JP2023143827A - Novel cyclic compound, angiotensin-converting enzyme inhibitor, method for producing the same, and applications thereof - Google Patents

Abstract

To provide an angiotensin-converting enzyme inhibitor including a novel cyclic compound, which has low environmental load, can be produced at low cost, and is highly safe and usable in pharmaceuticals, functional foods and the like, a method for producing the same, and applications thereof.SOLUTION: For example, the following compound 3153A or 3154A, or salts thereof or solvates thereof are illustrated.SELECTED DRAWING: Figure 1

Description

There is an application for exception to loss of novelty.

The present invention relates to angiotensin-converting enzyme inhibitors, and particularly relates to safe and inexpensive angiotensin-converting enzyme inhibitors, methods for producing the same, and uses thereof, and novel cyclic compounds thereof.

Lifestyle-related diseases are one of the serious problems in modern society. Among these, high blood pressure is a risk factor for serious diseases such as myocardial infarction and cerebral infarction, and depending on the condition, it can be life-threatening.

Angiotensin converting enzyme (ACE), which is one of the endogenous factors involved in blood pressure regulation, is an enzyme that produces angiotensin II, a physiologically active peptide that exhibits a pressor effect. Furthermore, it is involved in the decomposition of the bioactive peptide bradykinin, which has antihypertensive effects, so if ACE activity can be inhibited, it will lead to a reduction in blood pressure.

The drugs currently mainly prescribed as inhibitors of ACE activity (angiotensin-converting enzyme inhibitors) are synthetic compounds such as Captopril. However, synthetic compounds generally have safety issues, and pharmaceuticals such as captopril are manufactured using petroleum as raw materials, which places a large burden on the environment.

From this point of view, sesame protease degradation products derived from natural products have been proposed as conventional angiotensin-converting enzyme inhibitors (see Patent Document 1). In addition, elastase degradation products of elastin, which is a fibrous protein that mainly binds collagen, have been proposed as conventional angiotensin-converting enzyme inhibitors (see Patent Document 2).

Japanese Patent Application Publication No. 7-069922 Japanese Patent Application Publication No. 2012-056879

However, in conventional angiotensin-converting enzyme inhibitors, as in Patent Documents 1 and 2, the raw material proteolytic enzymes such as protease and elastase are themselves expensive. Therefore, angiotensin-converting enzyme inhibitors such as those disclosed in Patent Documents 1 and 2 have a problem in that their manufacturing costs are high and industrial mass production is not realistic.

Thus, there is a strong need for an angiotensin converting enzyme inhibitor that has a low environmental impact and can be produced at low cost, but such an excellent angiotensin converting enzyme inhibitor is currently unknown.

The present invention has been made to solve the above-mentioned problems, and is a highly safe angiotensin conversion method that has a low impact on the environment, can be manufactured at low cost, and can be used for pharmaceuticals, functional foods, etc. The object of the present invention is to provide an enzyme inhibitor and a method for producing the same.

As a result of intensive research into angiotensin-converting enzyme inhibitors, the present inventors discovered excellent effects as angiotensin-converting enzyme inhibitors from components derived from specific natural products, and among them, a novel cyclic compound was also identified. The present invention has now been completed. Furthermore, the inventors have also discovered the most suitable manufacturing method for manufacturing the angiotensin-converting enzyme inhibitor and its uses, thereby completing the present invention.

Thus, according to the present invention, an angiotensin converting enzyme inhibitor containing at least one active ingredient selected from the group consisting of a compound represented by the following general formula (I), a salt thereof, or a solvate thereof. agent is provided.

In the above formula, a and b are each independently a single bond or a double bond, and ring A is partially substituted with a linear or branched alkyl group having 1 to 10 carbon atoms and/or a hydroxyl group. R ₁ is a carbon atom or an oxygen atom, and R ₂ is a straight ring having 1 to 10 carbon atoms. A chain or branched alkylcarboxy group, an alkoxycarbonyl group, or a hydrogen atom, and R ₃ is a straight or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, or is represented by the following general formula (II) R ₄ is a substituent, and when a is a single bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, and when a is a double bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms. It is a branched alkylcarboxy group, R ₅ is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₆ is a straight-chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom; is a hydroxyl group or adenine, and when b is a double bond, it is an oxygen atom, and n is an integer of 0 to 3.

In the formula, R ₃₀₁ is a straight chain or branched alkyl group having 1 to 5 carbon atoms, R ₃₀₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms or a hydroxyl group, and R ₃₀₃ is , is a straight chain or branched alkylene group having 1 to 10 carbon atoms which may contain a carbon-carbon double bond, and R ₃₀₄ is a straight chain or branched alkyl group having 1 to 10 carbon atoms. .

In addition, the following compounds (I)-2-a, (I)-2-b, and (I)-3-a, or salts thereof, contained in the compound represented by the above general formula (I), or Also provided are novel cyclic compounds selected from the group consisting of solvates thereof.

1 shows a flowchart of a method for producing an angiotensin-converting enzyme inhibitor according to an embodiment of the present invention. A specific example of a flowchart related to a method for producing an angiotensin-converting enzyme inhibitor according to an embodiment of the present invention is shown. 1 shows a method for producing an angiotensin-converting enzyme inhibitor (isolation of active substance from microalgae of the genus Haematococcus) according to Example 1 of the present invention. 1 shows a method for producing an angiotensin-converting enzyme inhibitor (isolation of active substance from microalgae of the genus Haematococcus) according to Example 1 of the present invention. 1 shows an HPLC chart of an angiotensin-converting enzyme inhibitor according to Example 1 of the present invention (active substance isolation from Haematococcus microalgae). 1 shows a method for producing an angiotensin-converting enzyme inhibitor (isolation of active substance from microalgae of the genus Haematococcus) according to Example 1 of the present invention. 1 shows an HPLC chart of an angiotensin-converting enzyme inhibitor according to Example 1 of the present invention (active substance isolation from Haematococcus microalgae). 1 shows a method for producing an angiotensin-converting enzyme inhibitor (isolation of active substance from microalgae of the genus Myconastes) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) 1H NMR and (b) 13C NMR) of the angiotensin converting enzyme inhibitor (active substance of sample number 3153A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) COSY and (b) HMQC) of the angiotensin converting enzyme inhibitor (active substance of sample number 3153A) according to Example 1 of the present invention. 1 shows the NMR spectrum results of the angiotensin-converting enzyme inhibitor (active substance of sample number 3153A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) NOESY and (b) identified compound) of the angiotensin converting enzyme inhibitor (active substance of sample number 3153A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) 1H NMR and (b) 13C NMR) of the angiotensin converting enzyme inhibitor (active substance of sample number 3154A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) COSY and (b) HMQC) of the angiotensin converting enzyme inhibitor (active substance of sample number 3154A) according to Example 1 of the present invention. 1 shows NMR spectrum results of the angiotensin converting enzyme inhibitor (active substance of sample number 3154A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) NOESY and (b) identified compound) of the angiotensin-converting enzyme inhibitor (active substance of sample number 3154A) according to Example 1 of the present invention. 1 shows NMR spectrum results (COSY, 13CNMR, HMBC, HMQC) of the angiotensin converting enzyme inhibitor (active substance of sample number D36-3154A) according to Example 1 of the present invention. 1 shows NMR spectrum results (identified compound) of the angiotensin-converting enzyme inhibitor (active substance of sample number D36-3154A) according to Example 1 of the present invention. 1 shows the high-resolution mass spectrometry results of the angiotensin-converting enzyme inhibitor (active substances of sample numbers D36-35152 and D36-35153) according to Example 1 of the present invention. 1 shows NMR spectrum results (COSY, TOCSY, 1HNMR, HMBC, HMQC) of the angiotensin converting enzyme inhibitor (active substance of sample number D36-35153) according to Example 1 of the present invention. 1 shows NMR spectrum results (identified compound) of the angiotensin converting enzyme inhibitor (active substance of sample number D36-35153) according to Example 1 of the present invention. 1 shows NMR spectrum results (identified compound) of the angiotensin converting enzyme inhibitor (active substance of sample number D36-35153) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) 1H NMR and (b) COSY) of the angiotensin-converting enzyme inhibitor (active substance of sample number D35-333A) according to Example 1 of the present invention. 1 shows NMR spectrum results ((a) HMQC and (b) NOESY) of the angiotensin converting enzyme inhibitor (active substance of sample number D35-333A) according to Example 1 of the present invention. 1 shows NMR spectrum results (identified compound) of the angiotensin converting enzyme inhibitor (active substance of sample number D35-333A) according to Example 1 of the present invention. 2 shows the results of the angiotensin converting enzyme inhibitory activity of the angiotensin converting enzyme inhibitor (Haematococcus microalgae extract; water-soluble fraction, lipid fraction, organic layer fraction) according to Example 2 of the present invention. 2 shows the results of the angiotensin converting enzyme inhibitory activity of the angiotensin converting enzyme inhibitor (Haematococcus microalgae extract; sample number D36-3154A) according to Example 2 of the present invention. 2 shows the results of the angiotensin converting enzyme inhibitory activity of the angiotensin converting enzyme inhibitor (Myconastes microalgae extract; lipid fraction) according to Example 2 of the present invention. 2 shows the results of the angiotensin converting enzyme inhibitory activity of the angiotensin converting enzyme inhibitor (Myconastes microalgae extract; organic layer fraction) according to Example 2 of the present invention.

The angiotensin converting enzyme inhibitor according to the present embodiment contains at least one active ingredient selected from the group consisting of a compound represented by the following general formula (I), a salt thereof, or a solvate thereof. It is something.

In the above formula, a and b are each independently a single bond or a double bond, and ring A is partially substituted with a linear or branched alkyl group having 1 to 10 carbon atoms and/or a hydroxyl group. It is a 5-membered ring or a 6-membered ring which may have a carbon-carbon double bond.

R ₂ is a linear or branched alkylcarboxy group or alkoxycarbonyl group having 1 to 10 carbon atoms, or a hydrogen atom. R ₃ is a linear or branched alkyl group having 1 to 10 carbon atoms, a hydrogen atom, or a substituent represented by the following general formula (II).

In the above formula, R ₃₀₁ is a straight chain or branched alkyl group having 1 to 5 carbon atoms. R ₃₀₂ is a linear or branched alkyl group or hydroxyl group having 1 to 5 carbon atoms. R ₃₀₃ is a linear or branched alkylene group having 1 to 10 carbon atoms that may contain a carbon-carbon double bond, and preferably contains one carbon-carbon double bond. It is. R ₃₀₄ is a straight or branched alkyl group having 1 to 10 carbon atoms.

R ₄ is a straight chain or branched alkyl group having 1 to 10 carbon atoms when a is a single bond, and a straight chain or branched alkyl group having 1 to 10 carbon atoms when a is a double bond. It is the basis. R ₅ is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom. R ₆ is a hydroxyl group when b is a single bond, and is an oxygen atom when b is a double bond. n is an integer from 0 to 3; for example, n can be 0.

The above _R1 is not particularly limited, but is preferably an oxygen atom when the above ring A is a 5-membered ring, and a carbon atom or an oxygen atom when the above ring A is a 6-membered ring. .

For example, when the above ring A is a 6-membered ring and R ₁ is a carbon atom, the following general formula (I)-1 is, as an example, a 6-membered ring included in the general formula (I). Illustrated as a compound.

In the above formula, R ₂₁ is a linear or branched alkylcarboxy group or alkoxycarbonyl group having 1 to 10 carbon atoms, and R ₃₁ corresponds to R ₃ and is a linear or branched alkylcarboxy group having 1 to 10 carbon atoms. R 41 is a branched alkyl group or a hydrogen atom, and R ₄₁ corresponds to the case where a is a double bond for R ₄ , and is a linear or branched alkylcarboxy group having 1 to 10 carbon atoms; ₆₁ corresponds to the case where b is a single bond, and is a linear or branched alkylcarboxy group having 1 to 10 carbon atoms or a hydrogen atom, and R ₇₁ and R ₇₂ each independently have 1 to 10 carbon atoms. ~10 linear or branched alkyl groups or hydrogen atoms.

Further, for example, when the above ring A is a 5-membered ring and R ₁ is an oxygen atom, as an example, the following general formula (I)-2 is a 5-membered ring included in the general formula (I). It is exemplified as a ring compound.

In the above formula, R ₂₂ corresponds to a part of the above R ₂ and is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₃₂ corresponds to the above R ₃ and is a carbon R 42 is a linear or branched alkyl group or a hydrogen atom having 1 to 10 carbon atoms, and R ₄₂ corresponds to the case where a is a single bond for R ₄ , and R 42 is a linear or branched alkyl group having 1 to 10 carbon atoms. It is an alkyl group, and R ₅₂ corresponds to R ₅ above, and is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom.

Further, for example, when the above ring A is a 6-membered ring and R ₁ is an oxygen atom, as an example, the following general formula (I)-3 is a 6-membered ring included in the general formula (I). It is exemplified as a ring compound.

In the above formula, R ₃₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₄₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom. R ₅₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₆₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom.

In addition to the above, when the above ring A is a 5-membered ring and R ₁ is an oxygen atom, the following general formula (I)-4 is included in the general formula (I) as an example. It is exemplified as a 5-membered ring compound.

In the above formula, R ₃₄ is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom.

The above-mentioned active ingredient is not particularly limited, but is preferably an extract of microalgae. Microalgae are very small algae on the micrometer order, and they use sunlight to fix carbon dioxide and perform photosynthesis to synthesize carbohydrates.

This microalgae has a higher photosynthetic ability (ability to fix carbon dioxide) than terrestrial organisms and grows faster. From this, large-scale culture is easily possible as long as there is carbon dioxide and light, and in this embodiment, it is possible to realize a large-scale supply of angiotensin-converting enzyme inhibitor by ultra-large-scale culture on a scale of several hundred tons. Furthermore, since it uses substances produced by microalgae through photosynthesis, that is, algal biomass, the burden on the environment can be reduced.

The types of microalgae are not particularly limited, but preferably include Haematococcus, Mychonastes, Coccomyxa, Euglena, Arthrospira, Spirulina, etc. ), Chlorella, Dunaliella, and more preferably Haematococcus, and Mychonastes. can be mentioned.

For example, an active ingredient containing a compound included in the above general formula (I)-1 can be obtained from an extract of microalgae belonging to the genus Haematococcus. Examples include the following compounds (I)-1-a and (I)-1-b.

Further, for example, an active ingredient containing a compound included in the above general formula (I)-3 can also be obtained from an extract of microalgae belonging to the genus Haematococcus. An example thereof is the following compound (I)-3-a. This compound (I)-3-a is a novel, unknown compound identified by the present inventors.

Further, for example, an active ingredient containing a compound included in the above general formula (I)-4 can also be obtained from an extract of microalgae belonging to the genus Haematococcus. An example thereof is the following compound (I)-4-a.

Furthermore, for example, an active ingredient containing a compound included in the above general formula (I)-2 can be obtained from an extract of microalgae belonging to the genus Mychonastes. Examples include the following compounds (I)-2-a, (I)-2-b, and (I)-2-c. Among these, the following compounds (I)-2-a and (I)-2-b are novel, unknown compounds identified by the present inventors.

As described above, the angiotensin-converting enzyme inhibitor according to the present embodiment can be widely used for various purposes, and its uses are not limited. For example, a hypotensive agent containing the angiotensin converting enzyme inhibitor according to the present embodiment may be mentioned. Also included are pharmaceutical compositions containing the angiotensin-converting enzyme inhibitor according to the present embodiment and used for the prevention and/or treatment of hypertension.

Further, one of its uses is a food/drink composition containing the angiotensin converting enzyme inhibitor according to the present embodiment. Examples of such food and drink compositions include, but are not limited to, supplements, health foods, foods with health claims, foods for specified health uses, nutritionally functional foods, nutritional supplements, foods with functional claims, and foods for patients. Examples include food and health drinks.

When a microalgae extract is used as a raw material, the angiotensin-converting enzyme inhibitor according to the present embodiment has a blood pressure-lowering effect and prevents hypertension through the human endogenous angiotensin-converting enzyme inhibitory activity exhibited by the microalgae extract. It can be provided as a health food. Since these health foods use microalgae, they are extremely safe for consumption.

As for other uses, the angiotensin-converting enzyme inhibitor according to this embodiment can also be used as a constituent raw material for cosmetics. Examples of the form of such cosmetics include, but are not limited to, basic cosmetics, finishing cosmetics, skin cosmetics, hair cosmetics, special purpose cosmetics, makeup cosmetics, shampoos, body creams, cleansing agents, and the like. When the angiotensin-converting enzyme inhibitor according to the present embodiment is absorbed through the skin as a cosmetic ingredient, it easily exhibits blood pressure lowering and hypertension prevention effects, resulting in an excellent functional cosmetic with high added value.

(Production method)
The angiotensin converting enzyme inhibitor according to this embodiment is not particularly limited, but can be produced by the following production method.

As shown in FIG. 1, microalgae are roughly extracted with alcohol to obtain a hydroalcoholic crude extract (S1: crude extraction step). The alcohol used is not particularly limited, and for example, methanol or ethanol can be used, but it is more preferable to use ethanol, which is highly safe for food. For example, this hydroalcoholic crude extract can be obtained by extraction with 80% methanol or 80% ethanol for 14 days.

Next, as shown in FIG. 1, the hydroalcohol crude extract is subjected to liquid-liquid extraction with water and ethyl acetate, and an ethyl acetate fraction is collected (S2: collection step). That is, the obtained hydroalcoholic crude extract is partitioned between water/ethyl acetate and separated into a water-soluble fraction (aqueous layer) and an ethyl acetate fraction (ethyl acetate layer).

Next, as shown in FIG. 1, the ethyl acetate fraction is subjected to liquid-liquid extraction with alcohol and an alkane to separate the compound represented by the above formula (I), its salt, or its solvate ( S3: separation step). The alcohol and alkane are not particularly limited, but for example, hexane/methanol or hexane/ethanol can be used. That is, the above-mentioned ethyl acetate fraction (ethyl acetate layer) is partitioned, for example, with hexane/methanol, to obtain a lipid fraction (hexane layer) and an organic layer fraction (methanol layer).

The subsequent treatment of the lipid fraction and the organic layer fraction is not particularly limited, but for example, the lipid fraction may be sequentially eluted with hexane, ethyl acetate, chloroform, methanol, ethanol, etc. using silica gel chromatography. Additionally, multiple fractions can be obtained. For example, five additional fractions can be obtained by silica gel chromatography, sequentially eluting with hexane, hexane/ethyl acetate, ethyl acetate, methanol/chloroform, and methanol.

Further, the organic layer fraction can be sequentially eluted with methanol at a plurality of concentrations using a column to obtain a plurality of fractions. For example, five fractions can be obtained by sequential elution with 20% methanol, 40% methanol, 60% methanol, 80% methanol, and methanol using an ODS open column.

The method for isolating the compound (active substance) represented by the above formula (I) is not particularly limited, but examples include separation and fractionation using thin layer chromatography (PTLC) and high performance liquid chromatography (HPLC). can be isolated by performing this multiple times.

For example, the 20% methanol elution fraction and 40% methanol elution fraction of the ODS column of the organic layer fraction are collected and separated and fractionated by thin layer chromatography using a developing solvent of hexane:ethyl acetate = 2:1. The active fraction was further separated and fractionated by thin layer chromatography using a developing solvent of hexane: ethyl acetate = 1:2, and the active fraction was further separated by thin layer chromatography using an ODS column. It becomes possible to isolate the active substance as an angiotensin-converting enzyme inhibitor according to the form.

The above manufacturing method can be simply carried out using 80% methanol, hexane, and ethyl acetate, as shown in FIG. 2, for example. Note that this 80% methanol is given as an example for convenience, and 80% ethanol may also be used. When 80% ethanol is used, the production method is extremely safe for human consumption, and is more suitable for applications such as food and drink compositions.

First, as shown in FIG. 2, microalgae are roughly extracted with alcohol (80% ethanol) to obtain a hydroalcoholic crude extract (S1: crude extraction step). Next, this hydroalcohol crude extract is subjected to liquid-liquid extraction with water and ethyl acetate, and an ethyl acetate fraction is collected (S2: collection step). That is, the obtained hydroalcoholic crude extract is partitioned between water/ethyl acetate and separated into a water-soluble fraction (aqueous layer) and an ethyl acetate fraction (ethyl acetate layer).

Next, this ethyl acetate fraction was subjected to liquid-liquid extraction with alcohol and alkane (hexane/80% methanol), and the lipid fraction (hexane layer) and organic layer fraction (80% methanol layer) were extracted using the above formula (I ) or a salt thereof, or a solvate thereof is separated (S3: separation step).

EXAMPLES The present invention will be explained in more detail with reference to examples below, but the present invention is not limited to the following examples.

(Example 1)
Algal samples containing microalgae of the genus Haematococcus and microalgae of the genus Myconastes were extracted with 80% methanol for 14 days to obtain crude extracts. This was first partitioned between water/ethyl acetate and separated into an aqueous layer (water-soluble fraction) and an ethyl acetate layer. The ethyl acetate layer was further partitioned with hexane/80% methanol to obtain a hexane layer (lipid fraction) and an 80% methanol layer (organic layer fraction). As described below, the ACE inhibitory activity of the Haematococcus algae was measured at this stage.

The lipid fraction was subjected to silica gel chromatography and sequentially eluted with hexane, 50% hexane/50% ethyl acetate, ethyl acetate, 20% methanol/80% chloroform, and methanol to obtain five fractions. The organic layer fraction was collected using an ODS open column (50 x 150 mm) (manufactured) packed with a packing material (Cosmo Seal (registered trademark) 75C18-OPN, manufactured by Nacalai Tesque) in 20% methanol, 40% methanol, and 60% methanol. Five fractions were obtained by sequential elution with methanol, 80% methanol, and methanol. As described below, the ACE inhibitory activity of the Myconastes algae was measured at this stage.

(Isolation of active substances from Haematococcus microalgae) (1)
A mass-cultured algal sample, Haematococcus K1 strain, was immersed in water-containing methanol for two weeks and simultaneously extracted by ultrasonication for 30 minutes once a week to obtain a crude extract. The crude extract was partitioned between water/ethyl acetate and the ethyl acetate layer was further partitioned between 80% methanol/hexane.
That is, as shown in Figure 3, for the isolation of active substances from microalgae of the genus Haematococcus, the 20% methanol elution fraction (36.4 mg) and the 40% methanol elution fraction (6.1 mg) of the ODS column of the organic layer fraction were used. mg) were collected and separated and fractionated by thin layer chromatography (PTLC) (manufactured by Merck, product number 113894) (20 x 10 cm) using a developing solvent of hexane:ethyl acetate = 2:1. The active fraction was further separated and fractionated by thin layer chromatography (10×10 cm) using a developing solvent of hexane:ethyl acetate=1:4. The active fraction was further separated by high performance liquid chromatography (HPLC) (4.6 x 250 mm) using an ODS column with isocratic elution under 40% acetonitrile, and sample number 3153A (0.22 g) and sample number 3154A (0.08g) of active substance was obtained.

(Isolation of active substances from Haematococcus microalgae) (2)
Using the same procedure as above, as shown in Figure 4, collect only the 20% methanol elution fraction (14.4 mg) from the ODS column of the organic layer fraction, and add it to a developing solvent of hexane:ethyl acetate = 1:4. Separation and fractionation were performed by thin layer chromatography (PTLC) (manufactured by Merck, product number 113894) (20 x 10 cm). The active fraction was further separated and fractionated by thin layer chromatography (10×10 cm) using a developing solvent of chloroform:methanol=2:1. The active fraction was further separated using an ODS column using high-performance liquid chromatography (HPLC) (4.6 x 250 mm) with isocratic elution at 5% to 15% acetonitrile for 30 minutes, and the HPLC chart shown in Figure 5 was obtained. As shown above, 0.21 mg of the active substance of sample number D36-3154A was purified by elution using an ODS column at a retention time of approximately 18 minutes under the conditions of 5% → 15% acetonitrile (30 minutes).

(Isolation of active substances from Haematococcus microalgae) (3)
In the same procedure as above, as shown in Figure 6, only the methanol elution fraction (102 mg) of the ODS column of the organic layer fraction was collected, and the ODS column (manufactured by Nacalai Tesque, COSMOSIL (registered trademark) 75C18- OPN) with methanol 85%, 90%, and 95%, and thin layer chromatography (PTLC) (Merck, product number 113894) (20× The active fraction was further separated using an ODS column using isocratic elution using high performance liquid chromatography (HPLC) (4.6 x 250 mm) under 40% acetonitrile. As shown in the HPLC chart shown, sample number D36 was eluted with a retention time of 20 minutes (D36-35152) and 21 minutes (D36-35153) under the conditions of 43% → 43% acetonitrile (25 minutes) using an ODS column. -35152 (0.25 mg) and sample number D36-35153 (0.23 mg) of active substance were obtained.

(Isolation of active substances from microalgae of the genus Myconastes)
As shown in Figure 8, for the isolation of active substances from microalgae of the genus Myconastes, the 60% methanol fraction (9.68 mg) obtained with an ODS open column was separated by high performance liquid chromatography (HPLC) using an ODS column. ) and separated by gradient elution under 30-75% acetonitrile for 30 minutes to obtain the active substance of sample number D35-333 (0.42 mg). Here, D35 is the serial number of the sample, and 333 is the number of the fraction separated by column.

(NMR spectrum measurement)
(1) Active substance from microalgae of the genus Haematococcus NMR spectra were measured using the following device.
1H-NMR, 13C-NMR: Bruker AVANCE III 600MHz
(A) Sample numbers 3153A and 3154A
Regarding the active substance from microalgae of the genus Haematococcus, FIGS. 9 to 12(a) each show the NMR spectra of the active substance (sample number 3153A). Moreover, FIGS. 13 to 16(a) each show the NMR spectrum of the active substance (sample number 3154A).

Since the NMR spectra of the active substances of these sample numbers 3153A and 3154A showed very good agreement, they were considered to be analogs that are very similar to each other. In proton NMR, the signal at 5.74 ppm is presumed to be a proton bonded to the olefin, and the signal at 4.19 ppm is presumed to be a proton on the carbon to which the hydroxyl group is attached. The singlets at 1.75, 1.45, and 1.26 ppm suggest three methyl groups. Furthermore, the carbon NMR spectrum revealed that it has 11 carbon atoms. Furthermore, the signal at 174.7 ppm indicates the presence of carbonyl carbon. It is presumed that 113.3 ppm is derived from olefin carbon, 88.9 ppm is derived from methylene to which an oxygen atom is bonded, and the signals at 20 ppm to 70 ppm are derived from methyl and methylene chains. When we analyzed the planar structures of both from this information and the correlation in COSY (homogeneous nuclear shift correlation 2-dimensional NMR), HMQC (heterogeneous nuclear multi-quantum coherence 2-dimensional NMR), and HMQC, we found that they are carboxylic acids with a ring structure, and that both are carboxylic acids with a ring structure. It was suggested that they have the same planar structure.

On the other hand, for the active substance of sample number 3153A, correlations in NOESY spectra were observed between 1.75 ppm and 1.45 ppm, 5.74 ppm and 1.26 ppm, while for the active substance of sample number 3154A, correlations were observed between 4.11 ppm and 1.59 ppm, Correlation was observed in the NOESY (homogeneous NOE correlation two-dimensional NMR) spectra between the signals of 4.11 ppm and 1.29 ppm, 1.59 ppm and 1.29 ppm, and 5.77 ppm and 1.32 ppm. It was concluded that it was a mar.

From the above results, the active substances of sample numbers 3153A and 3154A, which are natural products showing ACE inhibitory activity in Haematococcus organic solvent extracts, are as follows as shown in FIG. 12(b) and FIG. 16(b). The compound was identified as

(B) Sample number D36-3154A
Using the same procedure as above, the active substance of sample number D36-3154A as a natural product showing ACE inhibitory activity in the Haematococcus organic solvent extract was extracted, and as shown in Figure 17, the NMR spectrum results (COSY, 13CNMR, HMBC, HMQC) were obtained. From the results in Figure 17, from the ¹ H NMR spectrum, the signals at 8.17 ppm and 8.31 ppm are derived from aromatic heterocycles containing heteroatoms, and the signals at 3.73 ppm, 3.88 ppm, 4.32 ppm, and 4.34 ppm are derived from carbon having a hydroxyl group next to it. It was thought that it was derived from hydrogen bonded to. Although ¹³ C NMR spectra could not be observed due to the small amount of sample, HMQC and HMBC spectra were obtained, and from the correlation of the COZY spectra, as shown in Figure 18, the active substance of sample number D36-3154A was detected. identified the following compound (adenosine):

(C) Sample numbers D36-35152 and D36-35153
The active substances of sample numbers D36-35152 and D36-35153 as natural products exhibiting ACE inhibitory activity in the Haematococcus organic solvent extract were extracted using the same procedure as above. The results of each high-resolution mass spectrometry are shown in FIG. 19. From this, it was shown that both are compounds represented by the same molecular formula: C ₂₅ H ₄₄ O ₁₀ .

Similarly to the above, the NMR spectrum results (COSY, TOCSY, 1HNMR, HMBC , HMQC). From the obtained results, the ¹ H NMR spectra of both are very similar, and both have the same molecular formula, so they are considered to be stereoisomers. Furthermore, by analyzing the correlation of various two-dimensional NMRs of COSY, TOCSY, HMQC, and HMBC, the plane structures of these two were determined as shown in FIG. 21. That is, the active substances of sample numbers D-36-35152 and D36-35153 were identified as the following unknown novel compounds, as shown in FIGS. 21 and 22.

(2) Active substance from microalgae of the genus Myconastes (sample number D35-333)
NMR spectra of the active substance from microalgae of the genus Myconastes (sample number D35-333) are shown in FIGS. 23 to 25. As shown in FIG. 23, from this NMR spectrum, the signal at 3.07 ppm is estimated to be a methoxy group. The signals at 1.90 ppm and 1.83 ppm both have 3H components, and are considered to be methyl groups bonded to olefins. The 1H-minute signals at 1.95 ppm and 1.77 ppm are considered to be methylene attached to an alkoxy group, and the signals at 2.21 ppm, 1.58 ppm, 1.30 ppm, 1.26 ppm, and 1.14 ppm are considered to be the methylene chain and methyl group that follow. The planar structure of this compound was determined from the correlations between these and COSY (homogeneous nuclear shift correlation two-dimensional NMR), HMQC (heteronuclear multi-quantum coherence two-dimensional NMR), and HMBC.

From the above results, the active substance as a natural product (sample number D35-333) showing ACE inhibitory activity in the Myconastes organic solvent extract was identified as the following unknown new compound as shown in Figure 25. did.

D35-333

D35-333

In addition, similar to the above compounds, the following compounds, which can be said to be analogs of the above compounds, were also identified as active substances as natural products that exhibit ACE inhibitory activity in organic solvent extracts of the genus Myconastes. Among these, in particular, the compound of chemical formula (I)-2-b was confirmed to be a novel compound that is not known to the public.

(Example 2)
(ACE inhibitory activity)
The ACE inhibitory activity of the microalgae extract containing each active substance obtained in Example 1 above was confirmed.

(1) Active substance from microalgae of the genus Haematococcus Regarding the active substance from microalgae of the genus Haematococcus, at the stage of obtaining the crude extract extracted with 80% methanol as described in Example 1, the aqueous solution shown in Figure 2 was prepared. The ACE inhibitory activity was measured for each of the sex fraction, lipid fraction, and organic layer fraction. The measurement results of the obtained ACE inhibitory activity (sample numbers 3153A and 3154A) are shown in FIG. 26. From the results obtained, very high ACE inhibitory activity was confirmed in all fractions. The active substances from Haematococcus microalgae identified in Example 1 (sample numbers 3153A and 3154A) were eluted in this lipid fraction with 20% methanol and 40% methanol as described in Example 1. contained in the lipid fraction obtained by

Furthermore, the measurement results of the ACE inhibitory activity obtained for the active substance from Haematococcus microalgae (sample number D36-3154A) confirmed in Example 1 are shown in FIG. The active substance from Haematococcus microalgae identified in this Example 1 (sample number D36-3154A) was obtained from this lipid fraction by elution with 20% methanol as described in Example 1. contained in the collected lipid fraction. The active substances from Haematococcus microalgae (sample numbers D36-35152 and D36-35153) confirmed in Example 1 were also confirmed to have very high ACE inhibitory activity.

From the above results, the extract of Haematococcus microalgae contains the active substances confirmed in Example 1 (sample numbers 3153A, 3154A, D36-3154A, D36-35152, D36-35153) as an active ingredient, and has a very high It was confirmed that high ACE inhibitory activity was exhibited.

(2) Active substance from microalgae of the genus Myconastes (2-1) Lipid fraction For the active substance from microalgae of the genus Myconastes (sample number D35-333), the hexane of Example 1 above, 50% hexane/50 ACE inhibitory activity was measured for five lipid fractions obtained by sequential elution with % ethyl acetate, ethyl acetate, 20% methanol/80% chloroform, and methanol.

The five lipid fractions were separated by column chromatography: the fraction eluted with 20% methanol (sample number D35-21), the fraction eluted with 40% methanol (sample number D35-22), and the fraction eluted with 60% methanol. It consists of the fraction eluted with 80% methanol (sample number D35-23), the fraction eluted with 80% methanol (sample number D35-24), and the fraction eluted with methanol (sample number D35-25).

The measurement results of ACE inhibitory activity obtained for each lipid fraction are shown in FIG. From the obtained results, ACE inhibitory activity was confirmed in all lipid fractions, and it was particularly high in the lipid fractions in which 20% to 80% methanol was eluted (sample numbers D35-21 to D35-24). ACE inhibitory activity was confirmed.

(2-2) Organic layer fraction In addition, five organic layer fractions (each , sample numbers D35-31, D35-32, D35-33, D35-34, D35-35) were measured for ACE inhibitory activity. The measurement results of ACE inhibitory activity obtained for these organic layer fractions are shown in FIG. 29. From the results obtained, very high ACE inhibitory activity was confirmed in all organic layer fractions. The active substance from Myconastes microalgae (sample number D35-333) confirmed in Example 1 was obtained from this organic layer fraction by elution with 60% methanol as described in Example 1. This corresponds to the organic layer fraction.

From the above results, it was confirmed that the extract of microalgae of the genus Myconastes contains the active substance confirmed in Example 1 (sample number D35-333) as an active ingredient and exhibits extremely high ACE inhibitory activity. .

Claims (13)

The following compounds (I)-2-a, (I)-2-b, and (I)-3-a, or salts thereof, contained in the compound represented by the general formula (I) below, or their salts. A cyclic compound selected from the group consisting of solvates.

(In the formula, a and b are each independently a single bond or a double bond, and ring A is partially substituted with a linear or branched alkyl group having 1 to 10 carbon atoms and/or a hydroxyl group. R ₁ is a carbon atom or an oxygen atom, and R ₂ is a straight ring having 1 to 10 carbon atoms. A chain or branched alkylcarboxy group, an alkoxycarbonyl group, or a hydrogen atom, and R ₃ is a straight or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, or is represented by the following general formula (II) R ₄ is a substituent, and when a is a single bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, and when a is a double bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms. It is a branched alkylcarboxy group, R ₅ is a linear or branched alkyl group having 1 to 10 carbon atoms, a hydrogen atom or a hydroxyl group, and R ₆ is a branched alkylcarboxy group, when b is a single bond, a hydroxyl group or adenine which may be a double bond, and when b is a double bond, it is an oxygen atom, and n is an integer from 0 to 3)

(In the formula, R ₃₀₁ is a straight chain or branched alkyl group having 1 to 5 carbon atoms, R ₃₀₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms or a hydroxyl group, and R ₃₀₃ is a straight chain or branched alkyl group having 1 to 5 carbon atoms. is a straight chain or branched alkylene group having 1 to 10 carbon atoms which may contain a carbon-carbon double bond, and R ₃₀₄ is a straight chain or branched alkyl group having 1 to 10 carbon atoms. be)

An angiotensin converting enzyme inhibitor characterized by containing at least one active ingredient selected from the group consisting of a compound represented by the following general formula (I), a salt thereof, or a solvate thereof.

(In the formula, a and b are each independently a single bond or a double bond, and ring A is partially substituted with a linear or branched alkyl group having 1 to 10 carbon atoms and/or a hydroxyl group. R ₁ is a carbon atom or an oxygen atom, and R ₂ is a straight ring having 1 to 10 carbon atoms. A chain or branched alkylcarboxy group, an alkoxycarbonyl group, or a hydrogen atom, and R ₃ is a straight or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, or is represented by the following general formula (II) R ₄ is a substituent, and when a is a single bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, and when a is a double bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms. It is a branched alkylcarboxy group, R ₅ is a linear or branched alkyl group having 1 to 10 carbon atoms, a hydrogen atom or a hydroxyl group, and R ₆ is a branched alkylcarboxy group, when b is a single bond, a hydroxyl group or adenine which may be a double bond, and when b is a double bond, it is an oxygen atom, and n is an integer from 0 to 3)

(In the formula, R ₃₀₁ is a straight chain or branched alkyl group having 1 to 5 carbon atoms, R ₃₀₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms or a hydroxyl group, and R ₃₀₃ is a straight chain or branched alkyl group having 1 to 5 carbon atoms. is a straight chain or branched alkylene group having 1 to 10 carbon atoms which may contain a carbon-carbon double bond, and R ₃₀₄ is a straight chain or branched alkyl group having 1 to 10 carbon atoms. be) The angiotensin converting enzyme inhibitor according to claim 2,
An angiotensin converting enzyme inhibitor, characterized in that R ₁ is an oxygen atom when ring A is a 5-membered ring, and is a carbon atom or an oxygen atom when ring A is a 6-membered ring. The angiotensin converting enzyme inhibitor according to claim 3,
Contains at least one active ingredient selected from the group consisting of compounds represented by any of the following general formulas (I)-1 to (I)-4, salts thereof, or solvates thereof. An angiotensin-converting enzyme inhibitor characterized by:

(In the formula, R ₂₁ is a straight chain or branched alkylcarboxy group or alkoxycarbonyl group having 1 to 10 carbon atoms, and R ₃₁ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or hydrogen R ₄₁ is a linear or branched alkylcarboxy group having 1 to 10 carbon atoms, and R ₆₁ is a linear or branched alkylcarboxy group having 1 to 10 carbon atoms or a hydrogen atom. , R ₇₁ and R ₇₂ are each independently a straight or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom)

(In the formula, R ₂₂ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₃₂ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom.) (R ₄₂ is a straight chain or branched alkyl group having 1 to 10 carbon atoms, and R ₅₂ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom)

(In the formula, R ₃₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₄₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom.) (R ₅₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, and R ₆₃ is a straight chain or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom)

(In the formula, R ₃₄ is a linear or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom) The angiotensin converting enzyme inhibitor according to claim 4,
An angiotensin converting enzyme inhibitor characterized by containing at least one active ingredient selected from the group consisting of the following compounds included in the general formula (I), salts thereof, or solvates thereof.

The angiotensin converting enzyme inhibitor according to any one of claims 2 to 5,
An angiotensin converting enzyme inhibitor, wherein the active ingredient is an extract of microalgae. The angiotensin converting enzyme inhibitor according to claim 6,
An angiotensin converting enzyme inhibitor, characterized in that the microalgae is selected from the group consisting of the genus Haematococcus and the genus Mychonastes. A hypotensive agent comprising the angiotensin-converting enzyme inhibitor according to any one of claims 2 to 7. A pharmaceutical composition comprising the angiotensin-converting enzyme inhibitor according to any one of claims 2 to 7 and used for the prevention and/or treatment of hypertension. A food or drink composition comprising the angiotensin converting enzyme inhibitor according to any one of claims 2 to 7. The food and drink composition according to claim 10,
A food/beverage composition characterized by being a supplement, a health food, a food with health claims, a food for specified health uses, a nutritionally functional food, a nutritional supplement, a food with functional claims, a food for the sick, or a health drink. A cosmetic product comprising the angiotensin converting enzyme inhibitor according to any one of claims 1 to 7. a step of roughly extracting microalgae with alcohol to obtain a hydrous alcohol crude extract;
A step of performing liquid-liquid extraction of the hydroalcoholic crude extract with water and ethyl acetate and collecting an ethyl acetate fraction;
Angiotensin is characterized by comprising a step of performing liquid-liquid extraction with alcohol and an alkane from the ethyl acetate fraction to separate a compound represented by the following formula (I), a salt thereof, or a solvate thereof. Method for producing converting enzyme inhibitor.

(In the formula, a and b are each independently a single bond or a double bond, and ring A is partially substituted with a linear or branched alkyl group having 1 to 10 carbon atoms and/or a hydroxyl group. R ₁ is a carbon atom or an oxygen atom, and R ₂ is a straight ring having 1 to 10 carbon atoms. A chain or branched alkylcarboxy group, an alkoxycarbonyl group, or a hydrogen atom, and R ₃ is a straight or branched alkyl group having 1 to 10 carbon atoms or a hydrogen atom, or is represented by the following general formula (II) R ₄ is a substituent, and when a is a single bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, and when a is a double bond, R 4 is a straight chain or branched alkyl group having 1 to 10 carbon atoms. It is a branched alkylcarboxy group, R ₅ is a linear or branched alkyl group having 1 to 10 carbon atoms, a hydrogen atom or a hydroxyl group, and R ₆ is a branched alkylcarboxy group, when b is a single bond, a hydroxyl group or adenine which may be a double bond, and when b is a double bond, it is an oxygen atom, and n is an integer from 0 to 3)

(In the formula, R ₃₀₁ is a straight chain or branched alkyl group having 1 to 5 carbon atoms, R ₃₀₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms or a hydroxyl group, and R ₃₀₃ is a straight chain or branched alkyl group having 1 to 5 carbon atoms. is a straight chain or branched alkylene group having 1 to 10 carbon atoms which may contain a carbon-carbon double bond, and R ₃₀₄ is a straight chain or branched alkyl group having 1 to 10 carbon atoms. be)

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