JP7642966B2 - Composition for enhancing adrenomedullin gene expression - Google Patents

Description

The present invention relates to a composition for enhancing adrenomedullin gene expression.
The present invention also relates to a composition having the effect of enhancing the expression of the adrenomedullin gene.

Adrenomedullin is a circulatory regulatory peptide with vasodilatory properties. It is produced by various organs, such as the circulatory and digestive systems, and has been reported to have important physiological activities, such as vasodilatory properties, angiogenesis, antibacterial properties, antienteritis properties, gastric mucosal protection, and inhibition of thrombus formation (Patent Document 1).

Patent Document 1 describes an adrenomedullin production enhancer that contains as active ingredients a compound derived from ginseng (= Korean ginseng) and a compound derived from dried ginger and/or a compound derived from Japanese pepper. Example 2 of Patent Document 1 describes that a combination of ginseng (= Korean ginseng) extract and dried ginger extract, a combination of ginseng extract and Japanese pepper extract, a combination of dried ginger extract and Japanese pepper extract, ginseng extract alone, dried ginger extract alone, and Japanese pepper extract alone were administered to rats, and VC (vascular conductance), an index of blood flow, was measured and the rate of increase was calculated. The combination of ginseng extract and dried ginger extract and the combination of ginseng extract and Japanese pepper extract showed a clearly higher increase in VC than each of the individual administrations, but no change in the rate of increase in VC was observed with the combination of dried ginger extract and Japanese pepper extract.

Non-Patent Document 1 describes that Daikenchuto (a type of herbal medicine containing Japanese pepper, dried ginger, and Korean ginseng) promotes the release of adrenomedullin from intestinal epithelial cells, thereby promoting blood flow in the intestine, and that Daikenchuto and one of its components, 6-shogaol, promote the release of adrenomedullin from intestinal epithelial cells, and that this effect is inhibited by an antagonist of TRPA1, a type of TRP channel.

Patent Document 2 describes an anti-inflammatory composition containing Japanese pepper, dried ginger, ginseng (= Korean ginseng) and glue candy. Patent Document 2 describes that this anti-inflammatory composition can treat and alleviate inflammatory symptoms in the digestive organs.

Patent Document 3 describes a gastrointestinal mucosa protective agent that contains Japanese pepper or an extract thereof as an active ingredient. Patent Document 3 also describes that the gastrointestinal mucosa protective agent may be blended with other medicinal active ingredients, such as dried ginger or an extract thereof.

Patent document 4 describes a hair tonic that contains five types of herbal extracts: Chinese crap, Japanese hawthorn, Japanese angelica, red pepper, and Chinese laurel. Patent document 4 describes that the five types of herbal extracts in the hair tonic increase peripheral blood flow by dilating the local peripheral blood vessels in the hair loss area, thereby promoting the activity of hair roots.

International Publication WO2009/104248 JP 2002-145791 A JP 2007-291008 A Japanese Unexamined Patent Publication No. 2-17117

Am J Physiol Gastrointest Liver Physiol 304:G428-G436, 2013

The objective of the present invention is to provide a composition that contains naturally occurring ingredients as active ingredients and has the effect of enhancing gene expression of adrenomedullin.

The present inventors have discovered that a composition containing 6-shogaol and an ethanol extract of Japanese pepper as active ingredients has the effect of synergistically enhancing the expression of the adrenomedullin gene, and have completed the present invention as described below.
(1) A composition for enhancing adrenomedullin gene expression, comprising 6-shogaol and an ethanol extract of Japanese pepper as active ingredients.
(2) The composition according to (1), wherein the ethanol extract of Japanese pepper contains one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool.
(3) A composition containing 6-shogaol and an ethanol extract of Japanese pepper.
(4) The composition according to (3), wherein the ethanol extract of Japanese pepper contains one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool.
(5) A composition containing 6-shogaol and one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool in a total amount of 2 to 10 moles per mole of 6-shogaol.
(6) The composition according to (5), comprising 2 to 10 moles in total of one or more selected from γ-sanshool and α-sanshool per mole of 6-shogaol.

The present invention provides a composition that contains naturally occurring ingredients as active ingredients and has the effect of enhancing the expression of the adrenomedullin gene.

FIG. 1 shows the relative mRNA expression level of adrenomedullin to β-actin in rat-derived small intestinal cell lines cultured in medium supplemented with 10 μM 6-shogaol, 25 μg/mL Japanese pepper ethanol extract, 50 μg/mL Japanese pepper ethanol extract, a combination of 10 μM 6-shogaol and 25 μg/mL Japanese pepper ethanol extract, or a combination of 10 μM 6-shogaol and 50 μg/mL Japanese pepper ethanol extract, with the expression level under control conditions taken as 1. FIG. 2 shows the results of the experiments using 10 μM 6-shogaol, 10 μg/mL or 20 μg/mL Fr. 3-1 (28 μM or 56 μM as HAS), a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-1, 10 μg/mL or 20 μg/mL Fr. 3-2 (31 μM or 62 μM as AS), a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-2, 10 μg/mL or 20 μg/mL Fr. 3-3 (28 μM or 56 μM as GS), or a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. The relative mRNA expression level of adrenomedullin to β-actin in a rat-derived small intestine cell line cultured in a medium supplemented with a combination of 3-3 is shown, with the expression level under control conditions taken as 1.

<6-Shogaol>
6-Shogaol may be derived from a plant such as ginger, or may be chemically synthesized, but is preferably derived from a plant.

6-Shogaol may be used in the form of an extract from a plant, such as ginger, that contains 6-shogaol, or it may be used in the form of a purified or concentrated compound.

When using 6-shogaol derived from ginger, the ginger may be dried ginger or ginger (shokyo), which are herbal medicines.

A plant extract containing 6-shogaol can be prepared by subjecting the plant material to an extraction operation using an extraction medium. As the extraction medium, an organic solvent, water, hot water, or a supercritical fluid such as supercritical carbon dioxide can be used, with a solvent being particularly preferred. As the solvent, an alcohol such as ethanol, an organic solvent such as ethyl acetate, hexane, or acetone, or a mixture thereof is particularly preferred, with ethanol and/or ethyl acetate being particularly preferred. When producing an extract by solvent extraction, the plant material is immersed in an appropriate amount of solvent (for example, 0.5 to 20 times the weight of the plant material) and appropriately stirred or allowed to stand to dissolve the solvent-soluble components into the solvent. The extraction time is not particularly limited, but can be 5 to 120 minutes, for example, 15 to 30 minutes. The extraction temperature is not particularly limited, but can be 0°C to 125°C, for example, 25°C to 125°C. After extraction, the solvent fraction containing the solvent-soluble components and the solid fraction such as cell walls are separated by solid-liquid separation means (centrifugation, filtration (diatomaceous earth filtration, etc.)), and the solvent fraction is obtained as an extract. The obtained extract may be used as it is as a 6-shogaol-containing extract, or if necessary, the extract may be further subjected to further purification means such as concentration, solvent fractionation, chromatography (column chromatography, gas chromatography, high performance liquid chromatography (HPLC), supercritical fluid chromatography, etc.) and/or recrystallization, and the resulting product may be used as a 6-shogaol-containing extract. The plant raw material used for extraction may be in its original form, or may be cut to an appropriate size or shape, or may be crushed, pulverized, or ground, or may be in the form of juice.

<Ethanol extract of Japanese pepper>
The part of Japanese pepper used for preparing the ethanol extract is not particularly limited, but the peel, fruit, seeds, leaves, stems, etc. can be used, with the peel being particularly preferred.

The method for preparing the ethanol extract of Japanese pepper is not particularly limited. For example, Japanese pepper is immersed in an appropriate amount of ethanol (for example, 0.5 to 20 times the amount by weight of Japanese pepper), and then appropriately stirred or left to dissolve soluble components in ethanol. The extraction time is not particularly limited, but can be 5 to 120 minutes, for example, 15 to 30 minutes. The extraction temperature is not particularly limited, but can be 60°C to 125°C, for example, 85°C to 100°C. After extraction, the solution fraction containing the ethanol-soluble components and the solid fraction such as the cell wall are separated by the solid-liquid separation means, and the solution fraction is obtained as the extract. The obtained extract may be used as it is as the ethanol extract, or if necessary, the extract may be further subjected to further purification means such as concentration, solvent fractionation, chromatography (column chromatography, gas chromatography, high-performance liquid chromatography (HPLC), supercritical fluid chromatography, etc.) and/or recrystallization, and the resulting product may be used as the ethanol extract. The form of the Japanese pepper used for extraction can be the original form, or it can be cut to an appropriate size or shape, or it can be crushed, pulverized, or ground, or it can be in the form of juice.

The Japanese pepper used in the preparation of the ethanol extract of Japanese pepper may be a water extraction residue obtained by performing a water extraction treatment before the above-mentioned extraction with ethanol, that is, Japanese pepper from which water-soluble components have been removed by water extraction. Here, the water extraction of Japanese pepper is performed, for example, by immersing Japanese pepper in an appropriate amount of water (for example, 0.5 to 20 times the amount of Japanese pepper by weight) and appropriately stirring or leaving it to elute the water-soluble components into the water. The extraction time is not particularly limited, but can be 5 to 120 minutes, for example, 15 to 30 minutes. The extraction temperature is not particularly limited, but can be 60°C to 125°C, for example, 85°C to 100°C. After the extraction, the aqueous solution fraction and the solid fraction are separated by the solid-liquid separation means, and the solid fraction is collected and dried as necessary to obtain a Japanese pepper water extraction residue, which can be used in the preparation of the ethanol extract.

The ethanol extract of Japanese pepper may be formulated into a desired form such as powder or granules. Appropriate excipients can be used for formulation.

The ethanol extract of Japanese pepper preferably contains one or more selected from gamma-sanshool, alpha-sanshool, and hydroxy-alpha-sanshool.

A preferred example of an ethanol extract of Japanese pepper contains γ-sanshool, preferably at a concentration of 50% by weight or more, more preferably at a concentration of 70% by weight or more. Another preferred example of an ethanol extract of Japanese pepper contains α-sanshool, preferably at a concentration of 50% by weight or more, more preferably at a concentration of 70% by weight or more. Yet another preferred example of an ethanol extract of Japanese pepper contains hydroxy-α-sanshool, preferably at a concentration of 50% by weight or more, more preferably at a concentration of 70% by weight or more.

<Composition for enhancing adrenomedullin gene expression>
One or more embodiments of the present invention relate to a composition for enhancing adrenomedullin gene expression, which contains 6-shogaol and an ethanol extract of Zanthoxylum japonicum as active ingredients. The composition according to this embodiment preferably does not contain one or more selected from ginseng and components derived from ginseng, and more preferably does not further contain hawthorn and/or components derived from hawthorn, Angelica acutiloba and/or components derived from Angelica acutiloba, and capsicum and/or components derived from capsicum.

The subject of the composition according to this embodiment is typically a human, but is not limited to humans and may be other non-human animals, such as mammals other than humans.

Adrenomedullin is a peptide that has physiological activities such as vasodilatation and angiogenesis. By enhancing the gene expression of adrenomedullin, it is possible to increase blood flow. Furthermore, by increasing blood flow through the enhancement of gene expression of adrenomedullin, it is possible to achieve effects such as antibacterial action, anti-enteritis, protection of the gastric mucosa, and inhibition of thrombus formation.

Patent Document 1 describes that a combination of a compound derived from Korean ginseng and a compound derived from dried ginger and/or a compound derived from Japanese pepper has an effect of enhancing adrenomedullin production, while it describes that there was no synergistic effect of increasing blood flow when dried ginger extract and Japanese pepper extract were combined. Non-Patent Document 1 describes that Daikenchuto and one of its components, 6-shogaol, have the effect of promoting the release of adrenomedullin from intestinal epithelial cells. Patent Document 2 describes an anti-inflammatory composition containing Japanese pepper, dried ginger, Korean ginseng, and glue candy, but does not describe the effect of a composition that does not contain Korean ginseng. Patent Document 3 describes that Japanese pepper or an extract thereof has an effect of protecting the digestive tract mucosa, and Patent Document 4 describes that a combination of five herbal extracts (ethanol extract in the example) of Chinese ginseng, Japanese cratae, Japanese angelica, red pepper, and Chinese bell pepper has a hair growth effect by increasing peripheral blood flow. Patent documents 1 to 4 and non-patent document 1 do not describe the combination of 6-shogaol and an ethanol extract of Japanese pepper.

The composition according to this embodiment contains 6-shogaol and an ethanol extract of Japanese pepper as active ingredients, and thus has an effect that could not be predicted from the matters described in Patent Documents 1 to 4 and Non-Patent Document 1, that is, it can synergistically enhance the expression of the adrenomedullin gene.

6-Shogaol and the ethanol extract of Japanese pepper are both components with strong pungent tastes. By combining the two, the expression of the adrenomedullin gene can be synergistically enhanced, so the total amount of 6-shogaol and the ethanol extract of Japanese pepper required to enhance the expression of the adrenomedullin gene can be reduced compared to when each is used alone. For this reason, the composition for enhancing the expression of the adrenomedullin gene according to this embodiment is suitable for use as a food or beverage with reduced pungent taste.

Enhancement of adrenomedullin gene expression can be confirmed by preparing cDNA from mRNA in cells taken from a subject who has ingested or administered the composition of this embodiment, or in cells cultured in the presence of the composition of this embodiment, and using the cDNA as a template, carrying out a nucleic acid amplification reaction using a primer set capable of specifically amplifying at least a portion of the cDNA base sequence of adrenomedullin (5' untranslated region, coding region and/or 3' untranslated region, preferably the coding region), and detecting the amount of amplified product from the reaction.

Furthermore, enhanced adrenomedullin gene expression can be confirmed by detecting the amount of adrenomedullin peptide in cells taken from a subject who has ingested or been administered the composition of this embodiment, or in cells cultured in the presence of the composition of this embodiment. When the amount of adrenomedullin peptide increases in the cells, it can be evaluated that adrenomedullin gene expression is enhanced.

The amino acid sequence of the precursor of human adrenomedullin (preproadrenomedullin) is shown in SEQ ID NO: 3, and the cDNA base sequence of human adrenomedullin is shown in SEQ ID NO: 4. In SEQ ID NO: 4, the coding region is from positions 179 to 736.

The content of 6-shogaol and ethanol extract of Japanese pepper in the composition according to this embodiment is not particularly limited, and for example, the composition may contain a total of 0.1 to 95% by mass of 6-shogaol and ethanol extract of Japanese pepper per total amount of the composition. The blending ratio of 6-shogaol and ethanol extract of Japanese pepper in the composition according to this embodiment is not particularly limited. For example, the amount of ethanol extract of Japanese pepper may be 2 to 100 parts by mass, preferably 3 to 20 parts by mass, per 1 part by mass of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing one or more selected from gamma-sanshool, alpha-sanshool, and hydroxy-alpha-sanshool, the content of one or more selected from gamma-sanshool, alpha-sanshool, and hydroxy-alpha-sanshool is preferably 2 to 10 moles in total, and more preferably 2 to 7 moles, per mole of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing one or more selected from γ-sanshool and α-sanshool, the content of one or more selected from γ-sanshool and α-sanshool is preferably 2 to 10 moles in total, more preferably 2 to 7 moles, per mole of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing γ-sanshool, the content of γ-sanshool is preferably 2 to 10 moles in total per mole of 6-shogaol, and more preferably 2 to 7 moles.

<6-Composition containing shogaol and ethanol extract of Japanese pepper>
Another embodiment of the present invention relates to a composition containing 6-shogaol and an ethanol extract of Zanthoxylum japonicum. The composition according to this embodiment preferably does not contain one or more selected from ginseng and ingredients derived from ginseng, and more preferably does not further contain crataegus and/or ingredients derived from crataegus, dong quiz and/or ingredients derived from dong quiz, and capsicum and/or ingredients derived from capsicum.

The subject of the composition according to this embodiment is typically a human, but is not limited to humans and may be other non-human animals, such as mammals other than humans.

The composition according to this embodiment has an effect that could not be predicted from the matters described in Patent Documents 1 to 4 and Non-Patent Document 1, that is, it can synergistically enhance the expression of the adrenomedullin gene.

The composition according to this embodiment is suitable for use as a food or beverage with reduced spiciness.

The content of 6-shogaol and ethanol extract of Japanese pepper in the composition according to this embodiment is not particularly limited, and for example, the composition may contain a total of 0.1 to 95% by mass of 6-shogaol and ethanol extract of Japanese pepper per total amount of the composition. The blending ratio of 6-shogaol and ethanol extract of Japanese pepper in the composition according to this embodiment is not particularly limited. For example, the amount of ethanol extract of Japanese pepper may be 2 to 100 parts by mass, preferably 3 to 20 parts by mass, per 1 part by mass of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing one or more selected from gamma-sanshool, alpha-sanshool, and hydroxy-alpha-sanshool, the content of one or more selected from gamma-sanshool, alpha-sanshool, and hydroxy-alpha-sanshool is preferably 2 to 10 moles in total, and more preferably 2 to 7 moles, per mole of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing one or more selected from γ-sanshool and α-sanshool, the content of one or more selected from γ-sanshool and α-sanshool is preferably 2 to 10 moles in total, more preferably 2 to 7 moles, per mole of 6-shogaol.

When the composition according to this embodiment contains an ethanol extract of Japanese pepper containing γ-sanshool, the content of γ-sanshool is preferably 2 to 10 moles in total per mole of 6-shogaol, and more preferably 2 to 7 moles.

<Composition containing 6-shogaol and one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool in a total amount of 2 to 10 moles per mole of 6-shogaol>
Yet another one or more embodiments of the present invention relate to a composition containing 6-shogaol and one or more selected from 2 to 10 moles in total of γ-sanshool, α-sanshool, and hydroxy-α-sanshool per mole of 6-shogaol. The composition according to this embodiment preferably does not contain one or more selected from ginseng and components derived from ginseng, and more preferably does not further contain hawthorn and/or components derived from hawthorn, dong qui and/or components derived from dong qui, and capsicum and/or components derived from capsicum.

The subject of the composition according to this embodiment is typically a human, but is not limited to humans and may be other non-human animals, such as mammals other than humans.

The composition according to this embodiment has an effect that could not be predicted from the matters described in Patent Documents 1 to 4 and Non-Patent Document 1, that is, it can synergistically enhance the expression of the adrenomedullin gene.

The composition according to this embodiment is suitable for use as a food or drink with reduced spiciness.
In the composition according to the present embodiment, the content of one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool is more preferably 2 to 7 moles in total per mole of 6-shogaol.

The composition according to this embodiment more preferably contains one or more selected from γ-sanshool and α-sanshool, and the content of one or more selected from γ-sanshool and α-sanshool is preferably 2 to 10 moles in total, more preferably 2 to 7 moles, per mole of 6-shogaol.

The composition according to this embodiment more preferably contains γ-sanshool, and the content of γ-sanshool is preferably 2 to 10 moles in total per mole of 6-shogaol, and more preferably 2 to 7 moles.

In the composition according to this embodiment, one or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool may be derived from a plant such as Japanese pepper or may be chemically synthesized, but is preferably derived from a plant.

One or more selected from γ-sanshool, α-sanshool, and hydroxy-α-sanshool may be used in the form of an extract from a plant such as Japanese pepper that contains it, or in the form of a purified or concentrated compound.

<Preferable Examples of Compositions>
More preferred examples of the compositions according to the above-described embodiments disclosed in this specification (hereinafter referred to as "the compositions disclosed in this specification") will be described below.

The subject of the composition disclosed herein is preferably a subject who requires or desires enhanced gene expression of adrenomedullin. The composition disclosed herein is also effective for healthy subjects.

The compositions disclosed herein may be in the form of pharmaceuticals, foods and beverages, cosmetics, etc. Food and beverages also include those in the form of functional foods, foods for specified health uses, nutritional supplements, etc.

The compositions disclosed herein are preferably compositions that are ingested or administered orally or nasally, and more preferably compositions that are ingested or administered orally.

The composition disclosed herein may also be a composition used to enhance adrenomedullin gene expression in cells ex vivo by coexisting with the cells. In this case, the composition disclosed herein is used by adding it to a medium for culturing the cells.

As used herein, the term "daily usage amount" refers to the total amount of the composition disclosed herein that is ingested, administered, or used in one day, and preferably refers to the total amount of the composition disclosed herein that is ingested, administered, or used in one day by one human, particularly one adult. Specific examples of "daily usage amount" include amounts of the composition disclosed herein that are 0.1 g to 500 g.

The compositions disclosed herein may be taken, administered or used continuously, or may be taken, administered or used on an as-needed basis.

The form of the composition disclosed herein is not particularly limited, and may be, for example, liquid, fluid, gel, semi-solid, or solid.

The compositions disclosed herein may further contain at least one other component in addition to the components described in each embodiment. The at least one other component that may be contained in the compositions disclosed herein is not particularly limited, but is preferably an ingredient that is acceptable in the final form of a pharmaceutical product, food or beverage, cosmetic product, etc.

Examples of such other ingredients include sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, water, etc. Furthermore, colorants, flavorings, preservatives, antiseptics, fungicides, and other biologically active substances may be added as necessary.

Sweeteners include monosaccharides and disaccharides such as glucose, fructose, sucrose, lactose, maltose, palatinose, trehalose, and xylose, isomerized sugars (glucose-fructose liquid sugar, fructose-glucose liquid sugar, mixed sugar isomerized sugar, etc.), sugar alcohols (erythritol, xylitol, lactitol, palatinit, sorbitol, reduced starch syrup, etc.), honey, and high-intensity sweeteners (sucralose, acesulfame potassium, thaumatin, stevia, aspartame, etc.).

Acidulants include citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, or salts thereof, and one or more of these may be used.

Vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, inositol, etc.

Minerals include calcium, magnesium, zinc, iron, etc.

Thickening agents include carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, karaya gum, agar, gelatin, pectin, soybean polysaccharides, carboxymethylcellulose (CMC), etc.

Emulsifiers include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, lecithin, plant sterols, saponin, etc.

Antioxidants include vitamin C, tocopherol (vitamin E), enzyme-treated rutin, etc.

The other ingredients can be appropriately blended in amounts within the ranges that are typically used by those skilled in the art in compositions such as food, beverages, and pharmaceuticals.

The compositions disclosed herein may contain only the components described in each embodiment as active ingredients involved in enhancing gene expression of adrenomedullin. In this case, the compositions disclosed herein may contain other ingredients as described above that are not involved in enhancing gene expression of adrenomedullin.

<6-Shogaol>
A commercially available reagent (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as 6-shogaol (6S).

<Japanese pepper ethanol extract>
A certain amount of ethanol was added to the residue of the hot water extraction of crushed Japanese pepper, and extraction was carried out for 60 minutes or more with stirring. The extract recovered by solid-liquid separation was used as the Japanese pepper ethanol extract.

<Fractions of Japanese pepper ethanol extract>
The ethanol extract of Japanese pepper was fractionated by medium pressure reversed phase chromatography (column: ODS, mobile phase: water/methanol, wavelength: 270 nm). The obtained eluates were dried using an evaporator to obtain fractions Fr. 3-1, Fr. 3-2, and Fr. 3-3. Fr. 3-1 contains 74% by weight of hydroxy-α-sanshool (HAS). Fr. 3-2 contains 78% by weight of α-sanshool (AS). Fr. 3-3 contains about 75% by weight of γ-sanshool (GS).

<Enhanced adrenomedullin gene expression 1>
A rat-derived small intestinal epithelial cell line (RIKEN) was suspended in DMEM (1 g/L glucose, 5% FBS, 4 μg/mL insulin) medium to give 1 × ¹⁰ cells per well, and the suspension was seeded onto a 24-well plate and cultured at 37°C under 5% _CO2 conditions.

24 hours after the start of the culture, the medium was replaced with DMEM (1 g/L glucose, 3% inactivated FBS) containing 10 μM 6-shogaol, 25 μg/mL Japanese pepper ethanol extract, 50 μg/mL Japanese pepper ethanol extract, a combination of 10 μM 6-shogaol and 25 μg/mL Japanese pepper ethanol extract, or a combination of 10 μM 6-shogaol and 50 μg/mL Japanese pepper ethanol extract, and cultured for another 6 hours.
As a control, a small intestinal epithelial cell line was cultured in the same manner except that a DMEM (1 g/L glucose, 3% inactivated FBS) medium containing neither 6-shogaol nor Japanese pepper ethanol extract was used.

After culturing, the cells were washed with phosphate-buffered saline (PBS), harvested, and RNA was extracted from the cells. Cell harvesting and RNA extraction were performed using RNeasy Mini Kit (QIAGEN) according to the attached procedure.

The obtained RNA was used to measure the mRNA expression levels of adrenomedullin and the internal standard β-actin by real-time PCR. This was done using One Step TB Green PrimeScript RT-PCR Kit II (Takara Bio) and following the attached protocol. The primer sequences are as shown in the following table.

The mRNA expression level of adrenomedullin was calculated as a relative value to the mRNA expression level of β-actin. Furthermore, the calculated mRNA expression level of adrenomedullin under each condition was expressed as a relative value to the calculated mRNA expression level of adrenomedullin under the control condition.

Measurements were performed for each condition with n=1. The results are shown in Figure 1.

The expression level of adrenomedullin in the small intestinal epithelial cell line was 4.17 when 10 μM 6-shogaol alone was added, 1.52 when 25 μg/mL Japanese pepper ethanol extract alone was added, and 11.39 when 50 μg/mL Japanese pepper ethanol extract alone was added, whereas it was 14.83 when 10 μM 6-shogaol and 25 μg/mL Japanese pepper ethanol extract were added in combination, and 25.63 when 10 μM 6-shogaol and 50 μg/mL Japanese pepper ethanol extract were added in combination. In all cases, a remarkable synergistic effect was confirmed that far exceeded the combined effect of each component.

<Enhanced adrenomedullin gene expression 2>
A rat-derived small intestinal epithelial cell line (RIKEN) was cultured in the same manner as in the above experiment, and 24 hours after the start of culture, the cells were treated with 10 μM 6-shogaol, 10 μg/mL or 20 μg/mL Fr. 3-1 (28 μM or 56 μM as HAS), a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-1, 10 μg/mL or 20 μg/mL Fr. 3-2 (31 μM or 62 μM as AS), a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-2, or 10 μg/mL or 20 μg/mL Fr. The medium was replaced with DMEM (1 g/L glucose, 3% inactivated FBS) medium containing 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-3 (28 μM or 56 μM as GS), or a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-3, and the cells were further cultured for 6 hours.

As a control, a small intestinal epithelial cell line was cultured in the same manner, except that DMEM (1 g/L glucose, 3% inactivated FBS) medium not containing 6-shogaol, Fr. 3-1, Fr. 3-2, or Fr. 3-3 was used.

The amount of adrenomedullin mRNA expression in the cultured cells was measured in the same manner as in the above experiment. The amount of adrenomedullin mRNA expression under each condition was expressed as a relative value to the amount of adrenomedullin mRNA expression under the control condition.

Measurements were performed for each condition (n = 2 or 3), and the average and standard deviation were calculated. The results are shown in Figure 2.

The adrenomedullin expression level in small intestinal epithelial cell lines cultured in a medium containing a combination of 10 μM 6-shogaol and 10 μg/mL or 20 μg/mL Fr. 3-1, Fr. 3-2, or Fr. 3-3 was significantly higher than the sum of the adrenomedullin expression level when only 10 μM 6-shogaol was added and the adrenomedullin expression level when only Fr. 3-1, Fr. 3-2, or Fr. 3-3 was added at each concentration, confirming a synergistic effect. The effect of synergistically improving the adrenomedullin expression level by combining with 6-shogaol was highest in Fr. 3-3 (high GS content), followed by Fr. 3-2 (high AS content), and then Fr. 3-1 (high HAS content).

Claims (2)

A composition for enhancing adrenomedullin gene expression, containing 6-shogaol and one or more selected from γ-sanshool and α-sanshool as active ingredients (however, the composition does not contain ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1, ginsenoside Rg2 and/or ginsenoside Rh1). The composition according to claim 1 , comprising 6-shogaol and at least one selected from 2 to 10 moles of γ-sanshool and α-sanshool per mole of 6-shogaol.