JP6289218B2 - Method for activating PPARα active composition - Google Patents

Description

The present invention relates to a method for activating a PPARα active composition, particularly as a method for activating a peroxisome proliferator-activated receptor (PPAR) active composition.

  PPAR is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily, like steroid receptors, retinoid receptors and thyroid receptors, and so far three isoforms (α Type, δ (or β) type, and γ type) have been identified in various animal species including humans (see Non-Patent Document 1). Among these, PPARα is expressed in the liver, kidney, skeletal muscle and the like having high fatty acid catabolism, and is particularly highly expressed in the liver (see Non-Patent Document 2), and is related to fatty acid metabolism and intracellular transport. By positively or negatively controlling the expression of genes (eg, acyl CoA synthase, fatty acid binding protein, lipoprotein lipase, etc.) and apolipoprotein (AI, AII, CIII) genes related to cholesterol and neutral lipid metabolism Yes. PPARδ is ubiquitously expressed in each tissue in the body centering on nerve cells. At present, the physiological significance of PPARδ is unknown. PPARγ is highly expressed in adipocytes and is involved in adipocyte differentiation (see Non-Patent Document 3). Thus, each isoform of PPAR performs a specific function in a specific organ or tissue.

  It is known that such a PPAR active composition is contained in a grass family plant. Specifically, an extract of a PPAR active composition contained in a sorghum plant is described in Patent Document 1. Yes. However, in general, even if a plant belongs to the same family or a plant of a different genus produces the same type of active substance, the active substance has an activity equivalent to that of an active substance obtained from a plant of another genus. The activity may not be inhibited by some factor. Therefore, when searching for such active substances, it is often necessary to develop extraction methods and separation methods peculiar to each plant, and active substances cannot be easily developed.

  Therefore, the present inventors have previously found a PPAR composition derived from sugarcane for the purpose of providing a peroxisome proliferator-responsive receptor (PPAR) active composition that can be efficiently obtained by a simple technique. (Patent Document 2).

JP 2004-161656 A JP 2013-193987 A

Proceedings of the National Academy of Sciences, 1992, 89, 4653-4657. Endocrinology, 1996, 137, 354-366 Journal of Lipid Research, 1996, 37, 907-925.

  However, according to the configuration of Patent Document 2, it is known that the sugarcane-derived PPAR composition contains an inhibitory substance, and the inhibitory substance suppresses the activity of the PPAR composition. Is required to be effectively reduced and the activity of the PPAR composition is improved.

  Therefore, an object of the present invention is to efficiently remove an inhibitor contained in the above-mentioned PPAR composition derived from a grass family such as sugarcane to obtain a highly active PPAR composition.

[Configuration 1]
Therefore, a characteristic configuration of the present invention for solving the above-described problem is that a grass plant extract obtained by extracting the top of a grass plant selected from sugarcane, sorghum, and corn with an extraction solvent containing acetone. It is a method for activating a PPARα active composition containing a product as a main component, which comprises a step of bringing the grass plant extract into contact with a carbon adsorbent.

[Operation effect 1]
As a result of intensive studies, the present inventors have clarified that sugarcane and sorghum contain sufficiently high PPAR active substances (see Patent Document 2). In particular, in sugarcane, PPARα activity was found in an extract extracted from a top of the treetop that was discarded during the sugar production process using an extraction solvent containing acetone .

  The PPAR active substance referred to in the present invention is a substance containing a compound having the ability (activity) to bind to the ligand binding region of PPAR, and the PPAR active composition contains such a substance as a component, It refers to a composition having the above activity. PPAR ligand activity can be determined by, for example, a reporter assay (Cell, 1995, 83, 803-812) for evaluating the binding of a PPAR ligand binding region to a GAL4 fusion protein by expression of luciferase, It can be measured by a competition binding assay (Cell, 1995, 83, 813-819) or the like using a protein containing the protein. In these assays, the activity of the sample is generally compared to the solvent control, and a sample that exhibits higher activity than the solvent control and is found to be dose-dependent is evaluated as “with PPAR activity”.

  Moreover, it is known that the obtained gramineous plant extract contains an activity inhibitory substance. Some PPAR active substances have a structure determined as a substance, but in reality, only the activity is confirmed, many structures are unknown, and the activity inhibitory substance also has any PPAR active substance. It is not clear what structure of the substance works as an activity inhibitor.

However the present inventors have made extensive research and the action of carbon adsorbent PPAR alpha active composition PPAR alpha active substance coexists with active inhibitor that improves the PPAR alpha activity of PPAR alpha active composition I found it. This, the active inhibitor is unstable with respect to carbon adsorbent, while a high adsorptivity, stable to carbon adsorbent PPAR alpha active substance compared to the active inhibitor, hardly adsorbed This is thought to be due to the nature of By utilizing this property, it is possible to remove the active inhibitors from grass extract, the activity of the PPAR alpha active substance is effectively draw.

  As the carbon adsorbent, various known carbon-based materials such as porous graphite carbon, activated carbon, carbon molecular sieve and the like can be used, and it is considered that the carbon adsorbent acts on the PPAR active substance and the activity inhibiting substance in the same manner.

As Lee Ne Plants, sugarcane, sorghum, when extracting PPAR active substance as a target corn, PPAR active substance by extracting the treetops head can be effectively extracted. For example, in sugarcane, the top of the treetop is a part that is discarded first in the sugar refining process, and can be easily secured as a resource. Further, if the PPAR active composition can be extracted from the waste that is simply discarded, the waste can be effectively used and the energy cost for disposal can be saved.

  The top of the treetop is the apex where the plant grows, and if it is sugar cane, it refers to a portion with a low sugar content of about two nodes from the tip. Sugarcane treetops are usually discarded.

  The PPAR active substance includes any of PPARα, PPARβ, and PPARγ, and the PPARα active substance is the most established substance at present. Therefore, from the embodiments described later, when the top of a gramineous plant selected from sugarcane, sorghum and corn is extracted with an extraction solvent containing acetone, the PPARα active substance is particularly efficiently extracted.

  By obtaining a fraction having PPARα activity, it is possible to isolate and identify a PPARα active substance and use it with higher activity. As a result, it can be used in a more active state.

[Configuration 2 ]
In addition, it is preferable to include a step of separating the Gramineae plant extract by a chromatographic method and removing a PPAR α activity inhibitory substance.

[Function and effect 2 ]
Fractions with PPAR alpha activity of the extract by separating by chromatography, it is possible to utilize a PPAR alpha active composition in a highly active state with no activity inhibition.

  Note that the step of removing the PPAR activity inhibitor by the chromatographic method may be performed prior to the step of contacting the carbon adsorbent or after the step of contacting the carbon adsorbent. In any case, the PPAR activity inhibitor can be removed to make the PPAR active composition highly active.

Therefore, it is possible to obtain a PPAR alpha active ingredient to be effective in small amounts from waste materials such as sugarcane, it is possible to use as a health food and beverage and pharmaceutical metabolic syndrome improved.

Graph showing PPARα activity of each fraction after the column separation step

  Below, the PPAR active composition of this invention is demonstrated. In addition, although suitable examples are described below, these examples are described in order to more specifically illustrate the present invention, and various modifications can be made without departing from the spirit of the present invention. The present invention is not limited to the following description.

[Extraction of PPAR active composition]
Extract the sugarcane treetop with an extraction solvent containing acetone (extraction process), and contact the resulting extract with a carbon adsorbent (adsorption process). (Column separation step). Thereby, a PPAR active composition can be extracted as a high methanol solvent extraction fraction.

<Extraction process>
A sugarcane treetop was selected, and the dried sample was chopped and ground to obtain a sample for extraction. 20 g of the sample was suspended in 400 mL of 70% acetone, stirred for 16 hours at room temperature, and then centrifuged to recover the supernatant. Furthermore, the operation of suspending in 200 mL of 70% acetone, centrifuging, and collecting the supernatant was repeated 10 times, and all the supernatants were combined into an extract. The extract was concentrated under reduced pressure, and 10 portions (200 g of sugarcane treetop) were concentrated together to 300 ml, acetone was removed, then extracted twice with 200 ml of diethyl ether, concentrated to dryness under reduced pressure, 2 .43 g of extract (Poaceae extract) was obtained. The extract was dissolved in 20 mL of chloroform to obtain an extract containing the PPAR active composition.

<Adsorption process>
0.5 ml of the obtained extract was added to the following graphite carbon column, and further eluted with 20 mL of methanol. The eluate was concentrated to dryness to obtain an extract.
The obtained extract was dissolved in 5 mL of chloroform per 0.5 mL of the extract used to obtain an extract containing the PPAR active composition.

(Graphite carbon column)
Column size: φ12mm x 0.5cm,
Filler: SampliQ Carbon SPE (Agilent carbon adsorbent)
Property: Non-porous Specific surface area: 100 m ² / g
Particle size: 37-125 μm
Amount used: 500 mg,
Filling solvent: methanol

  In this case, non-porous graphite carbon is used. In this case, the specific surface area is sufficiently large, which is preferable in terms of eliminating nonspecific adsorption of the low molecular weight compound of the PPAR active composition as compared to using a porous material.

  Moreover, since the pressure loss will become large and the elution rate will become slow when the particle size of graphite carbon is too small, when it is too large, the elution rate will become high and removal efficiency will fall, and 30-400 micrometers is preferable.

<Column separation process>
After diluting 1.0 mL of the extract 10 times, 1.0 mL was separated into 21 fractions using the solvent described in Table 1 by silica gel column chromatography. Each fraction was concentrated to dryness and then dissolved in 0100 μL of DMS to obtain an extract.

(Silica gel column)
Column size: φ12mm x 3cm,
Filler: Silica gel 60N (silica gel manufactured by Kanto Chemical Co., Inc.)
Properties: spherical, neutral,
Particle size: 40-50 μm
Amount used: 2.0 g
Filling solvent: Chloroform

[PPAR activity]
The results of examining PPARα activity for each fraction are shown in FIG. 1 (graphite carbon column treatment). In addition, the PPARα activity of each fraction of the extract (untreated) obtained by performing the column separation step without performing the adsorption step is shown. The activity was measured using EnBio RCAsforppARa (manufactured by Fujikura Kasei). The binding activity was expressed as a relative activity (%) when the activity of the positive control substance GW7467 (final concentration 500 nM) was defined as 100.

  As a result, the PPARα activity was high in the vicinity of the eleventh fraction (Fr.11 in the figure, correspondingly described below), and from the weight distribution of each of the concentrated and dried fractions shown in Table 2, the activity was also determined. It can be seen that high substances are eluted mainly in the vicinity of the seventh to fifteenth fractions.

  Here, referring to FIG. 1, it can be seen that the PPARα activity of the 13th to 15th fractions is greatly improved by the adsorption process. As a whole, it has been separately confirmed that the PPARα activity is improved about 3 times with and without the adsorption step. That is, even if it is estimated from the weight distribution that a substance having high PPARα activity is present, the 13th to 15th fractions having low PPARα activity are inhibited by some substance when the adsorption step is not performed. It is expected that the substance has been removed by the adsorption process. Similarly, other fractions may contain an activity inhibitory substance, but even if other fractions contain an activity inhibitory substance, the inhibitory substance may be removed by the column separation process. It is clear that it is effectively removed.

  Therefore, it was found that the elution components that cannot be separated only by the column separation step of separating the grass plant extract by the chromatographic method can be effectively removed by the adsorption step of contacting with the carbon adsorbent.

  In addition, when LC / MS analysis of the eleventh fraction after the adsorption step was performed, a fragment peak generated from a known PPARα active substance was not observed, and thus the extracted PPARα active substance is unknown as a PPARα active substance. It has become clear that this is a substance. In addition, since the inhibitory substance is unknown, the PPAR active composition mainly composed of a grass plant extract will be used in the future for finding that the adsorption process using a carbon adsorbent is useful for activity improvement. It is useful in the above.

[Others]
In the above examples, graphite carbon was used in the adsorption step, but graphite carbon is generally used as a column filler and is a carbonaceous material considered to exhibit adsorption / desorption performance for various substances. Therefore, it is not limited to this, and adsorption using a carbon adsorbent made of a carbonaceous material such as activated carbon and molecular sieve carbon, which exhibits the same adsorption / desorption performance as graphite carbon, to bring the grass plant extract into contact with the carbon adsorbent It is clear that similar effects can be expected by performing the process.

  In addition, sugarcane was used as the gramineous plant, but it is clear that the same gramineous plant contains the same PPAR active substance and inhibitor, so use other gramineous plants such as sorghum. Is also effective.

  In addition, the adsorption process in which the grass plant extract is brought into contact with the carbon adsorbent was separated by a chromatographic method and performed before the column separation process to remove the PPAR activity inhibitor, but the adsorption process and the column separation process are not necessarily performed. It is not necessary to perform in this order, and may be performed in reverse. Furthermore, the present invention does not prevent other separation steps from being included.

Therefore, it is possible to obtain a high PPAR alpha active ingredient the more active from sugar cane wastes, etc., for example, it is possible to use as a health food and beverage and pharmaceutical metabolic syndrome improved.

Claims (2)

It is a method for activating a PPARα active composition comprising as a main component a gramineous plant extract obtained by extracting the top of a gramineous plant selected from sugarcane, sorghum and corn with an extraction solvent containing acetone. A method for activating a PPARα active composition comprising the step of bringing the grass plant extract into contact with a carbon adsorbent. The method for activating a PPARα active composition according to claim 1, comprising a step of separating the Gramineae plant extract by a chromatographic method and removing a PPARα activity inhibitory substance.

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