JP7604841B2 - Composition for enhancing adrenomedullin gene expression - Google Patents

Description

The present invention relates to a composition for enhancing adrenomedullin gene expression.

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 a plant component as an active ingredient and has the effect of enhancing gene expression of adrenomedullin.

The inventors have discovered that a composition containing an aqueous extract of ginger and an aqueous extract of Japanese pepper as active ingredients has the effect of synergistically enhancing the expression of the adrenomedullin gene even without being used in combination with ginseng, contrary to the description in Patent Document 1, and have thus completed the present invention described below.
(1) A composition for enhancing adrenomedullin gene expression, which contains a water extract of ginger and a water extract of Japanese pepper as active ingredients, and does not contain one or more components selected from ginseng and components derived from ginseng.
(2) The composition described in (1), wherein the ginger is dried ginger.

The present invention provides a composition that contains a plant component as an active ingredient and has the effect of enhancing the expression of the adrenomedullin gene.

Figure 1 shows the relative mRNA expression level of adrenomedullin to β-actin in a rat-derived small intestinal cell line cultured under conditions in which different dilution ratios of dried ginger water extract and/or Japanese pepper water extract were added, with the expression level under control conditions taken as 1.

<Ginger water extract>
The part of ginger used to prepare the water extract is not particularly limited, but the rhizome is preferably used.

Ginger may be dried ginger or ginger (shokyo), which are herbal medicines, with dried ginger being particularly preferred.

The water extract of ginger is preferably a hot water extract of ginger.

The method for preparing the water extract of ginger is not particularly limited. For example, ginger is immersed in an appropriate amount of water (for example, 0.5 to 20 times the amount of ginger by weight) and appropriately stirred or left to stand to dissolve water-soluble components in 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 extraction, an aqueous fraction containing water-soluble components and a solid fraction such as cell walls are separated by solid-liquid separation means (centrifugation, filtration (diatomaceous earth filtration, etc.)), and the aqueous fraction is obtained as an extract. The obtained extract may be used as it is as an aqueous 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 an aqueous extract. The ginger used for extraction may be in its original form, or may be cut to a suitable size or shape, or may be crushed, pulverized or ground, or may be in the form of juice squeezed out.
The water extract of ginger may be formulated into a desired form such as powder, granules, etc. For the formulation, a suitable excipient can be used.

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

The water extract of Japanese pepper is preferably an extract of Japanese pepper using hot water.

The method for preparing the water extract of Japanese pepper is not particularly limited. For example, Japanese pepper is immersed in an appropriate amount of water (for example, 0.5 to 20 times the amount by weight of Japanese pepper) and appropriately stirred or left to stand to dissolve 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 extraction, the aqueous fraction containing the water-soluble components and the solid fraction such as the cell walls are separated by the solid-liquid separation means, and the aqueous fraction is obtained as the extract. The obtained extract may be used as it is as the aqueous 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 processed product may be used as the aqueous 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 water extract of Japanese pepper may be formulated into a desired form such as powder or granules. Appropriate excipients can be used for formulation.

<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 an aqueous extract of ginger and an aqueous extract of Japanese pepper as active ingredients, and does not contain one or more selected from ginseng and ingredients derived from ginseng. The composition according to this embodiment preferably does not contain hawthorn and/or ingredients derived from hawthorn, dong qui and/or ingredients derived from dong qui, 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.

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 gastrointestinal 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 specifically mention the combination of a water extract of ginger and a water extract of Japanese pepper.

The composition according to this embodiment contains an aqueous extract of ginger and an aqueous 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 even without being used in combination with ginseng.

The aqueous extract of ginger and the aqueous 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, and therefore the total amount of the aqueous extract of ginger and the aqueous 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 subject of the composition according to this embodiment is preferably a subject who requires or desires enhanced gene expression of adrenomedullin. The composition according to this embodiment is also effective for healthy subjects.

The composition according to this embodiment may be in the form of a pharmaceutical, food or beverage, cosmetic, etc. Food or beverage products also include those in the form of functional foods, foods for specified health uses, nutritional supplements, etc.

The composition according to this embodiment is preferably a composition that is ingested or administered orally or nasally, and more preferably a composition that is ingested or administered orally.

The composition according to the present embodiment may also be a composition for enhancing adrenomedullin gene expression in cells by coexisting with the cells outside of a living body. In this case, the composition according to the present embodiment is used by adding it to a medium for culturing the cells.

In this specification, the term "daily usage amount" refers to the total amount of the composition according to this embodiment ingested, administered or used in one day, and preferably refers to the total amount of the composition according to this embodiment ingested, administered or used in one day by one human, particularly one adult. Specific examples of the "daily usage amount" include an amount of the composition according to this embodiment of 0.1 g to 500 g.

The composition of this embodiment may be ingested, administered or used continuously, or may be ingested, administered or used when needed.

The content of the aqueous extract of ginger and the aqueous extract of Japanese pepper in the composition according to this embodiment is not particularly limited, and may be, for example, 0.1 to 95% by mass of the aqueous extract of ginger and the aqueous extract of Japanese pepper in total based on the total amount of the composition. The blending ratio of the aqueous extract of ginger and the aqueous extract of Japanese pepper in the composition according to this embodiment is not particularly limited. For example, the aqueous extract of Japanese pepper may be 10 to 1000 parts by mass per 100 parts by mass of the aqueous extract of ginger.

The shape of the composition according to this embodiment is not particularly limited, and may be, for example, any of liquid, fluid, gel, semi-solid, or solid forms.

The composition according to this embodiment may further contain at least one other ingredient in addition to the aqueous extract of ginger and the aqueous extract of Japanese pepper. The at least one other ingredient that may be contained in the composition according to this embodiment is not particularly limited, but is preferably an ingredient that is acceptable in the final form of a pharmaceutical product, food or beverage, cosmetic, 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 and beverages, pharmaceuticals, etc.

The composition according to this embodiment may contain only the aqueous extract of ginger and the aqueous extract of Japanese pepper as active ingredients involved in enhancing gene expression of adrenomedullin. In this case, the composition according to this embodiment may contain other ingredients as described above that are not involved in enhancing gene expression of adrenomedullin.

<Dried ginger water extract and Japanese pepper water extract>
Hot water in an amount 20 times that of the crushed dried ginger was added, and extraction was performed with stirring at 95° C. for 30 minutes, and the extract was concentrated to a Brix of 20° C. The concentrated extract was centrifuged (10,000 rpm, 25° C., 5 minutes), and the supernatant was used as the water extract of dried ginger.

Similarly, 20 times the amount of hot water was added to the crushed Japanese pepper, and extraction was carried out at 95°C for 30 minutes while stirring, and the extract was concentrated to a Brix of 20°. The concentrated extract was centrifuged (10,000 rpm, 25°C, 5 minutes), and the supernatant was used as the water extract of Japanese pepper.

<Enhanced adrenomedullin gene expression>
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.

After 24 hours from the start of the culture, the medium was replaced with the medium described below containing a water extract of dried ginger and/or a water extract of Japanese pepper, and culture was continued for another 6 hours.
Culture media containing the aqueous extract of dried ginger and/or the aqueous extract of Japanese pepper were prepared by adding the aqueous extract of dried ginger 400-fold or 200-fold and/or the aqueous extract of Japanese pepper 2000-fold or 1000-fold to DMEM (1 g/L glucose, 3% inactivated FBS) medium.

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 that did not contain the aqueous extract of dried ginger or the aqueous extract of Japanese pepper 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 (n=3), and the average and standard deviation were calculated. In addition, multiple comparisons were performed using one-way ANOVA and post-hoc tests were performed using the Turkey-Kramer test.

The results are shown in Figure 1. Conditions where a significant difference of p<0.05 was observed are indicated with different letters, and conditions where no significant difference was observed are indicated with the same letter.

Compared to the control condition where neither dried ginger water extract nor Japanese pepper water extract was added, when 400-fold or 200-fold diluted dried ginger water extract was added alone, and when 2000-fold or 1000-fold diluted Japanese pepper water extract was added alone, there was a tendency for adrenomedullin expression to increase, but no significant difference was confirmed.

In contrast, the adrenomedullin expression level when 400-fold or 200-fold diluted dried ginger water extract and 2000-fold or 1000-fold diluted Japanese pepper water extract were added in combination was significantly higher than the adrenomedullin expression level under control conditions. In addition, the increase in the adrenomedullin expression level when 400-fold or 200-fold diluted dried ginger water extract and 2000-fold or 1000-fold diluted Japanese pepper water extract were added in combination exceeded the sum of the increase in the adrenomedullin expression level when each dilution ratio extract was added alone from the adrenomedullin expression level under control conditions. This indicates that the expression of adrenomedullin was synergistically enhanced by adding the dried ginger water extract and Japanese pepper water extract in combination.

Claims (2)

A composition for enhancing adrenomedullin gene expression, which contains an aqueous extract of ginger and an aqueous extract of Japanese pepper as active ingredients, and does not contain one or more components selected from ginseng and ginseng-derived components. The composition according to claim 1 , wherein the ginger is dried ginger.

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