JP2017095374A - Lipid metabolism improving composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound having the action to inhibit fat accumulation in a liver, where the compound is stable even when blended in pharmaceuticals or food and drink in the production process.SOLUTION: The present invention provides a compound represented by formula I, or a composition comprising the compound and having the action to inhibit fat accumulation in a liver.SELECTED DRAWING: None

Description

  The present invention relates to a compound effective for suppressing fat accumulation in the liver, and a pharmaceutical composition and food additive containing the same.

  Fatty liver is a symptom caused by accumulation of neutral fat or the like in the liver due to excessive eating or drinking. Leaving fatty liver can lead to cirrhosis and liver cancer, and there is also a risk of increasing the risk of developing diabetes and promoting arteriosclerosis.

  So far, several substances having an effect of suppressing fat accumulation in the liver have been found, and examples of such substances include processed products of blueberry leaves, seaweed fermented foods, and soy protein ( Patent Documents 1 and 2, Non-Patent Document 1). In addition, it has been reported in the paper that tea, fish oil, unsaturated fatty acids, etc. also have an effect of suppressing the accumulation of fat in the liver. Furthermore, it has also been reported that onion or onion extract has a fatty liver inhibitory action and a body fat accumulation inhibitory action (Patent Documents 3 and 4).

  Onion is one of the vegetables widely eaten all over the world for a long time, and there are many reports regarding showing an effective physiological action on humans (Non-patent Document 2).

  Today, a lacrimatory factor synthase (hereinafter referred to as “LFS”) was discovered, and the mechanism by which propanthial S-oxide, a lacrimatory factor (hereinafter referred to as “LF”) of onion, is clarified. ing. That is, the enzyme alliinase acts on the precursor S-1-propenyl-cysteine sulfoxide (hereinafter referred to as “PRENCSO”) to form 1-propenylsulfenic acid, and 1-propenylsulfenic acid. Sulfenic acid is isomerized by LFS to produce LF (Non-patent Document 3).

  It was confirmed that the amount of thiosulfinate increased and the component called Zwiebelane isomer increased in the onion produced using RNAi technology with suppressed LFS compared to normal onion. (Non-Patent Document 4).

  As a component that increases in an onion in which LFS is suppressed, a compound that is Zwiebelane isomer (represented by Chemical Formula 1 below) has been found (Non-Patent Document 5), and the present inventors have so far described the compound. Clarified and reported that the compound has an activity to inhibit cyclooxygenase-1 (COX-1) and an activity to inhibit α-glucosidase (Patent Document 5) and that the compound has an action to suppress fat accumulation. (Patent Document 6).

JP 2008-189931 A JP 2003-02001 A JP 2009-089703 A JP 2007-326800 A JP 2012-111742 A JP 2014-136699 A

Ascencio C et al .; Soy Protein Affects Serum Insulin and Hepatic SREBP-1 mRNA and Reduces Fatty Liver in Rats. J. Nutr, 134 (3), 522-529, (2004) Griffiths G et al .; Onions-A Global Benefit to Health. Phytother. Res., 16, 603-615 (2002) Imai S et al .; An onion enzyme that makes the eyes water.Nature, 419, 685 (2002) Eady C. C. et al .; Silencing Onion Lachrymatory Factor Synthase Causes a Significant Change in the Sulfur Secondary Metabolite Profile.Plant Physiology, 147, 2096-2106 (2008) Aoyagi M et al .; Structure and Bioactivity of Thiosulfinates Resulting from Suppression of Lachrymatory Factor Synthase in Onion. J. Agric. Food Chem, 59 (20), 10893-10900 (2011)

  However, since the Zwiebelane isomer is unstable in an aqueous solution, there is a problem that it is easily decomposed in the production process and when it is included in pharmaceuticals, food additives, and foods and drinks.

  Accordingly, an object of the present invention is to provide a compound that is stable in the production process and also when included in pharmaceuticals, food additives, and foods and drinks and has an action of suppressing fat accumulation in the liver. To do.

  As a result of intensive research on the components in the low LFS onion extract and the components in the reaction solution of the PRENCSO alliinase decomposition reaction in the absence of LFS, the present inventors have conducted research on these extracts and reaction solutions. In order to complete the present invention, it is found that there is a compound having an action of suppressing the accumulation of fat in the liver, which is different from the Zwiebelane isomer, and that the compound has higher stability than the Zwiebelane isomer. It came.

（式中、
R₁は水素又は低級アルキル基であり、
R₂及びR₃は同一又は異なって低級アルキル基であり、
R₄は

であり、
R₅及びR₆は同一又は異なって低級アルキル基であり、
R₇は水素又は低級アルキル基であり、
ｎは3〜6の整数である。）
で表される、化合物又はその塩を含有することを特徴とする、肝臓への脂肪蓄積を抑制するための組成物。
［２］ 化合物が式１：

で表される、［１］の組成物。
［３］ 化合物が式２(a)：

、又は式２(b)：

で表される、［１］の組成物。
［４］ 医薬組成物である、［１］〜［３］のいずれかの組成物。
［５］ 食品添加剤である、［１］〜［３］のいずれかの組成物。
［６］ ［５］の組成物を含む飲食品。
［７］ 式Ｉ：

（式中、
R₁は水素又は低級アルキル基であり、
R₂及びR₃は同一又は異なって低級アルキル基であり、
R₄は

であり、
R₅及びR₆は同一又は異なって低級アルキル基であり、
R₇は水素又は低級アルキル基であり、
ｎは3〜6の整数である。）
で表される、化合物又はその塩を飲食品の材料に配合することを含む、肝臓への脂肪蓄積を抑制する作用を有する飲食品の製造方法。 The present invention includes the following inventions.
[1] Formula I:

(Where
R ₁ is hydrogen or a lower alkyl group,
R ₂ and R ₃ are the same or different and are lower alkyl groups,
R ₄ is

And
R ₅ and R ₆ are the same or different and are lower alkyl groups,
R ₇ is hydrogen or a lower alkyl group,
n is an integer of 3-6. )
The composition for suppressing fat accumulation to the liver characterized by containing the compound or its salt represented by these.
[2] The compound is represented by formula 1:

The composition of [1] represented by these.
[3] The compound is represented by formula 2 (a):

Or Formula 2 (b):

The composition of [1] represented by these.
[4] The composition according to any one of [1] to [3], which is a pharmaceutical composition.
[5] The composition according to any one of [1] to [3], which is a food additive.
[6] A food or drink comprising the composition of [5].
[7] Formula I:

(Where
R ₁ is hydrogen or a lower alkyl group,
R ₂ and R ₃ are the same or different and are lower alkyl groups,
R ₄ is

And
R ₅ and R ₆ are the same or different and are lower alkyl groups,
R ₇ is hydrogen or a lower alkyl group,
n is an integer of 3-6. )
The manufacturing method of the food / beverage products which have the effect | action which suppresses the fat accumulation to the liver including mix | blending the compound or its salt represented by these with the material of food / beverage products.

  ADVANTAGE OF THE INVENTION According to this invention, the compound which has the effect | action which suppresses the fat accumulation to a liver which is stable in the manufacturing process, and when it is included in a pharmaceutical, a food additive, and food-drinks is provided.

FIG. 1 shows the analysis results by HPLC of a solution to which methanol was added after 90 minutes from the start of the reaction of PRENCSO + alliinase. FIG. 2 shows the analysis result of the reaction solution (supernatant) of PRENCSO + alliinase using a large-scale preparative HPLC system. FIG. 3-1 shows the measurement results of MS / MS spectra of Peak 1 and Peak 2 in fraction 3. FIG. 3-2 shows the measurement results of MS / MS spectra of Peak 3 and Peak 4 in fraction 3. FIG. 4 shows the analysis result by HPLC of the solution to which methanol was added after 3 minutes or 90 minutes from the start of the reaction of PRENCSO + allylinase. FIG. 5 shows an m / z 203 extracted ion chromatogram in HPLC-Orbitrap analysis of a solution obtained by adding methanol to a reaction solution of garlic juice + heated onion juice. FIG. 6 shows the analysis results of the reaction solution (precipitate) of PRENCSO + allylinase by a large-scale preparative HPLC system. FIG. 7-1 shows the measurement results of MS spectra of Peak A and Peak B. FIG. 7-2 shows the measurement results of MS spectra of Peak C1 and Peak C2. Fig. 7-3 shows the measurement results of MS spectra of Peak D1 and Peak D2. FIG. 8 shows m / z 227 and m / z 259 extracted ion chromatograms in HPLC-Orbitrap analysis of a solution obtained by adding methanol to a reaction solution of garlic juice + heated onion juice. FIG. 9 is a graph showing changes in body weight over time in rats fed a high fat diet (control group) and rats fed a compound of formula 2 together with the high fat diet (test group). FIG. 10 is a graph showing changes in food intake over time in rats fed a high fat diet (control group) and rats fed a compound of formula 2 together with a high fat diet (test group). FIG. 11 is a graph showing the amount of triglyceride (TG) in the liver in rats fed a high fat diet (control group) and rats fed a compound of formula 2 together with the high fat diet (test group).

（式中、
R₁は水素又は低級アルキル基であり、
R₂及びR₃は同一又は異なって低級アルキル基であり、
R₄は

であり、
R₅及びR₆は同一又は異なって低級アルキル基であり、
R₇は水素又は低級アルキル基であり、
nは3〜6の整数である。）
で表される平面構造を有する。 I. Compounds in the present invention are compounds of formula I:

(Where
R ₁ is hydrogen or a lower alkyl group,
R ₂ and R ₃ are the same or different and are lower alkyl groups,
R ₄ is

And
R ₅ and R ₆ are the same or different and are lower alkyl groups,
R ₇ is hydrogen or a lower alkyl group,
n is an integer of 3-6. )
It has the planar structure represented by these.

  In the present specification, the “lower alkyl group” is preferably a linear or branched alkyl group having 1 to 6 carbon atoms, and a linear or branched alkyl group having 1 to 4 carbon atoms. Is more preferable. For example, methyl, ethyl, propyl, butyl, pentyl, hexyl groups and the like can be mentioned.

  A compound having a planar structure represented by Formula I has a plurality of optical isomers. The compound of this invention should just be a compound which has the planar structure represented by Formula I, and the structure of an optical isomer is not specifically limited.

  In the present invention, the “compound represented by formula I” means a mixture of a plurality of optical isomers of a compound having a planar structure represented by formula I, and individual optical isomers isolated or concentrated. Including both.

  In the present invention, the salt means a conventional one that is acceptable for use in medicines, foods and drinks, for example, alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt). Etc.), ammonium salts, organic amine salts, organic acid salts, inorganic acid salts, sulfonic acid salts and the like.

で表される平面構造を有する。式1で表される平面構造を有する化合物には、複数の光学異性体が存在する。本発明の化合物は、式1で表される平面構造を有する化合物であればよく、光学異性体の構造は特に限定されない。 In one embodiment, the compounds of the invention have the formula 1:

It has the planar structure represented by these. A compound having a planar structure represented by Formula 1 has a plurality of optical isomers. The compound of the present invention may be a compound having a planar structure represented by Formula 1, and the structure of the optical isomer is not particularly limited.

  In the present invention, the “compound represented by Formula 1” means a mixture of a plurality of optical isomers of a compound having a planar structure represented by Formula 1 and individual optical isomers isolated or concentrated. Including both.

  The compound represented by Formula 1 may be chemically synthesized based on the structural formula, or can be prepared based on a method for producing the compound described below. That is, from the reaction product of the degradation reaction of PRENCSO with alliinase under the condition where the activity of LFS is reduced or disappeared, and the pulverized product of low LFS onion, multiple optical compounds of the compound having a planar structure represented by Formula 1 A mixture of isomers can be obtained.

Specifically, the reaction product of the degradation reaction of PRENCSO with alliinase under the condition where the activity of LFS is reduced or eliminated, or the ground product of low LFS onion, the following conditions:
Column: Thermo Fisher Scientific ODS Hypersil 250 mm x 4.6 mm, 5 μm, column oven: 30 ° C, mobile phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 min (18% / 82%), 45 min (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 65 minutes (18% / 82%) (specifically, the gradient from 0 to 45 minutes the five minutes of the CH ₃ CN concentration from 18% to .45 and 50 minutes raised to 90% over, holding the CH ₃ CN 90%. from that after 51 minutes, CH ₃ CN concentration 18% And flowed up to 65 minutes), flow rate: 0.5 ml / min, detection 190-650 nm,
In the high-performance liquid chromatography (HPLC) chromatogram, the four major peaks (Peak1 to Peak4) appear in the retention time of about 13 minutes to about 17 minutes (Figure 1). A plurality of optical isomer compounds of the compound having These optical isomer compounds may be obtained separately from each other or may be obtained as a mixture of a plurality of optical isomers.

  In the chromatogram by HPLC under the above conditions, Peak 3 having the third longest retention time among the four main peaks appearing between retention times of about 13 minutes to about 17 minutes (from 27.5 to 28.5 in Example 1-3). The compound represented by the formula 1 in the fraction corresponding to (min) has the physicochemical characteristics shown in the following Example 1-3.

  The compound represented by Formula 1 may be provided as a compound in an isolated and purified form, or may be provided as a composition containing the compound represented by Formula 1. As a composition containing the compound represented by Formula 1, the compound is 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, 10% by mass or more based on the solid content of the composition, Examples thereof include compositions containing 30% by mass or more, 50% by mass or more, 70% by mass or more, or 90% by mass or more. Such a composition can be prepared in the manufacturing method of the compound mentioned later.

  The compound represented by Formula 1 may exist in the form of a hydrate with water or a solvate with a lower alcohol or the like. The hydrate or solvate is not particularly limited as long as it is a hydrate or solvate that is acceptable for uses such as pharmaceuticals and foods and drinks. That is, the “compound represented by the formula 1” in the present invention includes a hydrate or solvate form as a subordinate concept.

（式中、
R₁及びR₇は同一又は異なって水素又は低級アルキル基であり、
R₂, R₃, R₅ 及びR₆は同一又は異なって低級アルキル基であり、
ｎは3〜6の整数である。）
で表される平面構造を有する。式2で表される平面構造を有する化合物には、複数の光学異性体が存在する。本発明の化合物は、式2で表される平面構造を有する化合物であればよく、光学異性体の構造は特に限定されない。 Also, in one embodiment, the compounds of the invention have formula 2:

(Where
R ₁ and R ₇ are the same or different and are hydrogen or a lower alkyl group,
R ₂ , R ₃ , R ₅ and R ₆ are the same or different and are lower alkyl groups,
n is an integer of 3-6. )
It has the planar structure represented by these. A compound having a planar structure represented by Formula 2 has a plurality of optical isomers. The compound of the present invention may be a compound having a planar structure represented by Formula 2, and the structure of the optical isomer is not particularly limited.

  In the present invention, the “compound represented by the formula 2” means a mixture of a plurality of optical isomers of a compound having a planar structure represented by the formula 2 and individual optical isomers isolated or concentrated. Including both.

、又は式２(b)：

で表される平面構造を有する。式２(a)及び式２(b)で表される平面構造を有する化合物には、複数の光学異性体が存在する。本発明の化合物は、式２(a)及び式２(b)で表される平面構造を有する化合物であればよく、光学異性体の構造は特に限定されない。 Preferably the compound of formula 2 is of formula 2 (a):

Or Formula 2 (b):

It has the planar structure represented by these. A compound having a planar structure represented by Formula 2 (a) and Formula 2 (b) has a plurality of optical isomers. The compound of the present invention may be a compound having a planar structure represented by Formula 2 (a) and Formula 2 (b), and the structure of the optical isomer is not particularly limited.

  In the present invention, the “compound represented by formula 2 (a)” is isolated or enriched with a mixture of a plurality of optical isomers of the compound having a planar structure represented by formula 2 (a). Includes both individual optical isomers. In the present invention, the “compound represented by formula 2 (b)” refers to a mixture of a plurality of optical isomers of a compound having a planar structure represented by formula 2 (b), and isolation or increase in concentration. As well as individual optical isomers.

  The compound represented by Formula 2 may be chemically synthesized based on the structural formula, or can be prepared by a method for producing a compound described later. A mixture of compounds having a planar structure represented by Formula 2 is included in the reaction product of the degradation reaction of PRENCSO by alliinase under the condition in which the activity of LFS is reduced or eliminated, and the ground product of low LFS onion.

Specifically, the reaction product of the degradation reaction of PRENCSO with alliinase under the condition where the activity of LFS is reduced or eliminated, and the ground product of low LFS onion, the following conditions:
Column: Thermo Fisher Scientific ODS Hypersil 250 mm x 4.6 mm, 5 μm, column oven: 30 ° C, mobile phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 min (18% / 82%), 45 min (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 65 minutes (18% / 82%) (specifically, the gradient from 0 to 45 minutes the five minutes of the CH ₃ CN concentration from 18% to .45 and 50 minutes raised to 90% over, holding the CH ₃ CN 90%. from that after 51 minutes, CH ₃ CN concentration 18% And flowed up to 65 minutes), flow rate: 0.5 ml / min, detection 190-650 nm,
In the high-performance liquid chromatography (HPLC) chromatogram, six major peaks appear during the retention time of about 35 minutes to about 46 minutes (Peak A, Peak B, Peak C1, C2, Peak D1, D2), Among them, the peak having the shortest retention time and the peak having the next shortest retention time include a plurality of optical isomer compounds of the compound having a planar structure represented by Formula 2 (FIG. 1). These optical isomer compounds may be obtained separately from each other or may be obtained as a mixture of a plurality of optical isomers.

  In the chromatogram by HPLC under the above-mentioned conditions, Peak A (23 to 26 minutes in Example 3) has the shortest retention time among the six main peaks appearing between retention times of about 35 minutes to about 46 minutes. The corresponding fraction contains the compound represented by formula 2 (a).

  Further, in the chromatogram by HPLC under the above conditions, Peak B having the next shortest retention time among the six main peaks during the retention time of about 35 minutes to about 46 minutes (from 28.5 minutes to 31 minutes in Example 3). ) In the fraction corresponding to).

  The compound represented by the formula 2 (a) and the compound represented by the formula 2 (b) have the physicochemical characteristics shown in Example 3 below.

  The compound represented by Formula 2 may be provided in the form of an isolated and purified form, or may be provided as a composition containing the compound represented by Formula 2. As a composition containing the compound represented by Formula 2, the compound is 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, 10% by mass with respect to the solid content of the composition. Examples of the composition include 30% by mass or more, 50% by mass or more, 70% by mass or more, or 90% by mass or more. Such a composition can be prepared in the manufacturing method of the compound mentioned later.

  The compound represented by Formula 2 may exist in the form of a solvate with an organic solvent such as alcohol, ether, acetone, hexane, or chloroform. A solvate will not be specifically limited if it is a solvate accept | permitted for uses, such as a pharmaceutical and food-drinks. That is, in the “compound represented by Formula 2” in the present invention, the form of those solvates is also included as a subordinate concept.

II. Method for Producing Compound The production of the compound of the present invention is not particularly limited and may be chemically synthesized based on the structural formula, but can be preferably performed by a method including a decomposition reaction in which PRENCSO is treated with alliinase. . Although this decomposition reaction proceeds by grinding onion, LFS is contained in ordinary onion grinds, so the degradation product (1-propenylsulfenic acid) of PRENCSO alliinase is LF (tearing component) by LFS Therefore, the compound of the present invention hardly accumulates.

  Therefore, in the present specification, it is disclosed that the compound of the present invention is produced by a method including the following two methods. However, in order to industrially produce the compound, conditions under which the activity of LFS is reduced or eliminated The following second production method including the step of degrading PRENCSO with alliinase is preferable.

  The first method for producing the compound of the present invention or a composition containing the compound includes a step of pulverizing a low LFS onion having reduced LFS activity. The second production method includes a step of degrading PRENCSO with alliinase under conditions where the activity of LFS is reduced or eliminated.

The first manufacturing method will be described in detail.
A low LFS onion is an onion with reduced LFS activity compared to a normal onion and can be obtained by genetic recombination or mutation treatment. A preferred low LFS onion is an LFS activity that is 20 times or less, preferably 40 times or less, more preferably 1/160 or less, particularly preferably 1/400 or less of the LFS activity of the parent strain (normal onion). Have LFS activity can be measured by the following method.

<LFS activity measurement method>
LFS activity is quantified based on the peak area of the tear component (LF) generated by the enzymatic reaction. To 10 μl of the measurement sample (onion extract), 40 μl of 250 U / ml purified alliinase solution is added, and 20 μl of 20 mg / ml purified PRENCSO solution is further added to start the enzyme substrate reaction. After 3 minutes, inject 1 μl of the reaction solution into the HPLC and measure the amount of LF generated.

The following conditions can be applied as the HPLC conditions used here:
Column: Pegasil ODS 4.6 mmΦ × 25 cm (Senshu Kagaku), mobile phase: 7: 3 mixture of acidic water pH3.3 and methanol, temperature: 35 ° C., detection wavelength: 254 nm, flow rate: 0.6 ml / min. Using this condition, the LF peak is detected at a retention time of 9.6 minutes.

  A TFA solution can be used as the acidic water pH 3.3 used as the mobile phase. The manufacturing method is as follows. Pour 2 liters of distilled water into a sealed container and deaerate using an aspirator. Add 1 ml of TFA (special reagent for amino acid sequence analysis Nacalai Tesque 34902-11 in 1 x 5 ampoules) to 2 liters of degassed distilled water to make a stock solution (TFA solution (x10)). The stock solution can be diluted 10-fold with degassed distilled water to make acidic water pH 3.3. As the purified alliinase solution and the purified PRENCSO solution, those prepared by the methods described in Examples 1-1. And 1-2. Below can be used.

  In the present invention, as the low LFS onion, a bulb of the onion (generally called a “bulb” or “bulb”) or a sheath leaf extending from the bulb can be used.

  The low LFS onion can be pulverized by a normal pulverizer such as a homogenizer or a mixer. The low LFS onion pulverized product or a solution obtained by removing solids from the pulverized product can be used as a composition containing the compound of the present invention.

  The first production method may further include a step of extracting the compound of the present invention from a pulverized product of low LFS onion or a solution obtained by removing solids from the pulverized product. Since the compound of the present invention is gradually produced after treating PRENCSO with alliinase, the desired compound cannot be sufficiently obtained even after rapid extraction after pulverization. Therefore, it is preferable to extract the compound from the pulverized low LFS onion after 1 to 120 minutes, preferably 3 to 90 minutes after pulverization.

  Extraction of a compound of the present invention from a low LFS onion grind is obtained by removing water, methanol, ethanol, acetonitrile, acetone, ether, ethyl acetate, hexane, dichloromethane, a solution obtained by removing solids from the low LFS onion grind or ground grind. Extraction using one or a plurality of extraction solvents appropriately selected from chloroform and the like, and a solution obtained by removing solids from a pulverized product of low LFS onion are treated with a synthetic adsorbent to adsorb the compound onto the synthetic adsorbent. And then, the adsorbed compound is eluted with a solution containing a water-soluble organic solvent such as methanol, ethanol, propanol, acetonitrile, and acetone. In particular, since the compound of Formula 1 exhibits water-soluble properties, it can be extracted and eluted using water, methanol, ethanol, acetonitrile, acetone, or the like. In addition, the compound of Formula 2 can be extracted and eluted using, for example, methanol, ethanol, acetonitrile, acetone, ether, ethyl acetate, hexane, chloroform and the like. As the synthetic adsorbent, various adsorbents such as Diaion HP-20 (Mitsubishi Chemical Corporation) and Amberlite XAD-2 (organo Corporation) can be used. The extract thus obtained can be used as a composition containing the compound of the present invention.

  The first production method may further include a step of purifying the compound represented by Formula 1 and / or Formula 2 from the obtained extract. Purification can be performed by means of silica gel column chromatography, HPLC or the like.

を有する。ただし当該天然型とは異なるPRENCSOの立体異性体（例えばスルフォキシドの立体配置が異なる異性体）もまた原料PRENCSOとして利用可能である。 Next, the second manufacturing method will be described in detail.
S-1-propenyl-cysteine sulfoxide (PRENCSO) used as a raw material has the following structure when it is naturally derived:

Have However, a stereoisomer of PRENCSO different from the natural type (for example, an isomer having a different configuration of sulfoxide) can also be used as the raw material PRENCSO.

  PRENCSO used as a raw material may be synthesized or may be derived from onion or the like. Furthermore, a composition containing PRENCSO can be used as a PRENCSO raw material. For example, the onion is heat-treated to deactivate the enzyme or reduce the enzyme activity. Reduction or inactivation of enzyme activity by heat treatment is at least LFS activity is less than 1/20, less than 1/40, less than 1/160, less than 400 minutes compared to LFS activity in unheated onion Or less than 1/800, or less, or disappear. Next, juices and pastes (heated onion juice, heated onion paste) prepared by pulverizing the heat-treated onion are compositions containing PRENCSO and can be subjected to alliinase treatment as a PRENCSO raw material.

  Alliinase (EC4.4.1.4, S-alk (en) yl-L-cysteine sulfoxide lyase) is an enzyme that decomposes alkyl (alkenyl) cysteine sulfoxide into dehydroalanine and sulfenic acid. Named. It is now also called C-S lyase (Source: Food Function of Spice Components Kazuo Iwai, Nobuji Nakatani, Mitsuseikan P174-175 (1989)). The origin of alliinase that can be used in the second production method is not particularly limited, and in addition to onion, garlic, leek, leek, raccoon, etc., cruciferous broccoli and cauliflower, leguminous shrub Albizzia lophantha ( Sources derived from the same as above can be used.

  Conditions for treating PRENCSO with alliinase are not particularly limited. However, since the properties of alliinase vary depending on the origin, the degradation of PRENCSO can be made more efficient by adapting the characteristics of individual alliinase using the reaction conditions. For example, when alliinase is derived from garlic, since the optimum pH range is wide, the pH may be in the range of 5 to 8, and the temperature is optimally 37 ° C. On the other hand, when using an onion-derived alliinase, the enzyme activity is optimized when the pH is adjusted to 7.5 to 8.6, and when using an alliinase derived from the leaf of Welsh onion, the pH is optimized at 7.0 (source: spices) Ingredient food functions Kazuo Iwai, Enji Nakatani, edited by Mitsuseikan P174-175 (1989)). Since alliinase uses pyridoxal phosphate as a coenzyme, use of a buffer to which pyridoxal phosphate is added as in Example 1-2 also helps to optimize the activity of alliinase.

  The reaction solution obtained by decomposing PRENCSO with alliinase thus obtained can be used as a composition containing the compound of the present invention.

  The second production method may further include a step of extracting the compound of the present invention from a reaction solution obtained by decomposing PRENCSO with alliinase. Since the compound of the present invention is gradually produced after treating PRENCSO with alliinase, the target compound cannot be sufficiently obtained even after rapid extraction after the treatment. Therefore, it is preferable to extract the compound after at least 1 to 120 minutes, preferably 3 to 90 minutes after the treatment. In the extraction, the reaction product is centrifuged and separated into a supernatant and a precipitate, and the target compound can be extracted from each. That is, the compound represented by the above formula 1 can be extracted from the supernatant, and the compound represented by the above formula 2 can be extracted from the precipitate. Extraction of the compound represented by the above formula 1 from the supernatant can be performed using an extraction solvent such as water, methanol, ethanol, acetonitrile, and acetone. The extraction of the compound represented by the above formula 2 from the precipitate can be performed using an extraction solvent such as methanol, ethanol, acetonitrile, acetone, ether, ethyl acetate, hexane, chloroform, and the like. The obtained extract can be used as a composition containing a compound represented by each formula.

  The second production method may further include a step of purifying the compound represented by each formula from the extract. Purification can be performed by means of silica gel column chromatography, HPLC or the like.

III. Use Since the compound of this invention has the effect | action which suppresses accumulation | storage of the triglyceride in a liver, it is useful in suppressing onset of a fatty liver.

  The compound of the present invention is a component that is acceptable in a final form such as foods and beverages and pharmaceuticals, and can be in the form of a composition together with a component that can be taken orally.

  The composition containing the compound of the present invention may be in any form such as a food additive or a pharmaceutical composition (including a quasi-drug composition; the same applies hereinafter).

  The form of the pharmaceutical composition is not particularly limited, but is preferably a form suitable for oral administration. For example, solid compositions for oral administration (solid pharmaceutical preparations) include tablets (including sugar-coated tablets), pills, capsules, fine granules, granules and the like, and liquid compositions for oral administration (liquid pharmaceuticals). Formulations may take the form of emulsions, solutions, suspensions, syrups and the like. In these preparations, in addition to the active ingredients, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusters, and the like can be appropriately blended depending on the dosage form. And can be formulated according to conventional methods.

  The pharmaceutical composition may contain other functional ingredients such as vitamins in addition to the above-mentioned compound, which is an active ingredient, and the carrier or additive.

  A food additive contains the said compound, and can be used so that the effective amount of the said compound may be added to food-drinks. Here, the “effective amount” refers to an amount capable of obtaining an effect of suppressing fat accumulation in the liver when an amount normally eaten in each food or drink is ingested.

  The food additive which concerns on this invention can be mix | blended with food-drinks. “Food and beverage products” containing the food additive according to the present invention include health food, functional food, food for specified health use, food for the sick, and the like.

  The dose or intake of the above compound as an active ingredient can be appropriately determined according to the recipient, the age and weight of the recipient, symptoms, administration time, dosage form, administration method, drug combination, and the like.

Example 1. Isolation and purification of compound of formula 1 Example 1-1. Preparation of purified PRENCSO solution (1) Extraction of PRENCSO from heated onion 3 peels (about 1000g) of raw onion, peeled and wrapped into electrons Heated in the range for 12 minutes. The heated onion was put in a mixer, and after adding an equal amount of distilled water, it was roughly crushed, and the crushed liquid was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected, cation exchange resin IR120B (H type) was added and stirred, and then the resin was collected by suction filtration. Distilled water (1 L) was added to the recovered resin, and concentrated aqueous ammonia was added to adjust the pH to 8.5. The supernatant was recovered by suction filtration, and ammonia water having a pH of 8.5 was added to the remaining resin again. Again, the supernatant was collected by suction filtration. The resulting solution was neutralized to pH 7.0 by adding 1N hydrochloric acid. The solution was concentrated to dryness using an evaporator.

(2) Purification of PRENCSO 50 ml of distilled water was added to the residue and dissolved. The resulting crude PRENCSO solution was purified by medium pressure reverse phase chromatography. If necessary, further purification was performed by HPLC (column: ODS, mobile phase: acidic water pH 3.3, temperature: 35 ° C., UV: 230 nm). The eluate obtained from the column was dried using an evaporator and a freeze dryer to obtain purified PRENCSO powder (about 100 mg).

Example 1-2. Preparation of purified alliinase solution (1) Grinding and acid precipitation of garlic First, a mixer jug was placed in a refrigerator and allowed to cool. In addition, the rotor was set in a low-temperature centrifuge, and the temperature was set at 4 ° C. to be cooled. An equal amount of buffer A (described later) was added to garlic pieces (100 g) and pulverized with a mixer. A double gauze placed on the mouth of a mug placed on ice was fastened with a rubber band, and the crushed material was poured over the gauze and filtered. After a certain amount of filtrate was obtained, the ground material on the gauze was wrapped with gauze and squeezed. The obtained filtrate was centrifuged at 4 ° C. and 12000 rpm for 10 minutes, and the supernatant was recovered. While stirring the collected centrifugal supernatant on ice, acetic acid was added while monitoring the pH, and the pH was adjusted to 4.0. When the pH reached 4.0, it was allowed to stand for 5 minutes. The sample from which the precipitate appeared was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the centrifuged pellet was recovered (buffer A was used).

  The collected pellets were made 50 ml to 100 ml (buffer A) and allowed to stand at 5 ° C. for 30 minutes. The collected pellet suspension was centrifuged at 12000 rpm for 10 minutes at 4 ° C. The pH of the collected centrifugal supernatant was adjusted to 6.5 using 1N aqueous sodium hydroxide solution. This was used as the crude alliinase extract.

(2) Hydroxyapatite column treatment The above alliinase crude extract (centrifugal supernatant) was applied to a hydroxyapatite column equilibrated with buffer A. Alliinase was adsorbed as a yellow band on the hydroxyapatite column. The hydroxyapatite column to which the sample was applied was washed with 300 ml of buffer A. 300 ml of buffer C (described later) was passed through the hydroxyapatite column after washing to elute it. The eluate was fractionated by 10 ml using a fraction collector, and the fractions in which the yellow eluate was fractionated were collected.

(3) Purification of alliinase by ConA column A 20 mM volume of 20 mM calcium chloride and magnesium chloride solution was added to the collected fraction (20 ml) to increase the calcium ion and magnesium ion concentrations of the sample solution. After that, it was regenerated and applied to a ConA column equilibrated with starting buffer (recommended buffer described in the ConA Sepharose 4B manual). The ConA column after applying the sample was washed with 50 ml of starting buffer. 50 ml of ConA elution buffer (recommended buffer described in the ConA Sepharose 4B manual) was passed through the ConA column after washing to elute. The eluate was fractionated by 2 ml using a fraction collector, and the fractions in which the yellow eluate was fractionated were collected.

(4) Method for concentrating purified alliinase
10 ml of the yellow eluate (alliinase solution) obtained by the ConA column purification was placed in CENTRIPLUS CONCENTRATORS (up to 15 ml, No. 4421) (Millipore). CENTRIPLUS CONCENTRATORS was set in a centrifuge cooled to 4 ° C. and centrifuged at 3000 rpm for 30 minutes. After confirming the contents once, it was centrifuged again at 3000 rpm for 30 minutes. Concentrated alliinase was appropriately diluted with buffer D to 250 or 500 U / ml and stored at −80 ° C. until use.

[Buffer A]
The above buffer A (pH 7.0) (50 mM buffer A for purifying alliinase) was prepared as follows. A 50 mM dipotassium hydrogen phosphate solution (5.22 g dissolved in 600 ml) and a 50 mM potassium dihydrogen phosphate solution (3.4 g dissolved in 500 ml) were prepared. While monitoring the pH, both were mixed to adjust the pH to 7.0. Glycerol was added 1x vol to 9x vol of the finished buffer and mixed well. 5.3 mg of pyridoxal phosphate was added to and mixed with 1 L of the glycerol-containing buffer. The completed buffer was stored at 10 ° C.

[Buffer C]
The buffer C (pH 7.0) (500 mM buffer C for purifying alliinase) was prepared as follows. A 500 mM dipotassium hydrogen phosphate solution (43.6 g dissolved in 500 ml) and a 500 mM potassium dihydrogen phosphate solution (34.0 g dissolved in 500 ml) were prepared. While monitoring the pH, both were mixed to adjust the pH to 7.0. Glycerol was added 1x vol to 9x vol of the finished buffer and mixed well. 5.3 mg of pyridoxal phosphate was added to and mixed with 1 L of the glycerol-containing buffer. The completed buffer was stored at 10 ° C.

[Buffer D]
The above buffer D (pH 6.5) (100 mM buffer D for dilution of alliinase) was prepared as follows. A 100 mM dipotassium hydrogen phosphate solution (10.44 g dissolved in 600 ml) and a 100 mM potassium dihydrogen phosphate solution (6.8 g dissolved in 500 ml) were prepared. While monitoring the pH, both were mixed to adjust the pH to 6.5. Glycerol was added 1x vol to 9x vol of the finished buffer and mixed well. 5.3 mg of pyridoxal phosphate was added to and mixed with 1 L of the glycerol-containing buffer. The completed buffer was stored at 10 ° C.

Examples 1-3. Induction and isolation purification of the compound of formula 1
1200 μl of 250 U / ml alliinase solution was taken in a 2 ml microtube. The enzyme reaction was started by adding 800 μl of 20 mg / ml PRENCSO aqueous solution. Ninety minutes after the start of the reaction, the tube was centrifuged at 15000 rpm for 5 minutes, and the supernatant and the precipitate were collected.

About the obtained supernatant, the late reaction product-SUP was isolated and purified using a large-scale preparative HPLC system manufactured by Shimadzu Corporation. HPLC conditions were as follows: detection wavelength: 210 nm, column: GL Sciences Inertsil ODS-3 250 mm X 20 mm, 5 μm, mobile phase (CH ₃ CN / H ₂ O): 0 min (16% / 84%), 16 min ( 16% / 84%), 35 minutes (35% / 65%), 36 minutes (90% / 10%), 41 minutes (90% / 10%), 42 minutes (16% / 84%), 65 minutes ( 16% / 84%), flow rate: 15 ml / min.

  Of fraction 3 (Fr-3) from HPLC retention time 20 minutes to 32 minutes, Peak 3 (HPLC retention time 27.5 minutes to 28.5 minutes) was fractionated (FIG. 2). The fraction containing Peak 3 was concentrated and dried using an evaporator, and then analyzed using NMR and HPLC-Orbitrap. As a result, it was found that it had a planar structure of Formula 1.

  In addition, Peak 1, Peak 2 and Peak 4 in fraction 3 showed the same spectrum as Peak 3 as a result of MS spectrum analysis using HPLC-Orbitrap, confirming that it is an isomer fraction of Peak 3. (Figures 3-1 and 3-2).

The compound having the planar structure of Formula 1 that appears as Peak 3 has the following physicochemical properties.
(1) Appearance: colorless powder (2) HR MS [M + H] ⁺ : m / z 221.0661 (221.0664 calcd. For C ₉ H ₁₇ O ₂ S ₂ )
(3) Molecular formula: C ₉ H ₁₆ O ₂ S ₂
(4) Solubility: water, methanol, ethanol, acetonitrile (5) UV absorption spectrum: λ _max nm 230 (in 43% CH ₃ CN)
(6) ¹ H NMR spectrum: δ 6.53, 6.45, 5.19, 4.08, 2.31, 2.03, 1.93, 1.24, 1.05
(7) ¹³ C NMR spectrum: δ 139.10, 131.66, 84.39, 73.70, 53.52, 42.59, 17.51, 16.64, 12.43
(8) HPLC retention time: 15.5-15.9 minutes (peak top detection time: 15.67 minutes)
(HPLC conditions) HPLC: Thermo Fisher Scientific Ultimate 3000, column: Thermo Fisher Scientific ODS Hypersil 250 mm X 4.6 mm, 5 μm, column oven: 30 ° C., mobile phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 minutes (18% / 82%), 45 minutes (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 60 minutes (18% / 82%) ( Specifically, the CH ₃ CN concentration was increased from 18% to 90% over a gradient from 0 to 45 minutes, and CH ₃ CN was maintained at 90% for 5 minutes from 45 minutes to 50 minutes. Immediately after 51 minutes, CH ₃ CN concentration was switched to 18% and flowed up to 60 minutes), flow rate: 0.5 ml / min, detection 190-650 nm
(Orbitrap condition) Ionaization mode: Positive, Heater temperature: 200 ° C, Spray voltage: 3.5 kV, Capillary temperature: 300 ° C, Sheath gas: 50, Auxiliary gas: 15, Collision energy: 35

Example 2. Induction of a compound of formula 1 (1) From a mixture of alliinase and PRENCSO
30 μl of 250 U / ml alliinase solution was taken in a 1.5 ml microtube. 20 μl of 20 mg / ml PRENCSO aqueous solution was added thereto to start the enzyme reaction. Three minutes or 90 minutes after the start of the reaction, 150 μl of methanol containing 10 μg / ml formononetin was added and mixed for 30 seconds. The tube was centrifuged at 15000 rpm for 3 minutes, and the obtained supernatant was analyzed under the following conditions using HPLC-Orbitrap manufactured by Thermo Fisher Scientific.

[HPLC conditions]
HPLC: Thermo Fisher Scientific Ultimate 3000, column: Thermo Fisher Scientific ODS Hypersil 250 mm X 4.6 mm, 5 μm, column oven: 30 ° C, mobile phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 min (18 % / 82%), 45 minutes (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 60 minutes (18% / 82%) (specifically , at 0 to 45 minutes, 5 minutes from 18% to CH ₃ CN concentration with a gradient up to .45 and 50 minutes, which was raised to 90%, holding the CH ₃ CN at 90%. immediately after the 51 minutes , CH ₃ CN concentration was switched to 18% and flowed up to 60 minutes), flow rate: 0.5 ml / min, detection 190-650 nm
[Orbitrap condition]
Ionaization mode: Positive, Heater temperature: 200 ° C, Spray voltage: 3.5 kV, Capillary temperature: 300 ° C, Sheath gas: 50, Auxiliary gas: 15, Collision energy: 35

  The results are shown in FIG. A peak was observed at an HPLC retention time of 13-17 minutes, confirming that the compound of formula 1 was derived. In addition, it was confirmed that the compound of formula 1 was sufficiently present 90 minutes after the start of the reaction, whereas the Zwiebelane isomer was significantly reduced 90 minutes after the start of the reaction. This indicates that the compound of Formula 1 is a more stable compound in an aqueous solution than the Zwiebelane isomer.

(2) From a mixture of garlic juice and heated onion juice Garlic juice was prepared by crushing garlic bulbs and pods extending from bulbs. Moreover, the heated onion juice was prepared by heat-treating bulbs of onions and pods extending from the bulbs, followed by pulverization.

  30 μl of garlic juice was taken in a 1.5 ml microtube. 300 μl of heated onion juice was added thereto to start the enzyme reaction. 90 minutes after the start of the reaction, 990 μl of 10 μg / ml formononetin-containing methanol was added, and the tube was centrifuged at 15000 rpm for 5 minutes. The resulting supernatant was described in “(1) From the mixture of alliinase and PRENCSO” above. The analysis was performed under the following HPLC-Orbitrap conditions. However, for detection, ion chromatogram extraction was performed with m / z 203, which is the product ion of the compound of formula 1.

  The results are shown in FIG. A peak was observed at an HPLC retention time of 13-17 minutes, confirming that the compound of formula 1 was derived.

Example 3. Isolation and purification of compound of formula 2 The precipitate recovered in Example 1-3 was dissolved in 1 ml of methanol, and a late reaction product-PPT was prepared using a large volume preparative HPLC system of Shimadzu Corporation. Was isolated and purified. HPLC conditions are as follows: detection wavelength: 210 nm, column: GL Sciences Inertsil ODS-3 250 mm X 20 mm, 5 μm, mobile phase (CH ₃ CN / H ₂ O): 0 min (50% / 50%), 40 min (90% / 10%), 45 minutes (90% / 10%), 46 minutes (50% / 50%), 70 minutes (50% / 50%), flow rate: 10 ml / min.

  HPLC A retention time of Peak A from 23 to 26 minutes and Peak B from 28.5 to 31 minutes were fractionated (FIG. 6). About each fraction containing Peak A and Peak B which were fractionated, as a result of concentration and drying using an evaporator, and analysis using NMR and HPLC-Orbitrap, Peak A has a plan structure having the planar structure of Formula 2 (a). It was possible to specify that Peak B had a sulfide type structure, and that Peak B had a tetrasulfide type structure having a planar structure of Formula 2 (b).

The compound having a planar structure of the formula 2 (a) appearing as Peak A has the following physicochemical properties.
(1) Appearance: colorless oil (2) HR MS [M + HCOO] ⁻ : m / z 403.0205 (403.0205 calcd. For C ₁₃ H ₂₃ O ₄ S ₅ )
(3) Molecular formula: C ₁₂ H ₂₂ O ₂ S ₅
(4) Solubility: methanol, ethanol, acetonitrile, acetone, ether, ethyl acetate, hexane, chloroform (5) UV absorption spectrum: λ _max nm 280 (in 74.8% CH ₃ CN)
(6) ¹ H NMR spectrum: δ 5.18, 5.14, 4.49, 4.34, 2.12, 1.85, 1.84, 1.73, 1.18, 1.16, 1.10, 1.04
(7) ¹³ C NMR spectrum: δ 86.57, 83.41, 65.99, 64.19, 52.81, 51.20, 49.08, 47.25, 15.15, 14.79, 14.76, 12.68
(8) HPLC retention time: 35 to 36 minutes (peak top detection time: 35.65 minutes)
(HPLC conditions) HPLC: Thermo Fisher Scientific Ultimate 3000, column: Thermo Fisher Scientific ODS Hypersil 250mm X 4.6mm, 5μm, column oven: 30 ° C, mobile phase (CH ₃ CN / 10mM HCOONH ₄ H ₂ O): 0 min ( 18% / 82%), 45 minutes (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 60 minutes (18% / 82%) (specifically Increased the CH ₃ CN concentration from 18% to 90% with a gradient from 0 to 45 minutes, and maintained CH ₃ CN at 90% for 5 minutes from 45 minutes to 50 minutes. The CH ₃ CN concentration was switched to 18% and flowed up to 60 minutes), flow rate: 0.5 ml / min, detection 190-650 nm
(Orbitrap condition) Ionaization mode: Negative, Heater temperature: 200 ° C, Spray voltage: 2.5 kV, Capillary temperature: 275 ° C, Sheath gas: 50, Auxiliary gas: 15, Collision energy: 35

A compound having a planar structure of the formula 2 (b) that appears as Peak B has the following physicochemical properties.
(1) Appearance: colorless oil (2) HR MS [MH] ^- : m / z 388.9871 (388.9871 calcd. For C ₁₂ H ₂₁ O ₂ S ₆ )
(3) Molecular formula: C ₁₂ H ₂₂ O ₂ S ₆
(4) Solubility: methanol, ethanol, acetonitrile, acetone, ether, ethyl acetate, hexane, chloroform (5) UV absorption spectrum: λmax nm 300 (in 80.4% CH ₃ CN)
(6) ¹ H NMR spectrum: δ 5.19, 5.17, 4.52, 4.39, 2.15, 1.89, 1.86, 1.74, 1.20, 1.16, 1.11, 1.05
(7) ¹³ C NMR spectrum: δ 86.63, 83.55, 66.14, 64.13, 52.74, 51.28, 49.34, 47.54, 15.09, 14.92, 14.69, 12.69
(8) HPLC retention time: 38.5-39.5 minutes (peak top detection time: 38.96 minutes)
(HPLC conditions) HPLC: Thermo Fisher Scientific Ultimate 3000, column: Thermo Fisher Scientific ODS Hypersil 250mm X 4.6mm, 5 μm, column oven: 30 ° C, mobile phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 min (18% / 82%), 45 minutes (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 60 minutes (18% / 82%) (specific The CH ₃ CN concentration was increased from 18% to 90% with a gradient from 0 to 45 minutes, and CH ₃ CN was maintained at 90% for 5 minutes from 45 minutes to 50 minutes. From the minute, the CH ₃ CN concentration was switched to 18% and flowed up to 60 minutes), flow rate: 0.5 ml / min, detection 190-650 nm
(Orbitrap condition) Ionaization mode: Negative, Heater temperature: 200 ° C, Spray voltage: 2.5 kV, Capillary temperature: 275 ° C, Sheath gas: 50, Auxiliary gas: 15, Collision energy: 35

Example 4. Derivation of the compound of formula 2 (1) From a mixture of alliinase and PRENCSO The supernatant obtained in “(1) From a mixture of alliinase and PRENCSO” in Example 2 was obtained from Thermo Fisher Scientific. Analysis was performed under the same conditions using HPLC-Orbitrap.
[HPLC conditions]
HPLC: Thermo Fisher Scientific Ultimate 3000, Column: Thermo Fisher Scientific ODS Hypersil 250mm X 4.6mm, 5 μm, Column Oven: 30 ° C, Mobile Phase (CH ₃ CN / 10 mM HCOONH ₄ H ₂ O): 0 min (18% / 82%), 45 minutes (90% / 10%), 50 minutes (90% / 10%), 51 minutes (18% / 82%), 60 minutes (18% / 82%) (specifically, 0 The CH ₃ CN concentration was increased from 18% to 90% with a gradient from min to 45 min.CH ₃ CN was held at 90% for 5 min from 45 min to 50 min. ₃ CN concentration was switched to 18% and flowed up to 60 minutes), flow rate: 0.5 ml / min, detection 190-650 nm
[Orbitrap condition]
Ionaization mode: Negative, Heater temperature: 200 ° C, Spray voltage: 2.5 kV, Capillary temperature: 275 ° C, Sheath gas: 50, Auxiliary gas: 15, Collision energy: 35

  The results are shown in FIG. A peak was observed at an HPLC retention time of 35 to 46 minutes, confirming that the compound of formula 2 was derived. Further, like the compound of formula 1, it was confirmed that the compound of formula 2 was sufficiently present 90 minutes after the start of the reaction. This indicates that the compound of Formula 2 is a more stable compound in an aqueous solution than the Zwiebelane isomer. Peak C1, C2, Peak D1, D2 detected by HPLC-Orbitrap analysis may have a pentasulfide type or hexasulfide type structure as a result of compositional formula calculated from accurate mass and MS spectrum analysis. It was confirmed (Figs. 7-1, 7-2, 7-3).

(2) From the mixture of garlic juice and heated onion juice For the supernatant obtained in “(2) From the mixture of garlic juice and heated onion juice” in Example 2, the above “mixed solution of alliinase and PRENCSO” HPLC-Orbitrap analysis was performed under the same conditions as above. However, ion chromatogram extraction was performed at m / z 227 and 259 which are product ions of the compound of formula 2.

  The results are shown in FIG. A peak was observed at an HPLC retention time of 35-40 minutes, confirming that the compound of formula 2 (a) and the compound of formula 2 (b) were derived.

Example 5. Lipid metabolism improving effect by the compound of Formula 2 (1) Preparation of PRENCSO high-content solution Onion (peeled onion peeled off into thin strips of raw onion, cut into 4 equal parts along the fiber, and cut into 1/4 size) 7.9 kg) was immersed in 10 mM citrate buffer (pH 3.0) (7.9 L) and blanched at 70 ° C. for 2 hours.

  The crushed liquid obtained after blanching was cooled and crushed with a mixer was centrifuged at 8000 rpm for 10 minutes, and 13 L of the supernatant was recovered. The obtained supernatant was neutralized with 1N aqueous sodium hydroxide solution (200 mL) to pH 7.0 to obtain a PRENCSO-rich solution (13 L) in which alliinase and LFS were inactivated.

(2) Induction of the compound of Formula 2 To the PRENCSO-rich solution obtained in (1) above, 65 g of 1/200 equivalent raw garlic paste as an alliinase source was added and stirred to start the enzyme reaction. After 60 minutes, The reaction solution was collected. A part of the collected reaction solution was analyzed using HPLC-Orbitrap manufactured by Thermo Fisher Scientific, and it was confirmed that the compound of Formula 2 could be produced as a main product.

The reaction solution was frozen at −80 ° C. and then powdered using a freeze dryer. The obtained powder (about 1140 g) was stored in a freezer at −20 ° C. until use.
The operations (1) and (2) were repeated to produce the amount necessary for the following test.

(3) Evaluation of lipid metabolism improvement effect by compound of formula 2 (i) Method The lipid metabolism improvement effect of compound of formula 2 was evaluated using SD rats (male). That is, seven 5-week-old Slc: SD (SPF) males (Japan SLC Co., Ltd.) per group were reared for one week with free intake of commercially available solid feed CRF-1 (Oriental Yeast Co., Ltd.) and water. After that, the control group and the test group were allocated by “stratified randomized allocation” so that the body weight was uniform in each group.

  The control group was then fed a high fat diet mixed at a protein: fat: carbohydrate ratio of 21:59:20 three times a week.

On the other hand, the test substance was administered to the test group in addition to the high fat diet.
As the test substance, a suspension obtained by suspending the powder of the composition containing the compound of formula 2 prepared in the above (2) in a citrate buffer (pH 7.0) at 0.33 mg / mL was used.
The final citric acid concentration in the test substance was adjusted to 23.9 mM.

  In the control group, 23.9 mM citrate buffer (pH 7.0) was used as a test substance control substance.

  The test substance and the control substance citrate buffer were stored frozen and thawed in running water at the time of use.

  The dose of citrate buffer as the test substance and control substance was 10 mL / kg. The liquid volume for administration was set to the 0.1 mL unit by rounding off the second decimal place of the calculated value calculated based on the latest body weight.

  The test substance and the citrate buffer serving as a control substance were orally administered by gavage once a day between 9 am and 12 am using a disposable syringe and an oral sonde. The administration period was 56 days.

  During the test period, the general condition was observed once a day. From the start of administration, body weight and food consumption were measured at a frequency of 3 times a week. The animals were fasted from 16:00 on the 55th day after administration, and the animals were opened on the next day under 2% inhalation anesthesia with isoflurane. The collected liver was stored frozen at −80 ° C. until used for various measurements.

  The liver is crushed in a 4-fold volume of tetrahydrofuran: acetone (4: 1) mixed solvent, centrifuged at room temperature (15000 rpm for 5 minutes) to obtain a supernatant, which is filtered with a 0.2 μm mesh disk filter. The filtered product was designated as “test solution”.

  About the obtained liver test solution, the amount of triglyceride (TG) was quantified using HPLC-Orbitrap of Thermo Fisher Scientific.

(Ii) Results The changes in body weight and food intake of each group are shown in FIGS. 9 and 10, respectively. An increase in body weight and food consumption over time was observed in each group, and no significant difference was confirmed between the two groups.

  The amount of TG in the liver of each group is shown in FIG. The measured value was expressed as mean value ± standard error, and the statistical analysis used Dunnett's test because uniformity was recognized by the Bartlett test. The significance level was expressed as 5% (*).

  The amount of TG in the liver in the test group was confirmed to be significantly lower than that in the control group.

  From these results, it was shown that the intake of the compound of formula 2 can suppress an increase in the amount of TG in the liver even when a high fat diet is ingested, and the accumulation of fat in the liver is suppressed. The effect of the compound of Formula 2 was confirmed in rats at a dose of 3.3 mg / kg, but when this dose is converted to a human dose, it is calculated to be approximately 66 μg / kg. Therefore, it was shown that fat accumulation in the human liver can be suppressed by administering or ingesting the compound of Formula 2 to humans based on the amount.

Claims (7)

Formula I:

(Where
R ₁ is hydrogen or a lower alkyl group,
R ₂ and R ₃ are the same or different and are lower alkyl groups,
R ₄ is

And
R ₅ and R ₆ are the same or different and are lower alkyl groups,
R ₇ is hydrogen or a lower alkyl group,
n is an integer of 3-6. )
The composition for suppressing fat accumulation to the liver characterized by containing the compound or its salt represented by these. The compound is of formula 1:

The composition of Claim 1 represented by these. The compound is of formula 2 (a):

Or Formula 2 (b):

The composition of Claim 1 represented by these.   The composition according to any one of claims 1 to 3, which is a pharmaceutical composition.   The composition according to any one of claims 1 to 3, which is a food additive.   A food or drink comprising the composition according to claim 5. Formula I:

(Where
R ₁ is hydrogen or a lower alkyl group,
R ₂ and R ₃ are the same or different and are lower alkyl groups,
R ₄ is

And
R ₅ and R ₆ are the same or different and are lower alkyl groups,
R ₇ is hydrogen or a lower alkyl group,
n is an integer of 3-6. )
The manufacturing method of the food / beverage products which have the effect | action which suppresses the fat accumulation to the liver including mix | blending the compound or its salt represented by these with the material of food / beverage products.

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