Composition for enhancing adrenomedullin gene expression

Abstract

To provide a composition that contains a plant component as an active ingredient and has the action of enhancing adrenomedullin gene expression.SOLUTION: One embodiment of the present invention relates to a composition for enhancing adrenomedullin gene expression, containing water extract of ginger and ethanol extract of Japanese pepper as active ingredients and not containing one or more selected from Panax ginseng and Panax ginseng-derived components.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition for enhancing adrenomedullin gene expression. [Background technology]

[0002] Adrenomedullin is a circulatory regulatory peptide with vasodilatory properties. It is produced by various organs such as the circulatory system and digestive system, and has been reported to have important physiological activities such as vasodilatory properties, angiogenesis, antibacterial properties, anti-enteritis properties, gastric mucosa protection, and inhibition of thrombus formation (Patent Document 1).

[0003] Patent Document 1 describes an adrenomedullin production enhancer containing a compound derived from ginseng (= Korean ginseng) and a compound derived from dried ginger and / or a compound derived from Japanese pepper as active ingredients. 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 increase rate 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 single administrations, but no change in the increase rate of VC was observed in the combination of dried ginger extract and Japanese pepper extract.

[0004] Non-patent document 1 describes that Daikenchuto (containing Japanese pepper, dried ginger, and Korean ginseng), a type of herbal medicine, 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, have the effect of promoting the release of adrenomedullin from intestinal epithelial cells, and that this effect is inhibited by an antagonist of TRPA1, a TRP channel.

[0005] 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.

[0006] Patent Document 3 describes a gastrointestinal mucosa protecting agent containing Japanese pepper or an extract thereof as an active ingredient. Patent Document 3 also describes that the gastrointestinal mucosa protecting agent may be mixed with other medicinal active ingredients such as dried ginger or an extract thereof.

[0007] Patent Document 4 describes a hair tonic that contains five kinds of herbal extracts, namely, Chinese hawthorn, Japanese angelica, red pepper, and Chinese laurel. Patent Document 4 describes that the five kinds 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. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2009 / 104248 [Patent Document 2] JP 2002-145791 A [Patent Document 3] JP 2007-291008 A [Patent Document 4] Japanese Patent Application Publication No. 2-17117 [Non-patent literature]

[0009] [Non-Patent Document 1] Am J Physiol Gastrointest Liver Physiol 304:G428-G436,2013 Summary of the Invention [Problem to be solved by the invention]

[0010] An objective of the present invention is to provide a composition which contains a plant component as an active ingredient and has the effect of enhancing adrenomedullin gene expression. [Means for solving the problem]

[0011] 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 as 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. Effect of the Invention

[0012] According to the present invention, there is provided a composition which contains a plant component as an active ingredient and has the effect of enhancing the expression of the adrenomedullin gene. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows the relative mRNA expression level of adrenomedullin to β-actin in rat-derived small intestinal cell lines 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] <Ginger water extract> The part of ginger used to prepare the water extract is not particularly limited, but the rhizome is preferably used.

[0015] Ginger may be dried ginger or ginger (shokyo), which are herbal medicines, and dried ginger is particularly preferred.

[0016] The water extract of ginger is preferably a hot water extract of ginger.

[0017] 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 the water-soluble components are dissolved in the water by appropriately stirring or leaving it to stand. 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 fraction containing the water-soluble components and the solid fraction such as the cell wall 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 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 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.

[0018] <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.

[0019] The water extract of Japanese pepper is preferably an extract of Japanese pepper with hot water.

[0020] 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 of Japanese pepper 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, the aqueous fraction containing the water-soluble components and the solid fraction such as cell walls are separated by the solid-liquid separation means, 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 processed product may be used as an aqueous extract. The form of the Japanese pepper used for extraction may be in the original form, or cut to a suitable size or shape, or crushed, pulverized or ground form, or squeezed juice.

[0021] The water extract of Zanthoxylum japonicum may be formulated into a desired form such as powder, granules, etc. For formulation, a suitable excipient can be used.

[0022] <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.

[0023] 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, for example, mammals other than humans.

[0024] 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 by enhancing the gene expression of adrenomedullin, it is possible to achieve effects such as antibacterial action, antienteritis, gastric mucosa protection, and inhibition of thrombus formation.

[0025] 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 a combination of dried ginger extract and Japanese pepper extract did not have a synergistic effect of increasing blood flow. Non-Patent Document 1 describes that Daikenchuto and one of its components, 6-shogaol, have an 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 its extract has a gastrointestinal mucosa protecting effect, and Patent Document 4 describes that a combination of five herbal extracts (ethanol extract in the example) of Chinese ginseng, Japanese cratae, Japanese angelica, Japanese capsicum, 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 disclose a combination of a water extract of ginger and a water extract of Japanese pepper.

[0026] The composition of this embodiment contains a water extract of ginger and a water extract of Japanese pepper as active ingredients, and has an effect that cannot be predicted from the information described in Patent Documents 1 to 4 and Non-Patent Document 1, that is, it is able to synergistically enhance the expression of the adrenomedullin gene even without being used in combination with ginseng.

[0027] The water extract of ginger and the water extract of Japanese pepper are each a component with a strong pungent taste. By combining the two, the expression of the adrenomedullin gene can be synergistically enhanced, so that the total amount of the water extract of ginger and the water extract of Japanese pepper required to enhance the expression of the adrenomedullin gene can be reduced compared to the case of using each alone. Therefore, the composition for enhancing the expression of the adrenomedullin gene according to this embodiment is suitable for use as a food or drink with reduced pungent taste.

[0028] Enhanced gene expression of adrenomedullin 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, performing 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.

[0029] Furthermore, the enhanced expression of the adrenomedullin gene can be confirmed by detecting the amount of adrenomedullin peptide in cells taken from a subject who has taken or been administered the composition according to this embodiment, or in cells cultured in the presence of the composition according to this embodiment. When the amount of adrenomedullin peptide is increased in the cells, it can be evaluated that the expression of the adrenomedullin gene is enhanced.

[0030] The amino acid sequence of the precursor of human-derived adrenomedullin (preproadrenomedullin) is shown in SEQ ID NO: 3, and the cDNA base sequence of human-derived adrenomedullin is shown in SEQ ID NO: 4. In SEQ ID NO: 4, the coding region is from positions 179 to 736.

[0031] The subject of the composition according to this embodiment is preferably a subject who needs or desires to enhance the gene expression of adrenomedullin. The composition according to this embodiment is also effective for healthy subjects.

[0032] The composition according to the present embodiment may be in the form of a pharmaceutical, food or drink, cosmetic, etc. Food or drink includes those in the form of functional food, food for specified health use, nutritional supplement, etc.

[0033] 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.

[0034] The composition according to the present embodiment may also be a composition for enhancing adrenomedullin gene expression in cells by coexisting with the cells in vitro. In this case, the composition according to the present embodiment is used by being added to a medium for culturing the cells.

[0035] In the present specification, the term "amount used per day" refers to the total amount of the composition according to the present embodiment ingested, administered or used in one day, and preferably refers to the total amount of the composition according to the present embodiment ingested, administered or used in one day by one human, particularly one adult. A specific example of the "amount used per day" is 0.1 g to 500 g of the composition according to the present embodiment.

[0036] The composition according to the present embodiment may be ingested, administered or used continuously, or may be ingested, administered or used when needed.

[0037] The content of the water extract of ginger and the water extract of Japanese pepper in the composition according to the present embodiment is not particularly limited, and for example, the total amount of the composition may contain 0.1 to 95% by mass of the water extract of ginger and the water extract of Japanese pepper. The blending ratio of the water extract of ginger and the water extract of Japanese pepper in the composition according to the present embodiment is not particularly limited. For example, the water extract of Japanese pepper may be 10 to 1000 parts by mass per 100 parts by mass of the water extract of ginger.

[0038] The form of the composition according to the present embodiment is not particularly limited, and may be, for example, any of liquid, fluid, gel, semi-solid, and solid forms.

[0039] The composition according to the present embodiment may further contain at least one other component in addition to the water extract of ginger and the water extract of Japanese pepper. The at least one other component that the composition according to the present embodiment may contain is not particularly limited, but is preferably a component that is acceptable in the final form of a medicine, food or drink, cosmetic, etc.

[0040] Examples of such other components include sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, water, etc. Furthermore, colorants, flavors, preservatives, antiseptics, fungicides, and further physiologically active substances may be added as necessary.

[0041] Examples of 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 isomerized sugar, etc.), sugar alcohols (erythritol, xylitol, lactitol, palatinite, sorbitol, reduced starch syrup, etc.), honey, and high-intensity sweeteners (sucralose, acesulfame potassium, thaumatin, stevia, aspartame, etc.).

[0042] The acidulant may be citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, or a salt thereof, and one or more of these may be used.

[0043] Examples of vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, and inositol.

[0044] Examples of minerals include calcium, magnesium, zinc, and iron.

[0045] Examples of thickening agents include carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, karaya gum, agar, gelatin, pectin, soybean polysaccharides, and carboxymethylcellulose (CMC).

[0046] Examples of the emulsifier include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, lecithin, plant sterols, and saponin.

[0047] Antioxidants include vitamin C, tocopherol (vitamin E), enzyme-treated rutin, and the like.

[0048] The other ingredients can be appropriately blended in amounts within the ranges that are normally used by those skilled in the art in compositions such as foods, beverages, and pharmaceuticals.

[0049] The composition according to the present embodiment may contain only the water extract of ginger and the water extract of Japanese pepper as active ingredients involved in enhancing the gene expression of adrenomedullin. In this case, the composition according to the present embodiment may contain other ingredients as described above that are not involved in enhancing the gene expression of adrenomedullin. EXAMPLES

[0050] <Dried ginger water extract and Japanese pepper water extract> Twenty times the amount of hot water was added to the crushed dried ginger, and extraction was performed with stirring at 95°C for 30 minutes, 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 dried ginger.

[0051] Similarly, 20 times the amount of hot water was added to the crushed Japanese pepper, and extraction was carried out with stirring at 95°C for 30 minutes, 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.

[0052] <Enhancement of adrenomedullin gene expression> Rat-derived small intestinal epithelial cell line (RIKEN) was used at 1 × 10 per well. 5 The cells were suspended in DMEM (1 g / L glucose, 5% FBS, 4 μg / mL insulin) medium, the suspension was seeded onto a 24-well plate, and cultured at 37°C under 5% CO2 conditions.

[0053] 24 hours after the start of the culture, the medium was replaced with the following medium containing an aqueous extract of dried ginger and / or an aqueous extract of Japanese pepper, and cultured for an additional 6 hours. 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.

[0054] 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 containing neither the aqueous extract of dried ginger nor the aqueous extract of Japanese pepper was used.

[0055] After the culture, the cells were washed with phosphate buffered saline (PBS), and the cells were harvested and RNA was extracted from the cells. The cells were harvested and RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to the attached procedure.

[0056] The obtained RNA was used to measure the mRNA expression levels of adrenomedullin and the internal standard β-actin by real-time PCR. The One Step TB Green PrimeScript RT-PCR Kit II (Takara Bio) was used, and the procedure was carried out according to the attached protocol. The primer sequences are as shown in the following table.

[0057] [Table 1]

[0058] The expression level of adrenomedullin mRNA was calculated as a relative value to the expression level of β-actin mRNA. Furthermore, the calculated expression level of adrenomedullin mRNA under each condition was expressed as a relative value to the calculated expression level of adrenomedullin mRNA under the control condition.

[0059] Measurements were performed for each condition (n=3), and the average and standard deviation were calculated. Multiple comparisons were performed using one-way ANOVA and post-hoc tests were performed using the Turkey-Kramer test.

[0060] 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.

[0061] Compared to the control conditions in which 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 differences were confirmed.

[0062] In contrast, the adrenomedullin expression level under the condition where 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 the control condition. In addition, the increase in the adrenomedullin expression level under the condition where 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 under the condition where each dilution ratio extract was added alone from the adrenomedullin expression level under the control condition. This indicates that the expression of adrenomedullin was synergistically enhanced by adding the dried ginger water extract and the Japanese pepper water extract in combination.

Claims

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 according to claim 1 , wherein the ginger is dried ginger.

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