JP7338165B2 - Food composition for suppressing hepatic fat synthesis - Google Patents

Description

TECHNICAL FIELD The present invention relates to a food composition for suppressing hepatic fat synthesis.

Fatty liver is known to cause a vicious circle with diabetes and cardiovascular disease. An increase in hepatic lipogenesis plays an important role as a trigger for fatty liver, and liposynthetic enzymes are known as the control mechanism.
For the prevention and improvement of fatty liver, it is effective to suppress the synthesis of lipids in the liver and to suppress the accumulation of lipids by enhancing metabolism.

In recent years, various naturally-derived functional ingredients have been searched for, and there are reports of ingredients that act on lipid metabolism and can reduce the amount of lipids in the liver. Patent Documents 1 and 2 disclose that soybean protein or dried pulverized plant germ has the effect of reducing triglycerides in the blood. However, these documents do not confirm the effect of suppressing fat accumulation in the liver.

In addition, it has been found that intake of vegetable protein improves fatty liver through suppression of the expression of liposynthetic enzymes, and that peptide degradation products suppress the expression of liposynthetic enzymes in hepatocytes. (Non-Patent Document 1).

International publication WO2002/026243 JP-A-2006-69943

Watanabe H, Inaba Y, Kimura K, Asahara SI, Kido Y, Matsumoto M, Motoyama T, Tachibana N, Kaneko S, Kohno M, Inoue H. Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet -Induced, Nonalcoholic Fatty Liver Disease. J Nutr. 2017 Jan;147(1):52-60. doi: 10.3945/jn.116.231662.

The prior art does not disclose any component exhibiting an inhibitory effect on hepatic fat synthesis.
An object of the present invention is to provide a food composition containing an ingredient having hepatic fat synthesis inhibitory action.

The present inventors have made intensive studies to solve the above problems, and found that Ala-Gly, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp- We found a food composition for suppressing hepatic fat synthesis containing, as an active ingredient, one or more dipeptides selected from Lys, Tyr-Lys, Tyr-Ser and Tyr-Tyr, and completed the present invention. Ta.

That is, the present invention
(1) selected from Ala-Gly, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp-Lys, Tyr-Lys, Tyr-Ser or Tyr-Tyr A food composition for suppressing hepatic fat synthesis, containing one or more dipeptides as active ingredients,
(2) selected from Ala-Gly, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp-Lys, Tyr-Lys, Tyr-Ser or Tyr-Tyr hepatic lipogenesis inhibitors containing one or more dipeptides as active ingredients;
is.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a food composition containing, as active ingredients, 11 types of dipeptides exhibiting hepatic fat synthesis inhibitory effects.

1 is a reference diagram of a method for generating Fasn reporter mice in primary screening. FIG. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#1 to #50). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#51 to #100). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#101 to #150). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#151 to #200). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#201 to #250). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#251 to #300). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 is a diagram showing the results of measuring the Gaussia luciferase activity relative to a blank in primary screening of dipeptides in the dipeptide library (#301 to #336). A circle indicates the relative activity value of each sample, and a black circle indicates a sample showing a significantly lower value than the blank. FIG. 10 shows the results of measuring Fasn gene expression levels in secondary screening of 22 dipeptides. A black bar graph indicates a sample in which the Fasn gene expression level was significantly reduced compared to the blank (denoted as Veh). FIG. 10 shows the results of measuring Fasn gene expression levels in tertiary screening of 11 dipeptides. A black bar graph indicates a sample in which the Fasn gene expression level was significantly reduced compared to the blank (denoted as Veh). FIG. 10 shows the results of measuring Fasn gene expression levels in tertiary screening of 11 dipeptides. A black bar graph indicates a sample in which the Fasn gene expression level was significantly reduced compared to the blank (denoted as Veh).

(Food composition for suppressing hepatic fat synthesis)
The food composition for suppressing hepatic fat synthesis of the present invention contains 11 kinds of dipeptides (Ala-Gly, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp-Lys, Tyr -Lys, Tyr-Ser or Tyr-Tyr) as an active ingredient. One or more of these dipeptides may be contained in the food composition for suppressing hepatic fat synthesis.

The mechanism by which the dipeptide of the present invention exerts the hepatic lipogenesis inhibitory action is expression of the mRNA (Fasn) of Fatty acid synthase (FAS), an enzyme that synthesizes fatty acids that are synthesized de novo in the liver. is presumed to suppress fat synthesis by suppressing

In addition, the dipeptide of the present invention may be expressed using the symbols of amino acid abbreviations as follows.
Ala-Gly: AG, His-Asn: HN, His-Ser: HS, His-Thr: HT, His-Trp: HW, Val-Met: VM, Trp-Glu: WE, Trp-Lys: WK, Tyr- Lys: YK, Tyr-Ser: YS, Tyr-Tyr: YY.

The dipeptide of the present invention can be synthesized from amino acids or obtained by hydrolyzing proteins. Means for hydrolyzing proteins include hydrolysis by proteases, hydrolysis by acids, hydrolysis by alkalis, etc. Hydrolysis by proteases is preferred.
As raw materials for dipeptides, vegetable proteins and animal proteins can be used. For example, raw materials for vegetable proteins include legumes such as soybeans, peas, mung beans and chickpeas, grains such as rice, corn and wheat, and almonds. , cashew nuts, walnuts, pistachios, hazelnuts, macadamia nuts and the like, and examples of animal protein raw materials include beef, pork, chicken, eggs and milk.

(Protease)
In the case of protein hydrolysis by protease, the slurry or aqueous solution of the protein is used as a substrate and subjected to protease treatment. The protease used here is categorized as "metalloprotease", "acidic protease", "thiol protease" and "serine protease" in the classification of proteases, regardless of animal, plant or microbial origin, preferably "metalloprotease". It can be appropriately selected from proteases classified into "protease", "thiol protease", and "serine protease".

This classification of proteases is a classification method according to the type of amino acid in the active center, which is commonly used in the field of enzyme science. As representatives of each, "metalloprotease" includes Bacillus neutral protease, Streptomyces neutral protease, Aspergillus neutral protease, "Samoase", etc. "Acid protease" includes pepsin, Aspergillus acid protease, "Sumitum AP", etc. "Protease" includes bromelain, papain and the like, and "serine protease" includes trypsin, chymotrypsin, subtilisin, Streptomyces alkaline protease, "alcalase", "bioprase" and the like. Enzymes other than these can also be classified according to their working pH and reactivity with inhibitors. Enzymes with different active centers have significantly different sites of action on substrates, so that "leftovers" can be reduced and enzymatic degradation products can be efficiently obtained. Alternatively, by using enzymes of different origins (originating organisms) in combination, enzymatic degradation products can be produced more efficiently.

The reaction pH and reaction temperature for the protease treatment may be set according to the properties of the protease to be used, and usually the reaction pH should be around the optimum pH, and the reaction temperature should be around the optimum temperature. The reaction can be carried out at a reaction temperature of generally 20 to 80°C, preferably 40 to 60°C. After the reaction, the mixture is heated at a temperature sufficient to deactivate the enzyme (approximately 60-170°C) to deactivate the remaining enzyme activity.

The reaction solution after protease treatment can be used as it is or after being concentrated, but usually it can be used in the form of a dry powder after being sterilized and spray-dried, freeze-dried, or the like. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170°C, more preferably 130 to 170°C. The heating time is preferably 3 to 20 seconds. In addition, the reaction solution can be adjusted to an arbitrary pH, and precipitates and suspended matter generated during pH adjustment can be removed by centrifugation, filtration, or the like. Further, it can be purified with activated carbon or adsorption resin.

(Hydrolysis with acid or alkali)
Conditions such as substrate concentration, amount of enzyme, treatment temperature, pH, time, etc. in acid or alkali hydrolysis of protein hydrolysis can be appropriately set.

The food composition of the present invention can be used in the form of pharmaceuticals or in the form of food additives, by mixing with other raw materials if necessary. When provided as a pharmaceutical form, it can be used in various forms such as liquids, powders, tablets and capsules. When provided in the form of being mixed with food, it can be used by mixing with solid foods such as biscuits, cakes, bread, food bars, and meat products, and can be dissolved in water and used as a drink, or yogurt, There is no problem even if it is mixed with liquid or semi-solid food such as pudding. It can also be used as a supplement by mixing carbohydrates, vitamins, minerals, and the like.

The invention is illustrated by the following examples. It should be noted that % in the examples means the capacity standard unless otherwise specified.

(Mouse Breeding Method)
Mice for collecting hepatocytes were kept in a constant temperature environment under a 12-hour light-dark cycle under controlled lighting, with free access to water and food. Hepatocyte harvesting was performed on 8- to 12-week-old mice by collagenase perfusion under anesthesia under pentobarbital and isoflurane. Reporter mice were prepared as described below, and wild-type mice were C57BL/6J mice having no transgene purchased from Japan SLC Co., Ltd.

(Primary screening)
Among the 336 types of dipeptide library (manufactured by Anaspec), we will examine those that may be involved in the suppression of hepatic lipogenesis. In the primary screening, in order to roughly confirm whether there is an inhibitory effect on hepatic lipogenesis, cultured hepatocytes derived from a reporter mouse into which the Fasn (Fatty acid synthase) gene, a fatty acid synthase gene, was introduced were used. Fasn gene expression was assessed by measuring Fasn promoter activity. A detailed method is shown below.

(Generation of liver Fasn reporter mice)
YFP (yellow fluorescent protein) and poly A having loxP sequences (34 bp DNA sequences derived from the bacteriophage P1 genome) at both ends of the Fasn gene promoter were added to the full-length 240.2 kb DNA containing the Fasn gene locus. A gene cassette containing strands is inserted, followed by Gaussia luciferase (GLuc) and polyA strands to create mouse transfer DNA. This mouse-introduced DNA was introduced into a fertilized egg collected from a C57BL/6J mouse to generate mouse A. Mouse A was crossed with C57BL/6J mouse to obtain progeny, and then crossed with mouse B into which the rat albumin promoter and Cre recombinase were introduced. Create as a mouse. In this reporter mouse, a gene cassette containing YFP (yellow fluorescent protein) and poly A chain was shed specifically in the liver tissue by the Cre recombinase, which is expressed in a liver-specific manner by the rat albumin promoter.
Gaussia luciferase (GLuc) expression by the Fasn gene promoter is obtained in a liver tissue-specific manner (Fig. 1). This Fasn gene promoter causes an increase in the activity of Gaussia luciferase (GLuc) along with an increase in the expression of liposynthetic enzymes in the liver.

(evaluation)
Hepatocytes isolated from liver Fasn reporter mice were cultured in a 96-well plate in Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin and streptomycin and 10% fetal calf serum (FCS) for 8 hours. of Dulbecco's modified Eagle's medium (DMEM) containing penicillin and streptomycin, and starvation for 16 hours.
The medium was collected and cultured for 24 hours in a medium containing insulin, LXR (liver X receptor) agonist, amino acid mixture and each dipeptide (100 μg/ml in medium). In addition, 10% DMSO (dimethylsulfoxide) was added instead of the dipeptide and cultured for 24 hours in the same manner.
Addition of insulin, an LXR agonist, and an amino acid mixture to the medium and culturing activates the promoter that promotes the expression of the Fasn gene involved in fatty acid synthesis. When the promoter is activated, the Gaussia luciferase (GLuc) gene is expressed and its activity is measured.
Gaussia luciferase activity was measured using a luminometer LB941 (manufactured by Bertold) according to the method described in Biochem. Biophys. Res. Commun. 365, 96-101 (2007) using coelenterazine as a luminescent substrate.
The measured value of the dipeptide-added group or the measured value of only 10% DMSO addition (dipeptide-free group) is measured, and the measured value of the dipeptide-free group is set to 1, and the relative activity of the dipeptide-added group is calculated. Calculate and evaluate.
Statistical analysis software "StatView" (manufactured by SAS Institute) was used for the significance test, and Bonferroni method was used for comparison with the control.
The results are shown in Figures 2-8.

Statistically, 22 types of dipeptides were shown to inhibit Gaussia luciferase activity (FIGS. 2 to 8, Table 1). Therefore, in order to confirm in detail whether these 22 types of dipeptides actually have a lipogenesis inhibitory effect, we synthesized these dipeptides and performed a secondary screening.

(Table 1) 22 types of dipeptides that were effective in the primary screening

(Secondary screening)
The dipeptide used was synthesized using a Fuji Oil proprietary peptide synthesizer (ResPep SL (INTAVIS)). Table 2 shows the synthesized dipeptides.

In the secondary screening, isolated hepatocytes derived from wild-type mice (C57BL/6J mice) were used for evaluation by measuring the mRNA expression level of the Fasn gene.
Specifically, isolated hepatocytes from wild-type mice (C57BL/6J mice) were cultured in 12-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin and streptomycin and 10% fetal calf serum (FCS). ) for 8 hours, replaced with Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin and streptomycin, and starvated for 16 hours.
The medium was collected and cultured for 24 hours in a medium containing insulin, LXR (liver X receptor) agonist, amino acid mixture and each dipeptide (final concentration in medium: 100 μg/ml). In addition, 10% DMSO (dimethylsulfoxide) was added instead of the dipeptide and cultured for 24 hours in the same manner.
After removing the medium from each well, mRNA was extracted. mRNA extraction was performed with SV Total RNA Isolation System (Promega, Z3105), reverse transcription from RNA was performed with PrimeScript RT reagent kit (Takara, RR0037A), and quantitative PCR was performed with SYBR Select Master Mix kit (Life technologies, 4472908). was performed using a CFX384 Real-Time System and a C1000 Thermal Cycler. Methods followed the manuals of the relevant kits and equipment.
The measurement results are shown in FIG.

13 out of 22 dipeptides (Ala-Gly, Ala-His, Ala-Tyr, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp-Lys , Tyr-Lys, Tyr-Ser, Tyr-Tyr) significantly suppressed Fasn gene expression compared to the control.

(Tertiary screening)
In the secondary screening, 13 types of dipeptides that had an inhibitory effect on Fasn gene expression were added in varying amounts (final dipeptide concentrations of 10, 30, and 100 μg/ml), and the results were similar to those in the secondary screening. The expression level of the Fasn gene was evaluated. The measured results are shown in FIGS.

11 out of 13 dipeptides (Ala-Gly, His-Asn, His-Ser, His-Thr, His-Trp, Val-Met, Trp-Glu, Trp-Lys, Tyr-Lys, Tyr-Ser or Tyr-Tyr) significantly suppressed the expression of the Fasn gene compared to the control. From these results, it was found that 11 types of dipeptides have an inhibitory effect on hepatic lipogenesis.

Claims (2)

One or more dipeptides selected from Ala-Gly, His-Asn, His-Ser, His-Thr, Val-Met, Trp-Lys, Tyr-Lys, Tyr-Ser and Tyr-Tyr as an active ingredient A food composition for suppressing hepatic fat synthesis, containing as One or more dipeptides selected from Ala-Gly, His-Asn, His-Ser, His-Thr, Val-Met, Trp-Lys, Tyr-Lys, Tyr-Ser and Tyr-Tyr as an active ingredient Hepatic fat synthesis inhibitor containing as.