JP2014185154A - NATURALLY OCCURRING PPARδ ACTIVATOR AND SLOW MUSCLE INCREASING AGENT USING THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peroxisome proliferator-activated receptor δ (PPARδ) ligand agent which is derived from safety natural product, and to provide an increasing agent of slow muscle of muscle fiber types.SOLUTION: There is provided an increasing agent of slow muscle of muscle fiber types with a PPARδ activator containing any of at least one ethanol extracts of Hericium erinaceum, P. eryngii var. touliensis CJ.Mou, mandarin orange stigma, mandarin orange petal, and tomato leaves as active ingredients. The above slow muscle type increasing agent of the invention is safe and effective in improving meat quality in livestock. As an extractant, a mixed solution with water, n-hexane, or the like is preferable besides ethanol.

Description

  The present invention relates to a PPARδ activator and a slow muscle increasing agent derived from natural products.

  Activation of peroxisome proliferator-activated receptor delta (hereinafter referred to as PPARδ), which is a nuclear receptor for skeletal muscle, increases slow muscle type muscle fibers of skeletal muscle, activates skeletal muscle fat metabolism, Is known to decrease (Non-patent Document 1).

  From the viewpoint of meat, it is said that increasing slow muscle type muscle fibers increases taurine, carnitine, iron content, and the amount of free taste amino acids, and further increases the evaluation of softness and juiciness (non-patent literature). 2).

  It has been disclosed that Yamabushitake, an extract of Yamabushitake using an organic solvent, and the like have an effect of reducing body fat, visceral fat and liver fat and are safe lipid metabolism promoters (Patent Document 1). There is also an announcement that a sorghum ethanol extract (Non-patent Document 3) or a compound contained in Enju (Non-patent Document 4) acts on PPARδ.

  Thus, PPARδ activation leads to suppression of obesity, activation of fatty acid metabolism, promotion of burning of body fat, reduction of blood neutral fat, suppression of fatty liver, improvement of endurance, etc. These activators are considered to be effective as obesity prevention, fatty acid oxidation activators, body fat combustion promoters, and endurance improvers.

  Therefore, research and development of PPARδ activators have been actively conducted for such purposes, and many studies have been reported so far.

JP 2010-1111586 A

Y. Wang et al., PLoS Biol., 2 (10), e294 (2004) Y.K.Kang et al., Meat Sci., 89 (4), 384-389 (2011) S.Y.Cho et al., PLoS One, 7 (3), e33815 (2012) T.H. Quang et al., Phytother. Res., Oct 30. doi: 10.1002 / ptr.4871. [Epub ahead of print] (2012)

  Accordingly, an object of the present invention is to provide a highly safe natural product-derived PPARδ activator and a skeletal muscle slow muscle increasing agent that is safe and improves the meat quality of livestock.

  As a result of intensive studies, the present inventors have found that organic solvent extracts of Yamabushitake, Yukireitake, citrus stigma, mandarin petals, and tomato leaves are useful as fatty acid metabolism activation, body fat burning promotion, obesity suppression, fatty liver suppression, It was found to be a PPARδ activator.

Therefore, the present invention is as follows.
1. A peroxisome proliferator-responsive receptor δ (PPARδ) activator comprising at least one plant selected from the group consisting of Yamabushitake, Yukireitake, Citrus stigma, Citrus petals and Tomato leaves.
2. A PPARδ activator comprising, as an active ingredient, at least one plant extract selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petal and tomato leaf.
3. 3. The PPARδ activator according to 2 above, wherein the at least one plant extract selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petal and tomato leaf is an ethanol extract.
4). A slow muscle increasing agent comprising the PPARδ activator according to any one of items 1 to 3.
5. A livestock feed comprising the agent for increasing slow muscle according to item 4 above.
6). Production of a PPARδ activator characterized by extracting at least one plant selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petals and tomato leaves with water, ethanol or n-hexane solution Method.

With the slow muscle increasing agent of the present invention used as a health functional food, fat metabolism of skeletal muscle can be activated, and body fat can be reduced safely and efficiently with a small amount of use.
Moreover, by using it as a feed ingredient for livestock, it is possible to increase the slow muscle type of live muscles of livestock and increase the softness and juiciness of livestock meat. Furthermore, improvement in taste and nutritional value can be expected.

FIG. 1 is a graph showing PPARδ agonist activity of various crop extracts. 2 (a) and 2 (b) are graphs showing the PPARδ agonist activity (fractional fraction Fr1-11) of the silica gel chromatography fraction of Yamabushitake hexane extract. FIG. 3 is a diagram showing the PPARδ agonist activity of Yamabushitake n-hexane extract fractions Fr3- (2,3) -1 and Fr3- (2,3) -2. FIG. 4 is a diagram showing PPARδ agonist activity in rat myocytes (isolated muscle fibers) of Yamabushitake n-hexane extract. FIG. 5 is a graph showing PPARδ agonist activity in rat muscle cells (isolated muscle fibers) of a mandarin petal ethanol extract. FIG. 6 is a graph showing the PPARδ agonist activity of Yamabushitake n-hexane extract Fr3- (2,3) -1 in rat myocytes (isolated muscle fibers). 7 (a) and 7 (b) are diagrams showing the expression levels of PPARδ target genes PDK4 and UCP3 in the mouse gastrocnemius muscle after feeding for 24 hours with a 5% Yamabushitake diet. FIG. 8 is a diagram showing the muscle endurance of mice after feeding for 8 weeks with a diet containing 5% Yamabushitake. FIG. 9A shows a GC-EIMS chromatogram of Fr3. FIG. 9B is a diagram showing a presumed compound.

  In the present invention, any part of Yamabushitake, Yukireitake, citrus stigma, mandarin petal, wolfberry, green tomato hull (water extraction) and tomato leaf, or a combination thereof can be used.

  The raw material can be subjected to an extraction step as it is or freeze-dried and pulverized to obtain the target extract, but is preferably extracted after drying and pulverization. The size of the pulverized product and the size of the pulverized product are not particularly limited, but the particle size of the pulverized product is preferably 1 μm to 10 mm, more preferably 100 μm to 500 μm. The above products are derived from natural ingredients and are highly safe.

  As a solvent used for obtaining the organic solvent extract of the present invention, any of a polar solvent and a nonpolar solvent can be used, and these can also be mixed and used. Furthermore, the organic solvent may contain water.

  Examples of the organic solvent include alcohols such as methanol, ethanol, n-propanol, isopropanol and isobutanol; polyhydric alcohols such as ethylene glycol and propylene glycol; hydrocarbons such as n-hexane, cyclohexane and petroleum ether; Nitriles such as acetonitrile and propionitrile; ketones such as acetone and methyl ketone; esters such as methyl acetate and ethyl acetate; chain and cyclic ethers such as tetrahydroxyfuran and diethyl ether; ethylene glycol, diethylene glycol and polyethylene glycol Polyethers such as halogenated hydrocarbons such as dichloromethane and chloroform, and the like can be used alone or in combination of two or more.

  Among these organic solvents, alcohols, hexane and water / alcohol mixed organic solvents are preferable, and water / ethanol mixed organic solvents, ethanol and n-hexane are particularly preferable.

  Here, as alcohol, a C1-C5 lower alcohol is preferable and a C1-C3 lower alcohol is more preferable. In the present invention, it is preferable to first extract with ethanol, and further extract with n-hexane as necessary.

  The mixing ratio of water and alcohol is not particularly limited, but water / alcohol is preferably 90/1 to 1/100, more preferably 2/1 to 1/100 (mass ratio). is there.

  In the present invention, Yamabushitake, Yukireitake, mandarin stigma, mandarin petal, wolfberry, green tomato hull (water extraction), tomato leaf and the like are freeze-dried and pulverized, and then at room temperature (0 to 30 ° C.) or heated with an organic solvent. Extraction is obtained by extraction below.

It is preferable to use 10 parts by mass of an organic solvent with respect to 1 part by mass of the above raw material. The extraction temperature at this time is preferably 0 to 40 ° C., and the extraction time is preferably 24 to 72 hours.
In the extraction step, means such as solid-liquid extraction, dipping, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, or stirring can be used, and solid-liquid extraction with stirring is preferable.

  The obtained extract is subjected to separation and purification means such as activated carbon treatment, liquid-liquid distribution, column chromatography, liquid chromatography, gel filtration or precision distillation after removing the organic solvent, and then with an appropriate solvent. It may be used as a diluted diluent, or may be a concentrated extract, a dry powder, or prepared in a paste form.

  On the other hand, Yamabushitake, Yukireitake, citrus stigma, mandarin petal, wolfberry, green tomato hull (water extraction) or tomato leaf extract is fractionated into each fraction (Fr) by silica gel chromatography, and the next most active fraction It is preferable to fractionate at least twice, measure the PPARδ agonist activity by the RCAS method, and collect the fraction with the highest activity.

  The extract having PPARδ activating action can be used as a PPARδ activator, fatty acid metabolism activator, body fat combustion accelerator and slow muscle increasing agent, and is used for producing a PPARδ activator and the like. be able to.

  The PPARδ activator can be used as food, feed, pharmaceuticals or quasi drugs for humans or animals. In addition, the organic solvent extract can be applied to foods, functional foods and foods for specified health use because it has effects such as fatty acid metabolism activation, body fat burning promotion and fatty liver suppression.

  The clinical dose of the extract of the present invention estimated from the difference in the metabolic activity with humans and the dose in which the effect was seen in animal experiments using mice is the age, body weight, patient sensitivity and symptoms. Although it varies depending on the degree and the like, the effective dose is usually 10 to 50 g per day for adults, more preferably about 50 to 70 g. However, if necessary, an amount outside the above range can be used.

  As an administration form when an extract containing a PPARδ activator or the like is used as a pharmaceutical product or a quasi-drug, for example, it can be orally administered by tablet, capsule, granule, powder or syrup, or an injection or suppository. And parenteral administration using an inhalant, a transdermal absorption agent, an external preparation and the like.

  In order to prepare such various dosage forms, the organic solvent extract of the present invention is used alone or other pharmaceutically acceptable excipients, binders, extenders, surfactants. , Dispersants, buffers, preservatives, fragrances, diluents, and the like can be used in appropriate combinations.

  Of these dosage forms, the preferred form is oral administration, and when an oral liquid preparation is prepared, it can be produced by a conventional method with the addition of a buffer, a stabilizer and the like. In the case of transdermal administration, it can be administered as an ointment, emulsifier, lotion, suspension, etc. depending on the state of the target skin, etc. The formulation for transdermal administration used in such administration methods is usually The method used can be used.

  As a form when using a PPARδ activator or the like as a food, in addition to various foods such as dairy products, beverages or soups, forms similar to the above-mentioned oral administration preparations (tablets, capsules, syrups and the like) can be mentioned. .

  In that case, various additive components such as a sweetener, a sour agent, a preservative, a fragrance, a colorant, an excipient, a stabilizer, a wetting agent, an absorption enhancer, or a pH adjuster can be blended.

  The compounding amount of the organic solvent extract with respect to these varies depending on the form of use, but in the form of food, in the total composition, it is usually 0.0001 to 10% by mass, further 0.002 to 2% by mass in terms of dry matter It is preferable to do this.

  In the present invention, when an extract is added to livestock feed, the extract and meat such as fish, protein, cereals, bran, vitamins and minerals are generally used as feed materials, and more generally used in feed. A gelling agent, a shape-retaining agent, a pH adjuster, a nutritional reinforcing agent, and the like can be mixed and used.

Hereinafter, examples and test examples are given to specifically describe the present invention. However, the present invention is not limited to the examples.
Example 1
[Preparation of various crop extracts]
Yamabushitake, Yukireitake, mandarin stigma, mandarin petal, wolfberry, green tomato hull (water extraction) and tomato leaf were freeze-dried to prepare a dried body. Ethanol (10 mL of ethanol per 1 g of dried product) was added to the dried product, and the mixture was extracted by shaking for 1 day, and an ethanol extract was obtained by suction filtration. The extraction operation was performed twice more in the same manner. After removing the solvent from the ethanol extract using a rotary evaporator, it was air-dried in a fume hood and dried with a vacuum desiccator to obtain an ethanol extract. The extracted residue after extraction was air-dried in a fume hood and then dried in a vacuum desiccator.

  Ultrapure water was added to the obtained extraction residue, and the water extract extracted for 4 hours while shaking was centrifuged (3000 rpm, 10 minutes), and the supernatant was filtered with filter paper. This operation was repeated once more. The water extract was lyophilized to obtain a water extract. Further, regarding the fruit body of Yamabushitake, n-hexane was added to the ethanol extract, extracted by shaking for 1 day, and an n-hexane extract was obtained by suction filtration. The extraction operation was performed twice more in the same manner. The obtained n-hexane extract was dried in the same manner as the ethanol extract to obtain an n-hexane extract.

(Example 2)
[Examination of action of various crops as PPARδ agonists]
・ Yamabushitake (water extraction, ethanol extraction, n-hexane extraction)
・ Yukireitake (water extraction, ethanol extraction)
・ Enoki (water extraction, ethanol extraction)
・ Tangerine stigmas (water extraction, ethanol extraction)
・ Tangerine petals (water extraction, ethanol extraction)
・ Wolfberry (ethanol extraction)
・ Green tomato skin (water extraction)
・ Tomato leaves (ethanol extraction)
FIG. 1 shows the results of measuring the PPARδ agonistic activity of the protein with a nuclear receptor cofactor assay system [EnBio RCAS for PPARδ (Fujikura Kasei Co., Ltd.)]. The synthetic PPARδ agonist GW501516 was used as a positive control, and the relative activity was shown with the value of 6.25 μM GW501516 as 100%.

  Yamabushitake (water extract, ethanol extract), snow flake (water extract), citrus stigma (water extract), mandarin petal (water extract), wolfberry (ethanol extract), green tomato skin (water extract) Almost no activity was observed, but it was obvious in Yamabushitake (hexane extract), Yukitake (ethanol extract), citrus stigma (ethanol extract), mandarin petal (ethanol extract) and tomato leaf (ethanol extract) Activity was observed. In addition, no activity was observed in any mushroom, and almost no activity was observed in water extract and ethanol extract.

(Example 3)
[Examination of the action of the chromatographic fraction of Yamabushitake hexane extract as a PPARδ agonist]
The Yamabushitake hexane extract was fractionated into 11 fractions (Fr) by silica gel chromatography described below. 800 g of silica gel was slurried with n-hexane and packed in an open column (φ4.25 × 60.0 cm, 3409.2 cm ³ ). Using this column, a gradient of n-hexane and ethyl acetate was used to separate the column from Fr1 to Fr11. Next, Fr3 having the highest activity was fractionated by silica gel column chromatography again using the column described below.

100 g of silica gel was slurried with n-hexane and packed in an open column (φ 1.7 × 30.0 cm, 288.49 cm ³ ). N-hexane and chloroform were used to flow in a gradient. As a result, four fractions from Fr3-1 to Fr3-4 were obtained. Fr3-2 and 3-3 were mixed [Fr3- (2,3)], and the fraction was further fractionated by silica gel column chromatography. 5 g of silica gel was slurried with n-hexane and packed in an open column (φ 0.5 × 30.0 cm, 23.55 cm ³ ). A solvent in which n-hexane and chloroform were mixed at a ratio of 1: 1 was passed to obtain two fractions [Fr3- (2,3) -1, Fr3- (2,3) -2].

  The results of measuring the PPARδ agonist activity of the above fractions by the RCAS method are shown (Fr3-1 and 3-4 are not measured). As a result, as shown in FIGS. 2 (a) and 2 (b), among the 11 fractions first fractionated from the Yamabushitake hexane extract, Fr3, 4, 5 and 6 contained the active ingredient, and Fr3 was the most active. It was found that the activity was high. As shown in FIG. 3, Fr3- (2,3) -1, Fr3- (2,3) -2 showed higher activity in Fr3- (2,3) -1. In addition, the addition amount of each fraction of FIG. 2 (a), (b) and FIG. 3 was made into the quantity corresponded to a 0.05% hexane extract (before fractionation).

Example 4
[Examination of the effect of Yamabushitake n-hexane extract and mandarin petal ethanol extract as PPARδ agonists in myocytes (muscle fibers)]
Whether the activity observed with RCAS was also observed in muscle cells (muscle fibers), or examined using muscle fibers isolated and cultured from rats. First, the short flexor muscle, which is the muscle tissue of the sole of the hind limb of a 5-week-old Fischer 344 rat male (KBT Oriental), was isolated.

  The isolated muscle tissue was treated with a collagenase solution, muscle cells (muscle fibers) were loosened by pipetting, purified by gravity sedimentation, and then seeded on a dish. Yamabushitake hexane extract or mandarin petal ethanol extract was added to the isolated and cultured muscle fiber medium, and after 12 hours of culturing, the expression level of PDK4, the target gene of PPARδ, was examined by real-time RT-PCR. The expression level of PDK4 was corrected with a kind of housekeeping gene, hypoxanthine-guanine phosphoribyltransferase (HPRT).

  As a result, as shown in FIGS. 4 and 5, a significant increase in the expression level of PDK4 was observed in the group with low concentration of n-hexane extract of Yamabushitake and the group with citrus petal ethanol extract. As shown in FIG. 6, the expression level of PDK4 significantly increased to a level comparable to the synthetic agonist GW501516 in the experimental group to which 0.002% of Fr3- (2,3) -1 was added.

(Example 5)
[24-hour feed intake test with yamabushitake using mice]
In order to verify whether or not PPARδ agonist activity is observed against skeletal muscle in vivo since it was suggested that the extract of Yamabushitake acts as a PPARδ agonist on muscle fibers in the cell experiment of Example 4. Experiments were performed using mice.

  Eight 8-week-old C57BL / 6J mouse males (KBT Oriental) were preliminarily raised for 1 week, and then freely fed with a diet containing 5% dry Yamabushitake or a control diet for 24 hours (4 mice per group). Mice were raised in an environment at room temperature 22.0 ± 2.0 ° C. and humidity 55 ± 10%, and water was freely given. After 24 hours of feeding, the extensor muscle was extracted, RNA was extracted with TRIzol, and mRNA expression levels of PDK4 and UCP3, which are target genes of PPARδ, were examined by real-time RT-PCR. The expression level was corrected with HPRT, a kind of housekeeping gene.

  As a result, as shown in FIGS. 7 (a) and (b), a significant increase of PDKδ target genes PDK4 and UCP3 was observed in the Yamabushitake intake group.

(Example 6)
[8-week Yamabushitake mixed feed intake test using mice]
In the 24-hour feeding experiment of Example 5, it was suggested that ingestion of yamabushitake acts as a PPARδ agonist on the skeletal muscle of mice, so is there a change in muscle characteristics of mice after long-term ingestion? An experiment to investigate was conducted.

  After 8-week-old male C57BL / 6J mice (KBT Oriental) were preliminarily bred for 1 week, a feed containing 5% of dried yamabushitake or a control feed was fed in the same amount as the control group for 8 weeks (7 per group). -8). Mice were raised in an environment at room temperature 22.0 ± 2.0 ° C. and humidity 55 ± 10%, and water was freely given. After 8 weeks of breeding, the muscle function of mice under gas anesthesia was examined by forced muscle contraction by electrical stimulation of the tibial nerve.

  As a result, as shown in FIG. 8, a significant increase in muscle endurance was observed in the Yamabushitake intake group. Maximum muscle strength did not change. Increased muscle endurance is consistent with the characteristics of the slow muscle type.

(Example 7)
[Estimation of PPARδ agonist component contained in Yamabushitake hexane extract]
Among silica gel chromatography fractions of Yamabushitake hexane extract, Fr 3 having the highest activity was subjected to GC-EIMS analysis. FIG. 9A shows an analytical chromatogram. Although it is a preliminary and qualitative result, as shown in FIG. 9B, oleic acid and stearic acid which are long chain fatty acids and 13-methyltetradecanoic acid which is a branched chain fatty acid were listed as active candidate substances. .

  For the purpose of increasing the slow muscle type of skeletal muscle as it is, or with a conventional carrier or the like, any one or more ethanol extracts of Yamabushitake, Yukireitake, citrus stigma, citrus petal or tomato leaf of the present invention are various in the food field. It can be used in animal feeds including livestock, pets, etc., as a health functional food, etc.

Claims (6)

  A peroxisome proliferator-responsive receptor δ (PPARδ) activator comprising at least one plant selected from the group consisting of Yamabushitake, Yukireitake, Citrus stigma, Citrus petals and Tomato leaves.   A PPARδ activator comprising, as an active ingredient, at least one plant extract selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petal and tomato leaf.   3. The PPARδ activator according to claim 2, wherein at least one plant extract selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petal and tomato leaf is an ethanol extract.   The slow muscle increasing agent containing the PPAR (delta) activator of any one of Claims 1-3.   Livestock feed containing the slow muscle increasing agent according to claim 4.   Production of a PPARδ activator characterized by extracting at least one plant selected from the group consisting of Yamabushitake, Yukireitake, citrus stigma, citrus petals and tomato leaves with water, ethanol or n-hexane solution Method.

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