JP2022072041A - Composition for enhancing adrenomedullin gene expression - Google Patents

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

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

Adrenomedullin is a circulatory regulatory peptide with vasodilatory activity. Adrenomedullin is produced from various organs such as the circulatory system and digestive system, and has been reported to have important physiological activities such as vasodilation, angiogenesis, antibacterial activity, anti-enteritis, gastric mucosal protection, and suppression of thrombosis. (Patent Document 1).

Patent Document 1 describes an adrenomedullin production enhancer containing a compound derived from ginseng (= ginseng) and a compound derived from dried ginger and / or a compound derived from Japanese pepper as an active ingredient. In Example 2 of Patent Document 1, a combination of ginseng (= Koryo ginger) extract and ginger extract, a combination of ginseng extract and sansho extract, a combination of ginger extract and sansho extract, ginger extract alone, When VC (vascular condition), which is an index of blood flow, was measured by administering ginger extract alone and ginger extract alone to rats, and the rate of increase was determined, the combination of ginger extract and ginger extract, and The combination of ginger extract and sansho extract showed a clearly higher increase in VC than the respective single doses, but the combination of ginger extract and sansho extract did not show any change in the rate of increase in VC. Have been described.

In Non-Patent Document 1, daikenchuto (including sansho, ginger, and ginger), which is a kind of Chinese herbal medicine, promotes the release of adrenomedulin from intestinal epithelial cells, thereby promoting blood flow in the intestine. In addition, daikenchuto and one of its components, 6-shogaol, have the effect of promoting the release of adrenomedurin from intestinal epithelial cells, which is due to the antagonist of TRPA1 which is one of the TRP channels. It is stated that it is inhibited.

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

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

Patent Document 4 describes a hair restorer containing five kinds of crude drug extracts of hawthorn, hawthorn, angelica acutiloba, capsicum, and hawthorn. Patent Document 4 describes that the five crude drug extracts of the hair restorer aim to increase peripheral blood flow by local peripheral vasodilation of the hair loss portion, thereby promoting the activity of hair roots.

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

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

An object of the present invention is to provide a composition containing a plant component as an active ingredient and having an action of enhancing gene expression of adrenomedullin.

Unlike the description in Patent Document 1, the compositions containing the water extract of ginger and the water extract of Sansho synergistically express the adrenomedulin gene even if they are not used in combination with Koryo ginseng. It has been found that it has an action of enhancing the present invention, and the following invention has been completed.
(1) A composition for enhancing adrenomedulin gene expression, which contains a water extract of ginger and a water extract of Sansho as an active ingredient, and does not contain one or more selected from ginseng and components derived from ginseng.
(2) The composition according to (1), wherein the ginger is dried ginger.

According to the present invention, there is provided a composition containing a plant component as an active ingredient and having an action of enhancing the expression of an adrenomedullin gene.

FIG. 1 shows the relative mRNA expression levels of adrenomedulin to β-actin in rat-derived small intestinal cell lines cultured under the conditions of addition of dry ginger water extract and / or Sansho water extract at different dilution ratios. It is shown as a value where the expression level in 1 is 1.

<Water extract of ginger>
The site of ginger used for preparing the water extract is not particularly limited, but rhizomes can be preferably used.

Ginger may be a crude drug, ginger or ginger, and is particularly preferably ginger.

The water extract of ginger is preferably an extract of hot water 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 the mixture is appropriately stirred or allowed to elute the water-soluble component in water. The extraction time is not particularly limited, but may be 5 minutes to 120 minutes, for example, 15 minutes 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 solution fraction containing a water-soluble component and the solid fraction such as a cell wall are separated by a solid-liquid separation means (centrifugation, filtration (filtration of diatomaceous earth, etc.)), and the aqueous solution fraction is obtained as an extract. The obtained extract may be used as it is as a water extract, or if necessary, the extract is further concentrated, solvent fractionated, and chromatographic (column chromatography, gas chromatography, high-speed liquid chromatography (HPLC)). , Supercritical fluid chromatography, etc.) and / or a treated product subjected to further purification means such as recrystallization may be used as the water extract. The shape of ginger used for extraction can be the original shape, cut into appropriate dimensions or shapes, crushed, crushed or ground, or squeezed.
The water extract of ginger may be formulated into a desired form such as powder, granules and the like. Suitable excipients can be used for formulation.

<Water extract of Japanese pepper>
The site of Japanese pepper used for preparing the water extract is not particularly limited, but pericarp, fruits, seeds, leaves, stems and the like can be used, and pericarp is particularly preferable.

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

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 the mixture is appropriately stirred or allowed to elute the water-soluble component in the water. The extraction time is not particularly limited, but may be 5 minutes to 120 minutes, for example, 15 minutes 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 solution fraction containing a water-soluble component and the solid fraction such as a cell wall are separated by the solid-liquid separation means, and the aqueous solution fraction is obtained as an extract. The obtained extract may be used as it is as a water extract, or if necessary, the extract is further concentrated, solvent fractionated, and chromatographic (column chromatography, gas chromatography, high-speed liquid chromatography (HPLC)). , Supercritical fluid chromatography, etc.) and / or treated products subjected to further purification means such as recrystallization may be used as the water extract. The shape of Japanese pepper used for extraction can be the original shape, cut into appropriate dimensions or shapes, crushed, crushed, or ground, or squeezed juice.

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

<Composition for enhancing adrenomedullin gene expression>
One or more embodiments of the present invention contain ginger water extract and sansho water extract as active ingredients and do not contain one or more selected from ginseng and ginseng-derived components, adrenomedulin gene expression. With respect to the composition for enhancement. The composition according to the present embodiment preferably does not contain hawthorn and / or components derived from hawthorn, angelica acutiloba and / or components derived from angelica acutiloba, and components derived from capsicum and / or capsicum.

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

Adrenomedullin is a peptide having physiological activities such as vasodilation and angiogenesis. Increased blood flow can be achieved by enhancing gene expression of adrenomedulin. In addition, by increasing blood flow by enhancing the gene expression of adrenomedullin, effects such as antibacterial action, antienteritis, gastric mucosal protection, and suppression of thrombus formation can be achieved.

Patent Document 1 describes that a combination of a compound derived from ginseng and a compound derived from dried ginger and / or a compound derived from Japanese pepper has an adrenomedulin production enhancing effect, while the dried ginger extract and the Japanese pepper extract are described. It is described that there was no synergistic blood flow increasing effect when combined with. Non-Patent Document 1 describes that daikenchuto and one of its components, 6-shogaol, have an action of promoting the release of adrenomedullin from intestinal epithelial cells. Patent Document 2 describes an anti-inflammatory composition containing Japanese pepper, dried ginger, ginseng and candy, but does not describe the effect of a composition containing no ginseng. Patent Document 3 describes that Sansho or an extract thereof has a gastrointestinal mucosal protective effect, and Patent Document 4 describes five kinds of crude drug extracts (in Examples, Sanshiyo, Hawthorn, Touki, Togarashi, and Shiyoukiyo). It has been described that the combination of ethanol extract) has a hair-growth effect by increasing peripheral blood flow. Patent Documents 1 to 4 and Non-Patent Document 1 do not specifically describe the combination of the water extract of ginger and the water extract of Japanese pepper.

By containing the water extract of ginger and the water extract of Sansho as active ingredients, the composition according to the present embodiment can synergistically enhance the expression of the adrenomedulin gene even if it is not used in combination with ginseng. That is, it has an effect that cannot be expected from the matters described in Patent Documents 1 to 4 and Non-Patent Document 1.

The water extract of ginger and the water extract of Japanese pepper are each a strong pungent ingredient. Since the expression of the adrenomedullin gene can be synergistically enhanced by combining the two, the total amount of the water extract of ginger and the water extract of Sansho required to enhance the expression of the adrenomedullin gene is used. It can be reduced as compared with the case of using it alone. Therefore, the composition for enhancing the expression of the adrenomedullin gene according to the present embodiment is suitable for use as a food or drink with reduced pungency.

The enhanced gene expression of adrenomedulin is due to the fact that the mRNA in cells collected from the subject ingested or administered the composition according to the present embodiment or cultured in the presence of the composition according to the present embodiment. A cDNA can be prepared and used as a template to specifically amplify at least a part of the cDNA base sequence of adrenomedulin (5'untranslated region, coding region and / or 3'untranslated region, preferably coding region). It can be confirmed by performing a nucleic acid amplification reaction using a primer set and detecting the amount of amplification product due to the reaction.

In addition, the enhanced gene expression of adrenomedulin means that the cells collected from the subject who ingested or administered the composition according to the present embodiment or the cells cultured in the presence of the composition according to the present embodiment. It can be confirmed by detecting the amount of peptide of adrenomedulin. When the amount of adrenomedulin peptide is increased in the cell, it can be evaluated that the gene expression of adrenomedulin is enhanced.

The amino acid sequence of the precursor of human-derived adrenomedulin (preproadrenomedurin) is shown in SEQ ID NO: 3, and the cDNA base sequence of human-derived adrenomedulin is shown in SEQ ID NO: 4. In SEQ ID NO: 4, the coding region is in the 179th to 736th positions.

The subject of the composition according to this embodiment is preferably a subject that 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 a composition of each form such as a pharmaceutical product, a food or drink, and a cosmetic product. Food and beverages also include foods with functional claims, foods for specified health use, supplements for nutritional supplements, and the like.

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

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

As used herein, the term "daily use" means the total amount of the composition according to the present embodiment ingested, administered or used in one day, preferably by one person, particularly one adult. , Means the total amount of the composition according to this embodiment ingested, administered or used in one day. As a specific example of the "daily usage amount", 0.1 g to 500 g can be exemplified as the amount of the composition according to the present embodiment.

The composition according to this embodiment may be continuously ingested, administered or used, or may be ingested, administered or used when necessary.

The contents of the water extract of ginger and the water extract of Sansho in the composition according to the present embodiment are not particularly limited, and for example, 0.1 to 95% by mass of the water extract of ginger and the water extract of ginger in total per the total amount of the composition. It can contain a water extract of Sansho. The mixing 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 amount of water extract of Japanese pepper can be 10 to 1000 parts by mass with respect to 100 parts by mass of water extract of ginger.

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

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 composition according to the present embodiment may contain at least one other ingredient, which is not particularly limited, but is preferably an ingredient that is acceptable in the final form of pharmaceuticals, foods and drinks, cosmetics, and the like.

Examples of such other components include sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, water and the like. Further, if necessary, pigments, fragrances, preservatives, preservatives, fungicides, further physiologically active substances and the like may be added.

Sweeteners include simple sugars such as glucose, fructose, sucrose, lactose, maltose, palatinose, trehalose, and xylose, disaccharides, and isomerized sugars (high fructose corn syrup, fructose fructose syrup, sugar mixed isomerized sugar, etc.). , Sugar alcohols (erythritol, xylitol, lactitol, palatinit, sorbitol, reduced water candy, etc.), honey, high-fructose sweeteners (sclarose, acesulfam potassium, somatin, stevia, aspartame, etc.) and the like.

Examples of the acidulant include citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, salts thereof and the like, and one or more of these can be used.

Examples of vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, inositol and the like.

Examples of minerals include calcium, magnesium, zinc, iron and the like.

Examples of the thickener include carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, karaya gum, agar, gelatin, pectin, soybean polysaccharide, carboxymethyl cellulose (CMC) and the like.

Examples of the emulsifier include glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, lecithin, vegetable sterol, saponin and the like.

Examples of the antioxidant include vitamin C, tocopherol (vitamin E), enzyme-treated rutin and the like.

The other components can be appropriately blended in an amount within a range usually adopted by those skilled in the art for compositions such as foods and drinks and pharmaceuticals.

The composition according to the present embodiment may contain only a water extract of ginger and a water extract of Japanese pepper as an active ingredient involved in enhancing gene expression of adrenomedullin. In this case, the composition according to the present embodiment may contain other components as described above that are not involved in the enhancement of gene expression of adrenomedullin.

<Dry 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 at 95 ° C. for 30 minutes with stirring, and the extract was concentrated to Brix 20 °. The concentrated extract was centrifuged (10,000 rpm, 25 ° C., 5 minutes), and the supernatant was used as a water extract of dried ginger.

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

<Enhancement of adrenomedullin gene expression>
The rat ^- derived small intestinal epithelial cell line (Lab. The seeds were seeded in a 24-well plate and cultured at 37 ° C. under 5% CO ₂ conditions.

Twenty-four hours after the start of culturing, the medium was replaced with the following medium containing a water extract of dried ginger and / or a water extract of Japanese pepper, and the cells were further cultured for 6 hours.
The medium containing the water extract of dried ginger and / or the water extract of Sansho was DMEM (1 g / L glucose, 3% deactivated FBS) medium, 400 times or 200 times the water extract of dried ginger, and /. Alternatively, it was prepared by adding a water extract of Sansho so as to be diluted 2000 times or 1000 times.

As a control, the small intestinal epithelial cell line was cultured by the same method except that DMEM (1 g / L glucose, 3% deactivated FBS) medium containing no water extract of dried ginger and water extract of Sansho was used.

After culturing, the cells were washed with phosphate buffered saline (PBS), the cells were collected, and RNA was extracted from the cells. RNeasy Mini Kit (QIAGEN) was used for cell recovery and RNA extraction, and the procedure was followed.

Using the obtained RNA, the mRNA expression levels of adrenomedullin and internal standard β-actin were measured by real-time PCR. One Step TB Green PrimeScript RT-PCR Kit II (Takara Bio) was used and the procedure was performed according to the attached protocol. The primer sequence is 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 made at n = 3 for each condition, and the mean value and standard deviation were obtained. In addition, multiple comparison by one-way ANOVA and post-test by Turkey-Kramer test were performed.

The results are shown in FIG. The conditions in which a significant difference of p <0.05 was observed were represented by different characters, and the conditions in which no significant difference was observed were represented by the same characters.

Compared with the control condition without the addition of the dry ginger water extract and the Sansho water extract, the condition of adding the dry ginger water extract of 400-fold dilution or 200-fold dilution alone, and the condition of 2000-fold dilution or 1000-fold dilution. Under the condition that Sansho water extract was added alone, the expression of adrenomedulin tended to be enhanced, but no significant difference was confirmed.

On the other hand, the adrenomedullin expression level under the condition of adding the 400-fold diluted or 200-fold diluted dry ginger water extract and the 2000-fold or 1000-fold diluted Sansho water extract in combination was the adrenomedullin under the control condition. It was significantly higher than the expression level. Further, from the adrenomedullin expression level under the control condition, the adrenomedullin expression level under the condition of adding the dry ginger water extract diluted 400-fold or 200-fold and the Sansho water extract diluted 2000-fold or 1000-fold in combination. The increase range of the adrenomedullin expression level under the condition that the extract of each dilution ratio was added alone exceeded the total increase range from the adrenomedullin expression level under the control condition. This indicates that the expression of adrenomedullin was synergistically enhanced by adding the dry ginger water extract and the Japanese pepper water extract in combination.

Claims (2)

A composition for enhancing adrenomedulin gene expression, which contains a water extract of ginger and a water extract of Sansho as an active ingredient, and does not contain one or more selected from ginseng and components derived from ginseng. The composition according to claim 1, wherein the ginger is ginger.

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