JP7612440B2 - Composition for activating peroxisome proliferator-activated receptors - Google Patents

Description

The present invention relates to a composition for activating peroxisome proliferator-activated receptors (PPARs), which exhibits properties that promote the activation of PPARs in vivo.

Soybeans have long been a part of the traditional Japanese diet, and in recent years their use has been reconsidered as they have been found to contain many functional ingredients that contribute to maintaining and promoting health.
The health benefits of soybeans are well known for their lipid metabolism improving effect. For example, it has been shown that soybean isoflavones lower blood lipid levels, soybean dietary fiber (okara) lowers serum total cholesterol levels, soybean protein lowers blood cholesterol and neutral fat levels, inhibits body fat accumulation, and linoleic acid and α-linolenic acid lower serum cholesterol levels.

Recent research has also revealed that activation of the nuclear receptor PPAR contributes to the control of lipid metabolism. For this reason, active research and development of PPAR activators that activate lipid metabolism has been conducted. Examples that have been reported include gallic acid esters (Patent Document 1), galloyl tannins, catechin and epicatechin (Patent Document 2), organic solvent extracts of tempeh-fermented soybeans (Patent Document 3), and monoacylglycerols whose constituent fatty acids are oleic acid or linoleic acid (Patent Document 4).

Also, synthetic substances that activate PPAR, such as fibrate compounds, thiazolidine derivatives, fatty acids, leukotriene B4, indomethacin, ibuprofen, fenoprofen, and 15-deoxy-Δ12,14-prostaglandin J2, are known to activate PPAR.

JP 2002-80362 A JP 2011-105763 A JP 2012-171911 A JP 2001-354558 A

Under these circumstances, the inventors have conducted extensive research into a composition for activating PPAR that is suitable for ingestion by adding to foods and the like without affecting the taste or flavor, and is also safe and suitable for continuous ingestion.

The present invention aims to provide a composition for activating PPAR that is suitable for ingestion by adding to food, etc., and is also suitable for continuous ingestion due to its excellent safety.

In addition, the present invention focuses on soybeans, which have excellent lipid metabolism, and provides a PPAR activation composition made from soybeans, which has an improved PPAR activation effect.

The present inventors have conducted extensive research to solve the above problems. In the course of their research, they have unexpectedly discovered that sucrose fatty acid esters have a property of promoting PPAR activation, which was previously unknown. Sucrose fatty acid esters are suitable for ingestion by adding them to foods and the like because they do not affect the taste or flavor, and are also excellent in safety and suitable for continuous ingestion. Therefore, by containing sucrose fatty acid esters as an active ingredient, a composition for activating PPAR that is superior to conventional compositions can be obtained.
It has also been found that the PPAR activity of processed foods made from soybeans can be significantly improved by adding sucrose fatty acid esters to soybean raw materials.

That is, the gist of the present invention is the following [1] to [3].
[1] A composition for activating PPAR, comprising a sucrose fatty acid ester as an active ingredient.
[2] The composition for activating PPAR according to [1], wherein the constituent fatty acid of the sucrose fatty acid ester is stearic acid.
[3] The composition for activating PPAR according to [1] or [2], which is a mixture of the sucrose fatty acid ester and soybean flour.

The PPAR activating composition of the present invention contains sucrose fatty acid ester as an active ingredient, so it does not affect the taste or flavor and is suitable for ingestion by adding it to food, etc., and is also suitable for continuous ingestion due to its excellent safety.

Next, we will explain the form for implementing the present invention. However, the present invention is not limited to this form.

As described above, the composition for activating PPAR of the present invention contains a sucrose fatty acid ester as an active ingredient.
Sucrose fatty acid esters can be obtained by reacting the hydroxyl groups of sucrose (sucrose) with the carboxyl groups of fatty acids to form ester bonds. Since there are eight hydroxyl groups in one sucrose molecule, and there are various types of fatty acids, various sucrose fatty acid esters can be synthesized depending on the number of ester bonds and the type of fatty acid. Examples of the constituent fatty acids of the sucrose fatty acid esters include saturated and unsaturated fatty acids having 12 to 22 carbon atoms, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, behenic acid, and erucic acid. These can be used alone or in combination of two or more types. Of these, it is preferable to use stearic acid, a saturated fatty acid having 18 carbon atoms.

In the PPAR activating composition of the present invention, the above-mentioned sucrose fatty acid esters are used alone or in combination of two or more. In particular, by using two or more different sucrose fatty acid esters in combination, a PPAR activating composition that exhibits performance more suited to individual needs can be obtained.

When the composition for activating PPAR of the present invention is in liquid form, for example, from the viewpoint of the effectiveness of PPAR activity, the content of sucrose fatty acid ester is preferably in the range of 0.01 to 20 mg/ml, more preferably in the range of 0.01 to 10 mg/ml.
The composition for activating PPAR of the present invention may be in any form, such as solid, powder, liquid, etc., as long as it can be orally ingested.

In addition, when the PPAR activating composition of the present invention is a mixture of sucrose fatty acid ester and soy flour, it is preferable because it additively or synergistically enhances the PPAR activating effect of isoflavones and saponins contained in the soy flour. In the present invention, soy flour refers to powdered soybeans from which the seed coat has been removed.

The PPAR activation composition of the present invention thus obtained can be taken orally continuously to promote the activation of PPAR, thereby exerting excellent effects in the prevention and improvement of hyperlipidemia, inflammatory diseases (including skin inflammation), insulin resistance diseases (diabetes, arteriosclerosis, etc.), stroke, biological rhythm imbalance (biorhythm disorder diseases), obesity, promotion of body fat burning, reduction of blood triglycerides, suppression of fatty liver, improvement of stamina, differentiation of epidermal cells and differentiation of melanocytes (whitening effect by inducing differentiation of epidermal cells and inhibiting differentiation of melanocytes), promotion of keratinization, development and repair of the epidermis, etc.

The PPAR activating composition of the present invention can be orally ingested directly as a mixture of sucrose fatty acid ester and soybean flour as described above, but it is also possible to orally ingest an extract of the active ingredient dissolved or dispersed in a suitable liquid carrier, or mixed with a suitable powder carrier.

Examples of pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, solubilizers, suspending agents, tonicity agents, buffers, and soothing agents in liquid preparations.

When the PPAR activating composition of the present invention is formulated, various ingredients that are usually used in formulations are optionally used, such as starch, dextrin, lactose, corn starch, inorganic salts, etc. These may be used alone or in combination of two or more kinds.

The dosage forms of the above formulations include, for example, ampoules, tablets, capsules, granules, fine granules, powders, and drinks.

Furthermore, the PPAR activating composition of the present invention can also be provided in the form of a food or beverage containing the composition. Examples of the food or beverage include health foods (tablets, powders, granules, concentrated liquids), soft drinks, foods for specified health uses, lactic acid bacteria fermented foods (yogurt, etc.), drinks, tea, milk, puddings, jellies, candies, gum, chocolate, soups, cookies, snacks, wine, shochu, sake, dressings, boiled beans, tofu, natto, soy milk, roasted beans, dried beans, miso, etc. By continuously consuming these foods and beverages in the same way as with normal foods and beverages, it is possible to promote the activation of PPAR, and various effects such as those mentioned above (prevention and improvement of hyperlipidemia and obesity, development and repair of the epidermis, etc.) can be obtained.

The PPAR activating composition of the present invention is gentle to the human body without causing side effects, and can exert a PPAR activating effect and preventive and ameliorative effects on various diseases related thereto (particularly obesity and hyperlipidemia). Furthermore, the above effects can be obtained not only in humans but also in animals such as pets and livestock, and the dosage is appropriately set depending on the organism to which the composition is administered, and conditions such as the sex, weight, and age of the recipient. As described above, the PPAR activating composition of the present invention can also obtain obesity suppressing effects in animals such as pets and livestock, and therefore can also be provided in a form incorporated into pet food or feed.

Next, examples will be described together with comparative examples. However, the present invention is not limited to these examples. In the following description, "%" is based on weight unless otherwise specified.

<PPAR activation test>
A nuclear receptor (PPARα, PPARδ) activation test using a reporter gene assay was carried out by the following method.
The cell line used was CV-1 (distributed by the National Institute of Biomedical Innovation, Health and Nutrition). The above cell line was seeded in a 6-well plate at 2.0 x ¹⁰ cells/well and cultured for one day in Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum (FBS) and 2 mol/l L-glutamine).
Next, a chimeric protein expression plasmid (pGal4DBD/NR(LBD)) of the DNA binding domain of Gal4 gene (Gal4-DBD) and the ligand binding domain (NR-LBD) of various human nuclear receptors (PPARα, PPARδ), a reporter plasmid (pGal4-Luc) containing the Gal4 gene DNA response element and the firefly luciferase gene, and an internal standard plasmid (pGL4.75hRluc-CMV; manufactured by Promega) in which a gene constitutive expression promoter (CMV) is linked upstream of the Renilla luciferase gene as an internal standard were mixed at a weight ratio of 1:0.9:0.1 and dissolved in Opti-MEM medium (manufactured by GIBCO), to which was added a gene transfer reagent X-tremeGENE HP (manufactured by Roche), and the mixture was added to the cultured cells and cultured to transfer the gene.
Next, the transfected cells were peeled off with trypsin, washed, and then seeded again on a 96-well plate at 1.6 x 10 ⁴ cells/well. At this time, the culture medium was replaced with Dulbecco's modified Eagle medium (phenol red-free, 10% activated charcoal-treated FBS, manufactured by SIGMA) containing the test sample, and the cells were further cultured for 48 hours. The cells were washed with phosphate buffered saline (PBS), lysed using a dual luciferase assay system (manufactured by Promega), and then a substrate solution containing luciferin was added, and firefly luciferase activity and Renilla luciferase activity were measured using a plate reader (Ferrios AB-2350, manufactured by ATTO). In addition, the PPAR activation test was performed after confirming that no significant damage was observed in the cells (no significant decrease in Renilla luciferase activity value was observed).
The above procedure was carried out using three wells per sample (including a negative control), and the average value of the three wells was used as data.
The transcription activity (luciferase activity) of a nuclear receptor-dependent gene was defined as shown in the following formula (1).

Nuclear receptor-dependent transcriptional activity value (internal standard correction value) = (firefly luciferase activity by Gal4-Luc) / (Renilla luciferase activity by hRluc-CMV) ... (1)

[Example 1]
<Measurement of PPAR activity of sucrose fatty acid esters>
An ethanol extract of sucrose stearate was obtained as a test sample, and the PPAR activity of the test sample was measured by the method described in the above-mentioned <PPAR activation test>. The test sample was prepared as follows. That is, first, two types of sucrose stearate (Ryoto Sugar Ester S-370, Ryoto Sugar Ester S-770, manufactured by Mitsubishi Chemical Foods Corporation) were mixed in a weight ratio of 1:1, and a 10-fold amount of 70% ethanol solution was added, stirred at room temperature (23°C) for 3 hours, filtered through a 0.2 μm membrane filter, and freeze-dried. This was dissolved by adding dimethyl sulfoxide (DMSO) to a concentration of 80 mg/ml, and used as the test sample.
Each test sample was adjusted to a final concentration of 0.04 mg/ml, 0.16 mg/ml, or 0.40 mg/ml, and a 0.5% dimethyl sulfoxide (DMSO) solution was used as a negative control (NC).
The evaluation of PPARα activity and PPARδ activity was expressed as a ratio to the negative control (activity value in the sample/activity value in the negative control), and a value of 1.2 or more was defined as significant activity. The results are shown in Table 1 below.

From the results in Table 1 above, it can be seen that the activity of PPARα and PPARδ is improved in almost proportion to the concentration of sucrose stearate. Furthermore, even when the concentration of sucrose stearate is small (0.04 mg/ml), the activity of PPARα and PPARδ is improved.

[Example 2]
<Measurement of PPAR activity of soy flour containing sucrose fatty acid esters>
An ethanol extract of soybean flour containing sucrose stearate was obtained and used as a test sample to measure the PPAR activity of the test sample in accordance with the method described in Example 1. The PPARα activity and PPARδ activity were evaluated in the same manner as in Example 1.
The test sample was prepared as follows. First, two types of sucrose stearates (Ryoto Sugar Ester S-370 and Ryoto Sugar Ester S-770, manufactured by Mitsubishi Chemical Foods Corporation) were mixed in a weight ratio of 1:1, and soy flour was further mixed, stirred and dissolved in hot water, and homogenized with a homogenizer to prepare a soy liquid. This soy liquid was freeze-dried, and a 10-fold amount of 70% ethanol solution was added to the freeze-dried product obtained, and the mixture was stirred at room temperature for 3 hours, centrifuged, and the supernatant after centrifugation was filtered through a 0.2 μm membrane filter and freeze-dried. This was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 400 mg/ml and used as a test sample.
The final concentrations of the test samples were adjusted to 0.04 mg/ml for sucrose stearate and 2.0 mg/ml for soybean flour, and the negative control (NC) was the same as in Example 1. The results are shown in Table 2 below.

[Comparative Example 1]
<Measurement of PPAR activity in soy flour>
An ethanol extract of soybean flour was obtained and used as a test sample, and the PPAR activity of the test sample was measured in accordance with the method described in Example 1. The PPARα activity and PPARδ activity were evaluated in the same manner as in Example 1.
The test samples were obtained in the same manner as in Example 2, except that sucrose stearate was not used.
The final concentration of the test sample was adjusted to 2.0 mg/ml of soybean flour, and the negative control (NC) was the same as in Example 1. The results are shown in Table 2 below.

From the results in Table 2 above, it can be seen that the activity of PPARα and PPARδ in Example 2, in which soybean flour was used in combination with sucrose stearate, is significantly improved compared to the activity of PPARα and PPARδ when the concentration of sucrose stearate is 0.04 mg/ml, as shown in Table 1. In particular, in Example 2, a high synergistic effect is observed in the activity of PPARα.
In addition, comparative example 1, in which soybean flour was used alone without sucrose stearate, also showed relatively high PPARα activity and PPARδ activity, whereas example 2 shows that even a small amount of sucrose stearate (concentration of sucrose stearate: 0.04 mg/ml) increases the degree of PPARα and PPARδ activity when used in combination with soybean flour.

The effect of improving PPAR activity seen in Example 1 and Example 2 above can be considered to indicate an effect of improving PPAR activity when the PPAR activating composition of the present invention is orally ingested as a food, which is one embodiment of the composition. In addition, since the active ingredient of the composition, sucrose fatty acid ester, does not affect the taste or flavor, it is considered to be suitable for being added to food and ingested. Furthermore, from the results of the above experiments, it can be expected that the PPAR activating composition of the present invention, when used in combination with soy flour as in Example 2, will have a higher effect of improving PPAR activity when orally ingested as a food.

The PPAR activating composition of the present invention is suitable for ingestion by adding to food, etc., since the active ingredient, sucrose fatty acid ester, does not affect the taste or flavor, and is also suitable for continuous ingestion due to its excellent safety. Therefore, the PPAR activating composition of the present invention can be applied as food and drink, and can also be powdered, tableted, encapsulated, etc., and used as a supplement, medicine, food additive, etc., as needed.

Claims (3)

A composition for activating peroxisome proliferator-activated receptor δ, comprising a sucrose fatty acid ester as an active ingredient, and promoting the activation of peroxisome proliferator-activated receptor δ. The composition for activating peroxisome proliferator-activated receptor δ according to claim 1, comprising the sucrose fatty acid ester and soybean flour . 3. The composition for activating peroxisome proliferator-activated receptor .delta . according to claim 1 or 2 , wherein the constituent fatty acid of the sucrose fatty acid ester is stearic acid.