JP5860579B2 - Filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter - Google Patents

Description

  The present invention relates to a type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter containing a noni extract.

The epidermis and dermis of the skin are composed of epidermal cells, dermal fibroblasts, and an extracellular matrix such as collagen that is outside these cells and supports the skin structure. In young skin, the interaction between these skin tissues is kept constant, so that moisture retention, flexibility, elasticity, etc. are secured, and the skin is maintained in a fresh state with its tension and gloss. .
However, when there is an influence of external factors such as irradiation of ultraviolet rays (UV-A, UV-B), significant drying of air, excessive skin washing, contact with hydrogen peroxide, or aging progresses, collagen As a result, the production amount of the extracellular matrix decreases, and the elasticity decreases due to crosslinking. As a result, the skin's moisturizing function and elasticity are lowered, and the horny layer begins to exfoliate abnormally, so that the skin loses its tension and gloss, and exhibits aging symptoms such as roughness and wrinkles.
As described above, it is known that changes accompanying aging of the skin, that is, wrinkles, dullness, disappearance of texture, decrease in elasticity, and the like are associated with reduction or modification of dermal matrix components such as collagen.
Among collagens, type I collagen is the most abundant collagen contained in the body and is also abundantly contained in the dermis of the skin and is known to play a role in producing the strength of the skin.

Moreover, the amino acid which is a main component of natural moisturizing factors (NMF) is produced by degrading filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilagrin in epidermal keratinocytes present in the granule layer immediately below the stratum corneum. It is then phosphorylated immediately, accumulated in keratohyalin granules, decomposed to filaggrin through dephosphorylation and hydrolysis, transferred to the stratum corneum, increased the efficiency of keratin filament aggregation, and is involved in the internal construction of keratinocytes Has been reported (see Non-Patent Document 1).
In recent years, it has been reported that this filaggrin is very important and indispensable for moisture retention of the skin, and that the synthetic power of filaggrin is reduced by conditions such as drying and the amount of amino acids in the stratum corneum is reduced (non-) Patent Document 2).
Therefore, it is expected that the amount of amino acids in the stratum corneum can be increased by promoting the synthesis of filaggrin through promoting the expression of profilagrin mRNA in epidermal keratinocytes, and the water environment of the stratum corneum can be essentially improved.
For this reason, as a filaggrin synthesis promoter, for example, licorice extract (see Patent Document 1), liquiritin known as a flavanone glycoside contained in natural plants (see Patent Document 2), profilagrin and filaggrin protein production promotion As at least one of the agents, a plant extract or yeast extract belonging to the genus Citrus (see Patent Document 3) and the like have been proposed.

The epidermis has a protective function of protecting the skin from various stimuli from the outside by constantly creating new keratinocytes through the division and subsequent differentiation of keratinocytes. In particular, in the differentiation process of keratinocytes, a protein such as involucrin is expressed from the spinous layer to the granule layer, is crosslinked by the action of the enzyme transglutaminase-1, and is an insoluble cell membrane-like structure that wraps around keratinocytes. It forms a fied envelope (CE) and contributes to the stability of the keratinocyte cytoskeleton and structure.
However, if the amount of transglutaminase-1 or involucrin produced in the epidermis is reduced due to various factors, the formation of a confined envelope (CE) becomes incomplete and keratinization cannot be performed normally. As a result, it is considered that the keratin barrier function and the skin moisturizing function are lowered, and skin symptoms such as rough skin and dry skin are exhibited.
For this reason, by increasing the production of involucrin and transglutaminase-1 in the epidermis of keratinocytes and promoting the formation of a cornified envelope to normalize keratinization, external stimuli such as drying and ultraviolet rays can be used. It is considered that various skin symptoms such as dry skin and rough skin can be prevented and ameliorated by suppressing the decrease in the skin barrier function associated with.

Noni ( Morinda citrifolia ) extract is known to have a collagenase activity inhibitory action (see Patent Document 4) and a glycation inhibitory action (see Patent Document 5). It is not known to have filaggrin production promoting action, involucrin production promoting action, and transglutaminase-1 production promoting action.

  Thus, it is considered that substances that can promote the production of type I collagen, filaggrin, involucrin, and transglutaminase-1 are very useful. However, up to now, however, it has a production promoting action as described above and is highly safe, and therefore, an excellent substance that can be widely used as a component for external preparations for skin, cosmetic foods and drinks, research reagents, etc. Has not been provided yet, and there is a strong demand for prompt provision.

JP 2002-363054 A JP 2003-146886 A JP 2001-261568 A JP 2005-145945 A JP 2004-250445 A Fragrance Journal Extra Edition Vol. 17, p14-19, published in 2000 “Arch. Dermatol. Res.” Vol. 288, p. 442-446, issued in 1996

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the first object of the present invention is to provide a type I collagen production promoter that has an excellent type I collagen production promoting action and is highly safe.
Another object of the present invention is to provide a filaggrin production promoter that has an excellent filaggrin production promoting action and is highly safe.
A third object of the present invention is to provide an involucrin production promoter having an excellent involucrin production promoting action and high safety.
A fourth object of the present invention is to provide a transglutaminase-1 production promoter that has an excellent transglutaminase-1 production promoting action and is highly safe.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems. As a result, the extract of Noni ( Morida citrifolia ) has an excellent type I collagen production promoting effect, filaggrin production promoting effect, involucrin production promoting effect, and It has been found that it has a transglutaminase-1 production promoting action, and the present invention has been completed.

The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A type I collagen production promoter characterized by containing an extract of noni ( Morida citrifolia ).
<2> A filaggrin production promoter characterized by containing an extract of noni ( Morinda citrifolia ).
<3> An involucrin production promoter characterized by containing an extract of noni ( Morida citrifolia ).
<4> A transglutaminase-1 production promoter characterized by containing an extract of noni ( Morinda citrifolia ).

According to the present invention, conventional problems can be solved, and the object can be achieved.
That is, according to the present invention, firstly, a highly safe type I collagen production promoter having an excellent type I collagen production promoting action and high safety can be provided.
In addition, according to the present invention, secondly, a filaggrin production promoter having an excellent filaggrin production promoting action and high safety can be provided.
In addition, according to the present invention, thirdly, it is possible to provide an involucrin production promoter having an excellent involucrin production promoting action and high safety.
In addition, according to the present invention, fourthly, it is possible to provide a transglutaminase-1 production promoter that has an excellent transglutaminase-1 production promoting action and is highly safe.

(Type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter)
The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention contain an extract of noni ( Morida citrifolia ), and if necessary, other It contains.
The details of the substances contained in the noni extract that exhibit the above-described actions (type I collagen production promoting action, filaggrin production promoting action, involucrin production promoting action, and transglutaminase-1 production promoting action) are unknown. However, it has not been known so far that the extract of Noni has such an excellent action, which is a new finding by the present inventors.

Noni ( Morinda citrifolia ) is the Japanese name of Yaeyama Aoki and is a plant belonging to the Rubiaceae family. It is distributed from Okinawa to the Ogasawara Islands, Taiwan to China, India and other coastal areas, and is easily available from these regions. It is.
The part of the noni used as the extraction raw material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, flowers, persimmons, fruits, fruit juices, pericarps, seeds, seed coats, stems, leaves, branches , Branches, leaves, stems, bark, roots, rhizomes, root barks, mixtures thereof, and the like. Among these, fruits or fruit juices are preferable.
In addition, the said fruit juice may be used as an extraction raw material, and may be used as it is as an extract.

  The noni, which is an extraction raw material, can be subjected to compression extraction or solvent extraction, for example, after being collected, as it is or after being dried and pulverized using a crusher or the like. The drying may be performed, for example, in the sun or using a commonly used dryer. The noni may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. By performing pretreatment such as degreasing, the extraction treatment with the noni polar solvent can be performed efficiently.

  The noni extract can be easily obtained by utilizing a method generally used for plant extraction. Moreover, you may use a commercial item as said noni extract. The noni extract includes the noni extract, a diluted or concentrated solution of the extract, a dried product of the extract, or a crude product or a purified product thereof.

  There is no restriction | limiting in particular as a solvent used for the said extraction, According to the objective, it can select suitably, For example, water, a hydrophilic organic solvent, or these mixed solvents are room temperature or the temperature below the boiling point of a solvent. It is preferable to use it. Ingredients that exhibit type I collagen production promoting action, filaggrin production promoting action, involucrin production promoting action, and transglutaminase-1 production promoting action contained in the noni are easily extracted by an extraction process using a polar solvent as an extraction solvent. be able to.

  The water that can be used as the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose. For example, pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc. In addition, those subjected to various treatments are included. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include lower ones having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol. Alcohols: lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol, glycerin, etc., and a mixed solvent of the hydrophilic organic solvent and water Etc. can also be used. In addition, when using the mixed solvent of the said water and the said hydrophilic organic solvent, in the case of a lower alcohol, 1 mass part-90 mass parts with respect to 10 mass parts of water, and the water 10 in the case of a lower aliphatic ketone. It is preferable to use what mixed 1 mass part-40 mass parts with respect to the mass part. Moreover, in the case of polyhydric alcohol, it is preferable to use what mixed 1 mass part-90 mass parts with respect to 10 mass parts of water.

When extracting an extract having type I collagen production promoting action, filaggrin production promoting action, involucrin production promoting action, and transglutaminase-1 production promoting action from the noni, which is an extraction raw material, it is necessary to adopt a special extraction method. Rather, it can be extracted using any extraction device at room temperature or under reflux.
Specifically, the extraction raw material is put into a processing tank filled with an extraction solvent, and further, while stirring occasionally as necessary, it is allowed to stand for 30 minutes to 4 hours to elute soluble components, followed by filtration. The solid can be removed, and the extract can be obtained by evaporating the extraction solvent from the resulting extract and drying. The amount of extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material. The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Moreover, when the mixed solvent of water and ethanol is used as an extraction solvent, it is normally 30 minutes-about 4 hours at 40 to 80 degreeC. In addition, the extract obtained by extracting with a solvent is the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase of the present invention as long as the extraction solvent is highly safe. -1 production promoter can be used as an active ingredient.

  The noni extract obtained by extraction is diluted, concentrated, dried according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crudely purified product or purified product thereof. Treatment such as purification may be performed. The obtained noni extract can be used as an active ingredient of type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter as it is. The liquid or its dried product is easier to use. In obtaining the dried extract, a conventional method can be used, and carriers such as dextrin and cyclodextrin may be added to improve hygroscopicity. In addition, the noni, which is an extraction raw material, may have a specific odor and taste. Therefore, the extract of noni can be decolorized, deodorized, etc. within a range that does not cause a decrease in physiological activity. Although it is possible to carry out the desired purification, for example, when it is added to skin cosmetics, it is not used in large quantities. Specifically, purification can be performed by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like.

  The noni extract obtained as described above has excellent type I collagen production promoting action, filaggrin production promoting action, involucrin production promoting action, and transglutaminase-1 production promoting action, and based on these actions. The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention can be suitably used as active ingredients.

  The content of the noni extract in the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter is not particularly limited and is appropriately selected according to the purpose. The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter may be the noni extract itself.

Moreover, as other components other than the noni extract, which can be contained in the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter, There is no particular limitation as long as it does not impair the effect, and it can be appropriately selected according to the purpose. Examples thereof include a physiological saline solution for diluting the noni extract to a desired concentration. It is done.
The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter include, as the other components, type I collagen production promotion action, filaggrin production promotion action, involucrin production It may contain a natural extract other than the noni extract, which has a promoting action and a transglutaminase-1 production promoting action. The content of the other components in the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter is not particularly limited and is appropriately selected depending on the purpose. be able to.

  In addition, the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter can be formulated as necessary to form powder, granules, tablets, etc. It can be in any dosage form. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.

The type I collagen production promoter exerts a type I collagen production promotion effect by the action of a noni extract contained as an active ingredient.
The filaggrin production promoter exhibits filaggrin production promoting action by the action of noni extract contained as an active ingredient.
The involucrin production promoter exerts an involucrin production promoting action by the action of a noni extract contained as an active ingredient.
The transglutaminase-1 production promoter exhibits a transglutaminase-1 production promotion effect by the action of the extract of noni contained as an active ingredient.

  According to the type I collagen production promoter of the present invention, through an excellent type I collagen production promotion effect, for example, changes due to skin aging, that is, wrinkles, dullness, disappearance of texture, loss of elasticity, etc. are improved. It becomes possible to prevent and improve skin aging. However, the type I collagen production-promoting agent of the present invention can be used for all purposes that are meaningful for exerting the type I collagen production-promoting action in addition to these uses.

  According to the filaggrin production promoter of the present invention, it is possible to increase the amount of amino acids in the stratum corneum, improve the water environment of the stratum corneum, and prevent and improve skin aging through excellent filaggrin production promoting action. It becomes. However, the filaggrin production promoter of the present invention can be used for all purposes that are meaningful for exerting the filaggrin production promoting action in addition to these uses.

  According to the involucrin production promoter of the present invention, through excellent involucrin production promoting action, for example, the formation of a cornified envelope (CE), which is an insoluble cell membrane-like structure enveloping keratinocytes, is promoted to normalize keratinization. This prevents skin barrier function from decreasing due to external stimuli such as dryness and ultraviolet rays, and can prevent and improve various skin symptoms such as dry skin and rough skin, and prevent and improve skin aging. It becomes possible. However, the involucrin production-promoting agent of the present invention can be used for all purposes that are meaningful for exerting a filaggrin production promoting action in addition to these uses.

  According to the transglutaminase-1 production promoting agent of the present invention, through the excellent transglutaminase-1 production promoting action, for example, the formation of a cornified envelope (CE) that is an insoluble cell membrane-like structure that wraps around keratinocytes is promoted. By normalizing keratinization, it is possible to prevent the skin barrier function from decreasing due to external stimuli such as dryness and ultraviolet rays, and to prevent and improve various skin symptoms such as dry skin and rough skin. It becomes possible to prevent and improve aging. However, the transglutaminase-1 production promoter of the present invention can be used for all uses that are meaningful for exerting a transglutaminase-1 production promoting action in addition to these uses.

  The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention are suitably applied to humans. Can be applied to animals other than humans (for example, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.).

  The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention have an excellent action and excellent usability and safety when applied to the skin. Therefore, for example, it is suitable for blending into a skin external preparation. Here, the external preparation for skin is not limited in its category, and includes a wide range of skin cosmetics, quasi drugs, pharmaceuticals, and the like. Specifically, for example, ointments, creams, milky lotions, cosmetic liquids , Lotions, packs, foundations, lip balms, bath agents, hair nicks, hair lotions, soaps, body shampoos and the like.

  In addition, the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention have an excellent action and are also safe when taken orally. Since it is excellent, for example, it is suitable for blending into beauty foods and drinks. Here, the cosmetic foods and drinks are not limited in their classification, and include a wide variety of general foods, health foods, health functional foods, quasi drugs, pharmaceuticals, and the like that are taken orally.

  In addition, since the type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention have excellent actions, type I collagen, filaggrin, involucrin, and transglutaminase It can also be suitably used as a reagent for studying diseases related to -1.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Production Example 1: Production of noni fruit extract (freeze-dried product))
The juice was extracted from 200 g of noni fruit as an extraction raw material and filtered. The filtrate was dried with a freeze dryer to obtain 12 g of an extract.

(Production Example 2: Production of 50% ethanol extract of noni fruit)
200 g of noni fruit as an extraction raw material was put into 2,000 mL of 50% by mass ethanol, kept at 80 ° C. for 2 hours with gentle stirring, and then filtered. The filtrate was concentrated under reduced pressure at 40 ° C. and further dried with a vacuum dryer to obtain 20 g of an extract.

(Example 1: Type I collagen production promoting action test)
Using the noni extract of Preparation Example 1 as a test sample, the type I collagen production promoting effect was tested by the following test method.

Human normal fibroblasts (NB1RGB) were cultured in Dulbecco MEM containing 10% FBS at 37 ° C. under 5% CO ₂ , and then cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a concentration of 1.6 × 10 ⁵ cells / mL, then seeded at 100 μL per well in a 96-well microplate, and cultured overnight at 37 ° C. and 5% CO ₂ . After completion of the culture, the medium was removed, and 200 μL of a test sample dissolved in 0.25% FBS-containing Dulbecco MEM was added to each well (sample concentration: 100 ppm or 25 ppm) (ppm = μg / mL) at 37 ° C., 5% CO After culturing under ₂ for 3 days, 90 μL of the supernatant was transferred to an ELISA plate and adsorbed on the plate overnight at 4 ° C., then the solution was discarded and phosphate physiological buffer containing 0.05% Tween-20 Washing was performed with (PBS-T). Thereafter, a blocking operation was performed with a phosphate physiological buffer containing 1% bovine serum albumin. The solution was discarded, washed with a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20, and reacted with an anti-human collagen type I antibody (rabbit IgG; manufactured by Chemicon). Discard the solution, wash with phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20, react with HRP-labeled anti-rabbit IgG antibody, and then perform the same washing procedure to develop color. Reaction was performed.
The rate of type I collagen production promotion was calculated by performing the above ELISA using a standard product, creating a calibration curve, and setting the amount of type I collagen production when no sample was added as 100%. Table 1 shows the type I collagen production promotion rate (%) of each sample. In addition, the calculation method of a type I collagen production promotion rate is as follows.
Type I collagen production promotion rate (%) = A / B × 100
In the above formula, A represents the amount of type I collagen when the test sample is added, and B represents the amount of type I collagen when no test sample is added.

表１の結果から、ノニの抽出物が、試料濃度１００μｇ／ｍＬの時に有意なＩ型コラーゲン産生促進作用を有することが認められた。

From the results in Table 1, it was confirmed that the extract of Noni had a significant type I collagen production promoting effect when the sample concentration was 100 μg / mL.

(Example 2: Profilaggrin / filaggrin production promoting action test)
Using the noni extract of Production Example 1 as a test sample, the effect of promoting filaggrin production was tested by the following test method.

Normal human newborn skin epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte medium (KGM) at 37 ° C. under 5% CO ₂ in an 80 cm ² flask, and the cells were collected by a conventional method. The obtained cells were adjusted to 1.5 × 10 ⁵ cells / mL in the same medium, seeded on a 6-well collagen-coated plate by 2 mL, and cultured at 37 ° C. for 3 days under 5% CO ₂ . After culturing, the medium was replaced with 2 mL of KGM containing or not containing (control concentration) test sample (sample concentration: 50 μg / mL, 12.5 μg / mL) dissolved in 0.5 mass% DMSO, 37 ° C., 5% CO ₂ were cultured for 5 days under. After completion of the culture, total protein was prepared by a conventional method.
<Western blotting>
Samples prepared at 10 μg / row were developed by SDS-PAGE and transferred to a PVDF membrane. After blocking with PBS (−) containing 5% skim milk, anti-human filaggrin monoclonal antibody (manufactured by Harbor Bio-Products), biotin-labeled anti-mouse Ig (manufactured by Whole Ab, Amersham Biosciences), and streptavidin-peroxidase The complex (CALBIOCHEM) was diluted 1,000-fold with PBS (-) containing 0.1% Tween20 and 0.3% skim milk, and allowed to react sequentially. ECL Western blotting detection reagents (Amersham Bioscience) Profilaggrin and filaggrin were detected by luminescence from the company. The detected band was quantitatively measured with KODAK 1D Image Analysis Software EDAS290 Version 3.5.
As a result, the filaggrin production promoting effect of the test sample was evaluated using the value obtained by adding the net intensities (band intensities) of profilaggrin and filaggrin in 10 μg of the protein prepared from the cells cultured with and without the test sample added. Then, profilaggrin / filaggrin production promotion rate (%) was calculated based on the following formula. The results are shown in Table 2.
Profilaggrin / filagrin production promotion rate (%) = A / B × 100
In the above formula, A represents “Net intensity at the time of addition of test sample (total value of profilagrin and filaggrin)”, and B represents “Net intensity at the time of no test sample addition (control)”.

表２の結果から、ノニの抽出物が、フィラグリン産生促進作用を有することが認められた。

From the results in Table 2, it was confirmed that the extract of noni has a filaggrin production promoting action.

(Example 3: Involucrin production promoting action test)
Using the noni extract of Production Example 1 as a test sample, the involucrin production promoting effect was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured in human normal neonatal epidermal keratinocyte medium (KGM) at 37 ° C. under 5% CO ₂ , and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 ⁵ cells / mL, then seeded in a 48-well plate at 200 μL per well, and cultured overnight at 37 ° C. and 5% CO ₂ . After completion of the culture, the medium was removed, and 200 μL of a test sample dissolved in KGM (sample concentration: 50 μg / mL, 12.5 μg / mL) was added to each well, followed by culturing at 37 ° C. under 5% CO ₂ for 48 hours. . After completion of the culture, the medium was removed, the cells were fixed to the plate, and the amount of involucrin expressed on the cell surface was measured by an ELISA method using a monoclonal anti-human involucrin antibody (manufactured by SIGMA) (Plate reader manufactured by Bio-Tech Instruments). From the obtained measurement results, the involucrin production promotion rate (%) was calculated by the following formula. The results are shown in Table 3.
Involucrin production promotion rate (%) = A / B × 100
In the above formula, A represents the absorbance at a wavelength of 405 nm when the test sample was added, and B represents the absorbance at a wavelength of 405 nm when no test sample was added (control).

表３の結果から、ノニの抽出物が、インボルクリン産生促進作用を有し、また、濃度依存的に有意なインボルクリン産生促進作用を有することが認められた。

From the results in Table 3, it was confirmed that the extract of noni has an involucrin production promoting action and a significant involucrin production promoting action in a concentration-dependent manner.

(Example 4: Transglutaminase-1 production promoting action test)
Using the noni extract of Production Example 1 as a test sample, the transglutaminase-1 production promoting effect was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured at 37 ° C. under 5% CO ₂ using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 ⁵ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured at 37 ° C. under 5% CO ₂ for 2 days. After completion of the culture, 100 μL of a test sample dissolved in KGM (sample concentration: 50 μg / mL, 12.5 μg / mL) was added to each well and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After completion of the culture, the medium was removed, the cells were fixed to the plate, and the amount of transglutaminase-1 expressed on the cell surface was measured by an ELISA method using a monoclonal anti-human transglutaminase-1 antibody (Biomedical Technologies) (Bio- Teck Instruments plate reader). The results are shown in Table 4.
The calculation method of the transglutaminase-1 production promoting action is as follows.
Transglutaminase-1 production promotion rate (%) = A / B × 100
In the above formula, A represents the absorbance at a wavelength of 405 nm when the test sample was added, and B represents the absorbance at a wavelength of 405 nm when no test sample was added (control).

表４の結果から、ノニの抽出物が、試料濃度５０μｇ／ｍＬの時に有意なトランスグルタミナーゼ−１産生促進作用を有することが認められた。

From the results in Table 4, it was confirmed that the extract of Noni had a significant transglutaminase-1 production promoting action when the sample concentration was 50 μg / mL.

  The type I collagen production promoter, filaggrin production promoter, involucrin production promoter, and transglutaminase-1 production promoter of the present invention are excellent type I collagen production promotion action, filaggrin production promotion action, involucrin production promotion action, and Since it has a transglutaminase-1 production promoting action and is excellent in safety, it can be suitably used, for example, as a component in an external preparation for skin, a cosmetic food or drink, or a research reagent.

Claims (3)

The filaggrin production promoter characterized by including the pressing extract of the fruit of Noni (Morinda citrifolia). The involucrin production promoter characterized by including the pressing extract of the fruit of Noni (Morinda citrifolia). The transglutaminase-1 production promoter characterized by including the pressing extract of the fruit of Noni (Morinda citrifolia).