones in Ashitaba as an active ingredient.
Ashitaba (Ashitaba, Angelica keiskei) is a plant belonging to the genus Ashitaba in the Umbelliferae family. It is native to Japan and grows naturally from the Boso Peninsula to the Kii Peninsula and the Pacific coast of the Izu Islands.

When chalcones in Ashitaba are used as an active ingredient, whole plants may be used, or some of stems, roots, leaves, fruits, seeds, seed coats, flowers and the like may be used.
In addition, the stems, roots, and leaves of Ashitaba contain a large amount of yellow substances, and the characteristic is that yellow juice exudes from the fractured surface. This yellow juice contains a large amount of chalcones that satisfy the conditions of the present invention, and can be suitably used for the present invention.

The whole plant of Ashitaba, a part thereof or yellow juice may be used as it is, or may be processed and used. Examples of the processing treatment include grinding, drying, crushing, crushing, extraction, concentration and the like.

Specifically, for example, chalcones can be obtained by extracting with a solvent using raw materials such as whole herbs of Angelica keiskei, a part thereof, pulverized products thereof, and yellow juice.

The solvent used for the above extraction is not particularly limited as long as it is a solvent capable of extracting the target calcons, and examples thereof include water, methanol, ethanol, propanol, acetone, ethyl acetate, and methyl acetate. , Ethyl acetate is preferred. The extraction solvent may be used alone or in combination of two or more.

After extraction, filtration, centrifugation, concentration, etc. may be performed, if necessary.
Further, the extract may be separated and purified by a known purification method such as chromatography.
Further, the extract may be dried by a known method such as freeze-drying, vacuum drying, spray drying and the like to form a solid state.

The AChE inhibitor of the present invention may be used by either oral administration or parenteral administration, and may be used as a raw material for health foods (including beverages), food additives, supplements, pharmaceutical compositions and the like. can.
For example, whole herb of Ashitaba, a part thereof, crushed matter thereof, yellow juice, extract from them (liquid containing extraction solvent, concentrate thereof or solid form obtained by drying them, etc.), etc. Can be used as a raw material for health foods, food additives, supplements, pharmaceutical compositions and the like.

When the AChE inhibitor of the present invention is used as a raw material for various uses, it can be formulated in combination with a base material or a drug commonly used for various uses.

Hereinafter, the AChE inhibitor according to the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[Extraction of Ashitaba extract]
Yellow juice (1 kg) coming out from the fractured surface of the stem of Ashitaba was extracted three times with ethyl acetate (3 L) and then concentrated under reduced pressure to obtain an ethyl acetate extract (221.1 g) of Ashitaba.
The obtained extract was separated by column chromatography (n-hexane-ethyl acetate system) of silica gel (2 kg) to obtain a crude fraction of each component.
The obtained crude fraction was repeatedly purified by silica gel column chromatography (CH ₂ Cl ₂ -methanol type and n-hexane-ethyl acetate type) and octadecylated silica gel column chromatography (90% methanol) to obtain calcons. 4-Hydroxydelicin (12.8 g), xanthangelol (12.3 g), xanthangelol F (1.4 g), isobabacalcon (0.3 g), 4'-O-geranyl as flavanones Naringenin (1.8 g) and Prostrator F (0.3 g) were obtained.
For the obtained compounds, the structure of each compound was determined by analysis of spectral data including NMR.
The NMR spectrum was measured and confirmed using Agilent-NMR-vnml600. The spectral data of ¹ H-NMR and ¹³ C-NMR of each compound are shown below.

<4-Hydroxydericin>
As described above, 4-hydroxydelicin is represented by the following formula (2).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
¹ H-NMR (δppm, CDCl ₃ ): 13.5 (1H, s), 7.84 (1H, d, J = 15.4Hz), 7.80 (1H, d, J = 9.0Hz), 7.57 (2H, d, J = 8.5Hz), 7.48 (1H, d, J = 15.4Hz), 6.89 (2H, d, J = 8.5Hz), 6.50 (1H) , D, J = 9.0Hz), 5.78 (1H, brs), 5.23 (1H, tm, J = 7.6Hz), 3.92 (3H, s), 3.40 (2H, d) , J = 7.6Hz), 1.80 (3H, s), 1.69 (3H, s).
¹³ C-NMR (δppm, CDCl ₃ ): 193.4 (s), 164.0 (s), 163.5 (s), 159.2 (s), 145.0 (s), 132.6 ( s), 131.2 (d) x 2,129.9 (d), 127.8 (s), 122.5 (d), 118.3 (d), 118.0 (s), 116.6 (D) × 2,115.1 (s), 102.8 (d), 56.1 (q), 26.0 (q), 21.9 (q), 18.0 (q).

<Xant Angelol>
As described above, xanthangelol is represented by the following formula (3).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
¹ H-NMR (δppm, CDCl ₃ ): 13.81 (1H, s), 9.16 (1H, brs), 8.85 (1H, brs), 7.81 (1H, d, J = 15. 4Hz), 7.66 (1H, d, J = 8.8Hz), 7.52 (2H, d, J = 8.6Hz), 7.43 (1H, d, J = 15.4Hz), 6. 89 (2H, d, J = 8.6Hz), 6.47 (1H, d, J = 8.8Hz), 5.30 (1H, tm, J = 7.0Hz), 5.07 (1H, m) ), 3.41 (2H, d, J = 7.0Hz), 2.05 (2H, m), 2.01 (2H, m), 1.81 (3H, s), 1.65 (3H, s), 1.57 (3H, s).
¹³ C-NMR (δppm, CDCl ₃ ): 193.2 (s), 164.5 (s), 162.6 (s), 159.0 (s), 145.1 (d), 140.0 ( s), 132.6 (s), 131.2 (d) x 2,129.9 (d), 128.0 (s), 124.3 (d), 121.5 (d), 118.3 (D), 116.6 (d) × 2,114.8 (s), 114.4 (s), 108.6 (d), 40.0 (t), 26.6 (t), 25. 9 (q), 21.9 (t), 17.9 (q), 16.5 (q).

<Xant Angelol F>
As described above, xanthangelol F is represented by the following formula (4).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
¹ H-NMR (δppm, CDCl ₃ ): 13.50 (1H, s), 7.82 (1H, d, J = 15.4Hz), 7.78 (1H, d, J = 9.1Hz), 7.52 (2H, d, J = 8.6Hz), 7.45 (1H, d, J = 15.4Hz), 6.87 (2H, d, J = 8.6Hz), 6.49 (1H) , D, J = 9.1Hz), 6.10 (1H, brs), 5.24 (1H, tm, J = 6.9Hz), 5.07 (1H, m), 3.90 (3H, s) ), 3.40 (2H, d, J = 6.9Hz), 2.05 (2H, m), 2.01 (2H, m), 1.79 (3H, s), 1.63 (3H, s), 1.56 (3H, s).
¹³ C-NMR (δppm, CDCl ₃ ): 193.3 (s), 164.0 (s), 163.6 (s), 158.9 (s), 144.8 (d), 136.0 ( s), 131.7 (s), 131.1 (d) x 2,129.8 (d), 128.1 (s), 125.0 (d), 122.4 (d), 118.5 (D), 118.5 (s), 116.5 (d) × 2,115.1 (s), 102.7 (d), 56.2 (q), 40.1 (t), 26. 9 (t), 25.9 (q), 21.9 (t), 17.9 (q), 16.3 (q).

<Isobaba chalcone>
Isobaba chalcone is represented by the following equation (5).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
^{1 1} H-NMR (δppm, CDCl ₃ ): 13.86 (1H, s), 7.84 (1H, d, J = 15.2Hz), 7.73 (1H, d, J = 8.9Hz), 7.56 (2H, d, J = 8.7Hz), 7.46 (1H, d, J = 15.2Hz), 6.88 (2H, d, J = 8.7Hz), 6.42 (1H) , Brs), 5.51 (1H, brass), 5.30 (1H, tm, J = 7.1Hz), 3.48 (2H, d, J = 7.1Hz), 1.84 (3H, d) , J = 1.5Hz), 1.77 (3H, d, J = 1.5Hz).
¹³ C-NMR (δppm, CDCl ₃ ): 192.2 (s), 163.9 (s), 161.5 (s), 158.0 (s), 144.0 (d), 135.9 ( s), 130.5 (d) x 2,129.2 (d), 127.7 (s), 121.1 (d), 118.1 (d), 116.0 (d) x 2,114 .1 (s), 114.0 (s), 107.7 (d), 25.8 (q), 21.8 (t), 17.9 (q).

<4'-O-geranyl naringenin>
4'-O-geranyl naringenin is expressed by the following formula (6).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
^{1 1} H-NMR (δppm, CDCl ₃ ): 12.04 (1H, s), 7.36 (2H, d, J = 8.8Hz), 6.96 (2H, d, J = 8.8Hz), 6.24 (1H, brass), 6.00 (1H, d, J = 2.2Hz), 5.97 (1H, d, J = 2.2Hz), 5.49 (1H, tm, J = 6) .6Hz), 5.36 (1H, dd, J = 13.0, 3.1Hz), 5.09 (1H, tm, J = 6.9Hz), 4.56 (1H, d, J = 6. 6Hz), 3.10 (1H, dd, J = 17.2, 13.0Hz), 2.78 (1H, dd, J = 17.2, 3.1Hz), 2.06-2.16 (2H) , M), 1.74 (3H, d, J = 1.4Hz), 1.68 (3H, d, J = 1.4Hz), 1.60 (3H, d, J = 1.4Hz).
¹³ C-NMR (δppm, CDCl ₃ ): 196.2 (s), 164.7 (s), 164.3 (s), 163.3 (s), 159.3 (s), 141.6 ( s), 131.8 (s), 130.1 (s), 127.7 (d) x 2,123.7 (d), 119.1 (d), 115.0 (d) x 2,103 .1 (s), 96.7 (d), 95.5 (d), 79.0 (d), 65.0 (t), 43.0 (t), 39.5 (t), 26. 3 (t), 25.7 (q), 17.7 (q), 16.7 (q).

<Prostrator F>
Prostrator F is expressed by the following equation (7).

The spectral data of ¹ H-NMR and ¹³ C-NMR are as follows.
^{1 1} H-NMR (δppm, CDCl ₃ ): 7.45 (1H, d, J = 8.8Hz), 7.31 (2H, d, J = 8.5Hz), 6.90 (2H, d, J) = 8.5Hz), 6.79 (1H, brass), 6.56 (1H, d, J = 8.8Hz), 6.38 (1H, brass), 5.39 (1H, dd, J = 12) 9.9, 2.9Hz), 5.24 (1H, tm, J = 7.0Hz), 5.04 (1H, tm, J = 6.9Hz), 3.42 (2H, d, J = 7. 0Hz), 3.02 (1H, dd, J = 17.0, 12.9Hz), 2.83 (1H, dd, J = 17.0, 2.9Hz), 2.07 (1H, m), 2.01 (1H, m), 1.71 (3H, d, J = 1.2Hz), 1.65 (3H, d, J = 1.2Hz), 1.57 (3H, d, J = 1) .2Hz).
¹³ C-NMR (δppm, CDCl ₃ ): 192.7 (s), 162.1 (s), 161.1 (s), 156.2 (s), 138.7 (s), 131.9 ( s), 130.8 (s), 127.7 (d) x 2,126.5 (d), 123.8 (d), 120.9 (d), 115.6 (d) x 2,114 .8 (s), 114.6 (s), 110.8 (d), 79.3 (d), 43.7 (t), 39.7 (t), 26.4 (d), 25. 6 (q), 22.2 (t), 17.7 (q), 16.2 (q).

[Measurement of AChE inhibitory activity]
The AChE inhibitory activity of each sample was measured using 4-hydroxydelicin, xanthangelol, xanthangelol F, isobabacalcon, 4'-O-geranylnaringenin, and prostratol F obtained above as samples. The negative control sample was dimethyl sulfoxide (DMSO), and the positive control sample was galantamine hydrobromide. As other reference data, the AChE inhibitory activity was also measured for naringenin (purchased from LKT Laboratories, Inc.) and geraniol (purchased from Wako Pure Chemical Industries, Ltd.).

The specific measurement method is shown below.
The sample was dissolved in dimethyl sulfoxide (DMSO). As the buffer solution for the reaction, 50 mM Tris-hydrochloric acid buffer solution (pH = 7.8) was used.
180 μl of the buffer solution was placed in a 96-well microplate, and 5 μl of the sample was added thereto. Further, 10 μl of electrophorus-derived acetylcholinesterase (2units / ml) prepared with a buffer solution and 10 μl of 1-naphthyl acetate (18 mM) dissolved in DMSO were added, and the mixture was reacted at 37 ° C. for 1 hour.
After the reaction, 25 μl of a 5% sodium dodecyl sulfate aqueous solution was added to stop the reaction, and then 25 μl of a 2 mM Fast Blue B aqueous solution was added, and the absorbance at 595 nm was measured with a microplate reader.
The inhibition rate (%) was calculated by the following formula.
Inhibition rate (%) = 100-{[(absorbance with sample / enzyme)-(absorbance with sample / without enzyme)] / [(absorbance with negative control sample / enzyme)-(absorbance with negative control sample / enzyme) Absorbance without)] × 100}

<Results and discussion>
The results of the above measurements are shown in Table 1 below.

From the above test results, it can be seen that the 2', 4-dihydrochalcone skeleton has a great influence on the inhibitory activity.
However, looking at the results of isovaba chalcone, it can be seen that sufficient inhibitory activity cannot be obtained just by having a 2', 4-dihydrochalcone skeleton.
That is, as shown in the results of 4-hydroxydelicin, xanthangelol, and xanthangelol F, it has a 2', 4-dihydrochalcone skeleton and a geranyl group at the 3'position or a 4'position. The effect is enhanced by having the methoxy group of. And, especially by satisfying both elements at the same time, the effect is further enhanced.
Xanthangelol F, which has a geranyl group at the 3'position and a methoxy group at the 4'position and exhibits the highest inhibitory activity, had a strong inhibitory effect comparable to that of galantamine hydrobromide which is clinically applied. ..

Claims (1)

An acetylcholinesterase inhibitor containing xanthangelol F as an active ingredient.