JP6998212B2 - A β-secretase inhibitor containing turmeron as an active ingredient, and foods and drinks containing the inhibitor. - Google Patents

Description

The present invention relates to a β-secretase inhibitor containing a compound derived from the rhizome of turmeric as an active ingredient, and foods and drinks containing the inhibitor.

With the advent of the rapidly aging society in recent years, senile dementia has become a serious medical and social problem, and as a result, not only the development of effective anti-dementia drugs but also senile dementia The development of a drug that can effectively prevent dementia is strongly desired ( Non-Patent Document 1).

Histopathological studies suggest that amyloid plaques are deposited, which causes nerve cells to drop out and cause brain atrophy. Β-Amyloid, which is the main component of the amyloid plaque, has a cytotoxic effect (Non-Patent Documents 2 to 5). β-Amyloid is produced from amyloid precursor protein (APP) by the action of an enzyme called β-secretase . Research on compounds having an inhibitory effect on β -secretase has been actively conducted in recent years (Patent Document 1).

Such a compound having an inhibitory activity against β-secretase may inhibit not only the production of β-amyloid but also the reaction in other living organisms. Highly safe β-secretase inhibitors are required because other substances that inhibit the reaction in vivo may cause serious side effects to patients even if they inhibit the production of β-amyloid. Has been done.

As an example of searching for a highly safe β-secretase inhibitor, the invention described in Patent Document 2 by the present inventors can be mentioned.

In Patent Document 2, in order to obtain a highly safe β-secretase inhibitor, a β-secretase inhibitor is extracted from a natural product generally used as a spice such as pepper, sesame, and turmeric. Patent Document 2 further specifies a compound that exhibits β-secretase inhibitory activity among the compounds contained in the extract.

However, there is a demand for β-secretase inhibitors that have higher inhibitory activity and can be obtained at lower cost.

Japanese Unexamined Patent Publication No. 2008-137914 Japanese Unexamined Patent Publication No. 2013-189385 Special Table 2009-530305 Gazette Japanese Unexamined Patent Publication No. 2011-225478

Alzheimer, A (1907) Central Bl. Nervenheilk. Phychiatr. 30, 177-179 Yankner, B. et al. Science, 245, 417-429 (1989) Cai, X., Gold, T. & Younkin, S. Science, 259, 514-516 (1993) Rose, A. Nature Med. 2, 267-269 (1996) Scheuner, D. et al. Nature Med. 2, 864-869 (1996)

As another example of searching for a highly safe β-secretase inhibitor, the present inventors in Japanese Patent Application No. 2015-146208, sesamolin, ethyl 4-methoxycinnamate and pipataline extracted from sesame, galangal and long pepper, respectively. We are proposing foods and drinks having a β-secretase inhibitor containing the above as an active ingredient.
The invention described in Japanese Patent Application No. 2015-146208 specifies the above three compounds as compounds having β-secretase inhibitory activity, and shows specific inhibitory activity by calculating the IC50 value of each compound.
However, there is a demand for β-secretase inhibitors that have higher inhibitory activity and can be obtained at lower cost.

The present invention provides a β-secretase inhibitor having high safety and an even higher inhibitory activity on β-secretase activity, and foods and drinks containing the inhibitor.

The invention according to claim 1 comprises β-secretase inhibition that inhibits the activity of β-secretase, which comprises only one or more compounds selected from the group consisting of AR-termeron, α-termeron and β-termeron as an active ingredient. Regarding the agent.

The present invention also relates to the β-secretase inhibitor according to claim 1, wherein AR-turmeric, α-turmeric and β- turmeric are hexane extracts or ethyl acetate extracts of turmeric.

The invention according to claim 2 relates to the β-secretase inhibitor according to claim 1 , which comprises AR-termeron, α-termeron and β-termeron.

The invention according to claim 3 relates to a food or drink for inhibiting β-secretase, which comprises the β-secretase inhibitor according to claim 1 or 2 , and inhibits the activity of β-secretase.

According to the invention of claim 1, it is possible to have a higher inhibitory activity on β-secretase activity.

Further, according to the present invention, since AR-turmeric, α-turmeric and β-turmeric are hexane extracts or ethyl acetate extracts of turmeric (Curcuma longa), they are highly safe for the human body, and further, from turmeric. Since the method for extracting the compound is simple, it can be produced at low cost.

According to the invention of claim 2 , it is possible to have a higher inhibitory activity on β-secretase activity.

According to the invention of claim 3 , since it is a food or drink containing a β-secretase inhibitor having an excellent inhibitory effect on β-secretase activity, the β-secretase inhibitor can be easily ingested on a daily basis. ..

Hereinafter, the β-secretase inhibitor according to the present invention and foods and drinks containing the inhibitor will be described.

The β-secretase inhibitor of the present invention contains one or more compounds selected from the group consisting of AR-turmeron, α-termeron and β-termeron as an active ingredient.

The AR-turmeron has a CAS number 532-65-0 and has a structure represented by the following formula (formulation 1).

The α-turmeron has CAS No. 82508-15-4 and has a structure represented by the following formula (Chemical formula 2).

The β-turmeron has a CAS number 82508-14-3 and has a structure represented by the following formula (formulation 3).

In the present invention, standard products can be used as AR-termeron, α-termeron and β-termeron. Further, those obtained from an extract such as curry spice can be used, and in particular, those obtained from an extract of turmeric can be used.

Turmeric (English name: Turmeric, scientific name: Curcuma longa) is a monocotyledonous plant of the genus Turmeric in the family Zingiberaceae. It is a perennial plant native to India and has a well-developed rhizome. The rhizome is used as a raw material and dye for curry powder.

As the β-secretase inhibitor of the present invention, those containing AR turmeron, α-termeron and β-termeron as active ingredients can be used.
AR-turmeric, α-turmeric and β-turmeric are preferably extracted from the rhizome of turmeric by hexane. The reason is that by extracting the rhizome of turmeric with hexane, a large amount of AR-turmeric, α-turmeric and β-turmeric can be eluted.

The above compounds may be used alone or in admixture of two or more. As described in detail in the following examples, since the above compounds have strong β-secretase inhibitory activity, the β-secretase inhibitor of the present invention containing these as an active ingredient can be used for various purposes. can.

For example, when the β-secretase inhibitor of the present invention is used as a pharmaceutical preparation, it is used as a preventive agent for senile dementia in mammals (particularly humans), particularly as a preventive agent for Alzheimer-type dementia.

The β-secretase inhibitor of the present invention is a tablet, a powder, a fine granule, a granule, a coated tablet, a capsule, a syrup, a troche, an inhalant, a suppository, an injection, or an ointment by a conventional method. , Ophthalmic ointment, eye drops, nasal drops, ear drops, bops, lotions and the like can be formulated into pharmaceutical preparations.

Excipients, binders, lubricants, colorants, flavoring agents, and optionally stabilizers, emulsifiers, absorption enhancers, surfactants, pH regulators, preservatives, anti-formulations commonly used for formulation. An oxidant or the like can be used, and an ingredient and a blending amount generally used as a raw material for a pharmaceutical preparation are appropriately selected and formulated by a conventional method.

When the pharmaceutical product of the present invention is administered, its form is not particularly limited, and any commonly used method may be used, and oral administration or parenteral administration may be used. As the dose of the pharmaceutical preparation according to the present invention, a pharmaceuticalally effective amount can be appropriately selected according to the degree of symptoms, age, sex, body weight, administration form, specific type of disease and the like. As an example of the dose, in the case of oral administration, the amount of the active ingredient is usually about 0.001 to 1000 mg / kg in adults, and this can be administered once to several times a day. good.

The β-secretase inhibitor of the present invention can be used as a food additive for various foods and drinks such as soft drinks, dairy products (processed milk, yogurt), confectionery (jelly, chocolate, biscuits, gum, tablets) or supplements. It can also be blended with.

When used as a food additive, the amount of the additive is not particularly limited and may be appropriately determined according to the type of food. As an example, the above-mentioned extract may be added so that the dry weight of the extract is in the range of 0.0005 to 50% by weight.

In addition to the β-secretase inhibitor of the present invention, the above-mentioned food and drink contains excipients, taste agents, colorants, preservatives, thickeners, stabilizers, gelling agents, antioxidants and the like. May be good.
Of these, the excipient is not limited to these, for example, powders such as fine particle silicon dioxide, sucrose fatty acid ester, crystalline cellulose / sodium carboxymethyl cellulose, calcium hydrogen phosphate, wheat starch, rice starch, corn starch. , Potato starch, dextrin, cyclodextrin and other starches, crystalline celluloses, lactose, glucose, sugar, reduced malt sugar, water candy, fructo-oligosaccharides, galactooligosaccharides, soybean oligosaccharides, isomaltoligosaccharides, xylooligosaccharides, malto-oligosaccharides, milk fruits Examples thereof include sugars such as oligosaccharides and sugar alcohols such as sorbitol, eristol, xylitol, lactitol and mannitol. These excipients can be used alone or in combination of two or more.

The taste agent is not limited to these, for example, bontan extract, lychee extract, apple juice, orange juice, yuzu extract, peach flavor, ume flavor, sweetener Acesulfam K, eristol, oligosaccharide. , Mannose, xylitol, isomerized sugar, green tea, oolong tea, vanaba tea, tochu tea, iron Kannon tea, honeybee tea, amachazuru tea, makomo tea, kelp tea, yogurt flavor and the like.

Hereinafter, the effects of the present invention will be clarified by explaining the embodiments of the present invention. However, the present invention is not limited to the following examples.

(Preparation of hexane extract)
1 kg of turmeric rhizome was pulverized, stirred and extracted with 5000 ml of hexane at 40 ° C. for 1 hour, and 4200 ml of the supernatant was filtered through a filter paper.
Then, the residue was stirred and extracted with 2500 ml of hexane at 40 ° C. for 0.5 hours, and filtered through a filter paper. The solvent contained in the obtained extract was distilled off with an evaporator (40 ° C.) to obtain a turmeric hexane extract.

(Preparation of ethyl acetate extract)
The residue after hexane extraction of turmeric was dried, stirred and extracted with 5000 ml of ethyl acetate at 40 ° C. for 1 hour, and 4800 ml of the supernatant was filtered through a filter paper. The solvent contained in the obtained extract was distilled off by an evaporator (40 ° C.) to obtain an ethyl acetate extract of turmeric.
The yields of the hexane extract and the ethyl acetate extract are shown in Table 1 below.

(Measurement of β-secretase inhibition rate)
The enzyme inhibitory activity of the hexane extract and the ethyl acetate extract extracted as described above was examined by the following method. Also, Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe (Sta: (3S, 4S) -4-amino-3-hydroxy-6-methyl- Heptanoic acid; manufactured by Peptide Institute) was used as a positive control.
Specifically, first, for each extract and positive control, 2 μl of DMSO solution containing each is mixed with 78 μl of 0.02 M sodium acetate buffer (pH 4.5, 0.1% Triton X-100) in a 0.6 ml sample tube. I put it in. As a control, 2 μl DMSO was placed in a 0.6 ml sample tube with the 78 μl 0.02 M sodium acetate buffer described above.
Then, 10 μl of an enzyme solution (β-secretase, 17.4 μg protein / ml) was added and mixed, and precultured at 37 ° C. for 10 minutes.
Add 10 μl of MOCAc-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys (Dnp) -Arg-Arg-NH ₂ (manufactured by Peptide Institute) 0.1 mmol / l solution as a substrate. Was mixed and cultured at 37 ° C. for 1 hour. After the reaction, 50 μl of 2.5 M sodium acetate solution was added to stop the reaction.

100 μl of the reaction solution was added to a vial containing 900 μl of water and subjected to fluorescent HPLC analysis under the following conditions.
・ Column; L-column ODS (4.6 id x 250 mm)
・ Mobile phase; water / 0.1% formic acid: acetonitrile (9: 1 v / v) → 17.50 min (11: 9, v / v) → 17.51 min (1:19, v / v) → 22.50 min (1: 1: 19, v / v) → 22.51 min (9: 1, v / v) → 27.50 min (STOP)
・ Column temperature; 40 ℃
・ Detection; Ex. 325 nm / Em. 395 nm
・ Injection amount; 20 μl
From the peak area value of the generated fluorescent peptide fragment, the inhibition rate (%) was calculated from the following formula.
β-Secretase inhibition rate (%) = 100- [(Peak area value from each extract / Peak area value from control) x 100]

As a result of calculating the β-secretase inhibition rate of each hexane extract and ethyl acetate extract from the above calculation, the results were as follows.

From the above inhibition rate, it was found that the turmeric extract has a very high β-secretase inhibitory activity.

Hexane extracts were analyzed using GCMS under the following conditions.
-Column: Intertcap Pure Wax (0.25 id x 0.25 μm x 60 m)
・ Injection amount: 1.0 μl
・ Temperature rise; hold at 50 ℃ for 2 minutes, temperature rise at 2.5 ℃ / min → up to 240 ℃ ・ Vaporization room temperature; 250 ℃
・ Carry gas flow rate; helium 1.2 ml / min ・ split ratio; 1/80
From the results of GCMS analysis, the compounds were estimated to be AR-turmeron, α-termeron and β-turmeron by the attached library search (wiley7n and NIST08).
The hexane extract contained 9.9% AR-turmeron, 30.4% α-termeron, and 11.5% β-termeron.

The hexane extract was fractionated under the following HPLC conditions.
・ Column; L-column ODS (20 id x 250 mm)
・ Mobile phase; water: acetonitrile (20: 80, v / v)
・ Flow velocity: 18.9 ml / min
-Column temperature; room temperature-detection; UV 243 nm
・ Injection amount; 10 μl
Colorless oily substances were obtained from the fractions of RT7.5, 10.5 and 11.0 as the active ingredient of the hexane extract. Conventionally, α-turmeron and β-turmeron have been known as substances that are difficult to separate, but it is noteworthy that they could be extracted by the above-mentioned simple method.

The colorless oily substance was analyzed under the GCMS conditions described in the above paragraph [0050], and the compounds were obtained from the results using the attached library search (wiley7n and NIST08) and NMR analysis, respectively. And β-termeron (Hideji Itokawa, Fusayoshi Hirayama, Kazuko Funakoshi and Koichi Takeya Chem. Pharm.Bull.33 (8) 3488-3492 (1985) and Shin-ichi Uehara, Ichiro Yasuda, Koichi Takeya, It was confirmed by comparison with the literature values of Hideji Itokawa Yakugaku zasshi 112 (11), 817-823).

The enzyme inhibitory activity of the standard products of the compounds identified above (AR-termeron, α-termeron and β-termeron) was examined by the same method as above (measurement of β-secretase inhibition rate), and each standard product was examined. As a result of calculating the IC50 value, it was as follows.
AR-Turmeron: 92 μM
α-Termeron: 39 μM
β-Termeron: 62 μM
From the above IC50 value, it was found that AR-termeron, α-termeron and β-termeron have β-secretase inhibitory activity.
This IC50 value is the IC50 value of sesamolin, pipataline and ethyl 4-methoxysinamate (sesamolin: 0.14 mM, ethyl 4-methoxysinamate: 0) previously found by the present inventors and described in Japanese Patent Application No. 2015-146208. It was significantly lower than that of .676 mM, pipataline: 0.304 mM), and it was revealed that AR-turmeron, α-termeron and β-termeron have very high β-secretase inhibitory activity.
Further, since the extraction method of the present invention is simpler than the extraction method described in Japanese Patent Application No. 2015-140208, the production cost can be suppressed to 1/10 or less.

The present invention can be suitably used as an additive for various foods and drinks such as soft drinks , dairy products, confectionery or supplements.

Claims (3)

A β-secretase inhibitor that inhibits the activity of β-secretase and contains only one or more compounds selected from the group consisting of AR-turmeron, α-termeron and β-termeron as an active ingredient . The β-secretase inhibitor according to claim 1 , which comprises AR-termeron, α-termeron and β-termeron. A food or drink for inhibiting β-secretase that inhibits the activity of β-secretase, which comprises the β-secretase inhibitor according to claim 1 or 2 .