JP2014125468A - Fibroblast growth promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel fibroblast growth promoter.SOLUTION: This invention relates to a fibroblast growth promoter containing a water extract of a pericarp of Garcinia mangostana L. and hyaluronic acid as an active ingredient. This invention also relates to a fibroblast growth promoter containing macrulin or glycoside thereof and polyphenol, and hyaluronic acid.

Description

  TECHNICAL FIELD The present invention relates to a fibroblast growth promoter containing macululin or a glycoside thereof, polyphenol, and hyaluronic acid. The present invention also relates to a fibroblast growth promoter containing a water extract of mangosteen peel and hyaluronic acid.

  The skin has a three-layer structure consisting of epidermis, dermis, and subcutaneous tissue. The epidermis layer is a very thin membrane with an average thickness of about 0.2 mm and is present on the outermost side of the skin. It acts as a barrier that prevents foreign substances from entering from the outside and the body's moisture transpiration, and protects the inside. The dermis layer is filled with fibers, capillaries, lymph vessels, etc., which support the epidermis layer, send oxygen and nutrients, and carry away waste products. The dermis layer is inside the epidermis layer and occupies most of the skin tissue. Collagen is the major part of the dermis and, together with hyaluronic acid and elastin, works to support skin tension and elasticity. Fibroblasts present in the dermis produce these components. The subcutaneous tissue is the innermost tissue of the three-layer structure of the skin, and plays a role of supporting the epidermis and dermis. The subcutaneous tissue is mostly subcutaneous fat, and the arteries and veins that pass through it deliver nutrients to the skin tissue and carry waste. In addition, the subcutaneous tissue serves to relieve external stimuli and shocks, to act as heat insulation and heat retention, and to store energy in the form of fat.

  When skin is damaged by various internal and external stimuli such as aging, lifestyle, and ultraviolet rays, the proliferation of fibroblasts existing in the dermis layer decreases, collagen production decreases and collagen degradation occurs, As a result, various symptoms such as skin tension and reduced elasticity occur. A composition that promotes the proliferation of fibroblasts is considered to be useful as a functional food or cosmetic, and development of a material that is highly safe and effective is desired.

  Hyaluronic acid has a repeating structure in which N-acetylglucosamine and glucuronic acid are linked in a linear form with disaccharide units. It is said that it exists in the living body as a very high molecular weight and has a molecular weight of 1 million or more. In vivo, it exists widely in extracellular matrix such as joints, vitreous body, skin and brain.

  Hyaluronic acid is attracting attention for its various functions including its excellent water retention and viscoelasticity, and is used in the form of cosmetics, pharmaceuticals, and foods. It has been reported that high molecular weight hyaluronic acid having a molecular weight of about 950,000 or 1,000,000 has a fibroblast proliferation promoting action (Non-patent Document 1). In addition, it has been reported that hyaluronic acid (HA4) composed of tetrasaccharides has a cytoprotective action (Non-patent Document 2). However, it is not known that low molecular weight hyaluronic acid has a fibroblast proliferation promoting action.

  Mangosteen is an evergreen small Takagi of the genus Gutiferae, native to Southeast Asia, and has been used as a traditional medicine since ancient times, and has been known for its antibacterial, anti-inflammatory, antioxidant, and nourishing tonic effects. Of the fruits of mangosteen, the edible flesh is one-fourth, characterized by its light acidity and refined sweetness, and is counted among the world's three great fruits along with mango and cherimoya. Mangosteen has been reported to be present in mangosteen, but its physiological action is not known (see Patent Documents 1 and 2).

Special table 2008-520585 gazette JP-T-2006-504790

J Cell Sci., 90, 265-273, 1988. J Biol Chem. 10; 277 (19): 17308-17314, 2002.

  The main object of the present invention is to provide a novel fibroblast proliferation promoter.

  As a result of studies to solve the above-mentioned problems, the present inventor found that the composition of mangosteen peel water extract and hyaluronic acid has a fibroblast proliferation promoting action and completed the present invention. In addition, the present inventor has found that a composition containing macululin or a glycoside thereof, polyphenol, and hyaluronic acid has a fibroblast proliferation promoting action.

Examples of the present invention include the following.
(1) A fibroblast proliferation promoter containing a water extract of mangosteen peel and hyaluronic acid as active ingredients.
(2) A fibroblast growth promoter containing as an active ingredient macululin or a glycoside thereof, polyphenol, and hyaluronic acid.
(3) The fibroblast proliferation promoter according to the above (2), wherein the McLullin or glycoside thereof and the polyphenol are derived from an aqueous extract of mangosteen peel.

  Since the fibroblast proliferation promoter according to the present invention (hereinafter referred to as the present growth promoter) promotes the proliferation of fibroblasts and promotes the production of collagen and hyaluronic acid, it reduces collagen and hyaluronic acid. It is useful for suppressing wrinkles and sagging of the skin, dullness and aging of the skin, and dry skin. Moreover, since this invention growth promoter is derived from an edible component or a biological component, it is highly safe.

It is a figure which shows a liquid-liquid distribution scheme. is a diagram showing an HPLC chart of the liquid-liquid partition in n- hexane (Hexane) Water (H ₂ O) layer. The vertical axis represents detection intensity (mAU), and the horizontal axis represents retention time (minutes). The arrow indicates the peak of the McLullin glycoside. Is a diagram illustrating an HPLC chart of the liquid-liquid partition with ethyl acetate (EtOAc) Water (H ₂ O) layer. The vertical axis represents detection intensity (mAU), and the horizontal axis represents retention time (minutes). The arrow indicates the peak of the McLullin glycoside. It is a figure which shows the HPLC chart of a water saturated butanol (BuOH) layer. The vertical axis represents detection intensity (mAU), and the horizontal axis represents retention time (minutes). The arrow indicates the peak of the McLullin glycoside. ODS column Fraction No. It is a figure which shows the HPLC chart of 15-18. The vertical axis represents detection intensity (mAU), and the horizontal axis represents retention time (minutes). It is a figure which shows the HPLC chart of a mcculin glycoside. The vertical axis represents detection intensity (mAU), and the horizontal axis represents retention time (minutes). It is a figure which shows the light absorption spectrum of a mcculin glycoside. The vertical axis represents the detection intensity (mAU), and the horizontal axis represents the wavelength (nm). It is a figure which shows the fibroblast proliferation promoting effect of the water extract of mangosteen peel and hyaluronic acid. The horizontal axis shows the cell growth rate (%). It is a figure which shows the fibroblast growth promotion effect | action of a maclerin glycoside, epicatechin, and hyaluronic acid. The horizontal axis shows the cell growth rate (%). It is a figure which shows the fibroblast phase-proliferation effect | action of a macululin glycoside, procyanidin B2, and hyaluronic acid. The horizontal axis shows the cell growth rate (%). It is a figure which shows the fibroblast proliferation promoting effect of a macululin glycoside, catechin, and hyaluronic acid. The horizontal axis shows the cell growth rate (%).

Mangosteen peel water extract The mangosteen peel water extract according to the present invention (hereinafter referred to as “the extract of the present invention”) is obtained by extracting mangosteen peel (Garcinia mangostana L.) with water. The extraction method can be performed by a commonly used method.

Specifically, it can be produced by cutting mangosteen peel as it is or by cutting it into an appropriate size, blanching or drying as necessary, and extracting with water. The blanching can be performed, for example, by immersing the mangosteen peels raw or frozen and then thawed in hot water at 60 ° C. or higher for 5 minutes or less.
The amount of water used for extraction varies depending on the extraction conditions, but the weight ratio is suitably in the range of 1: 2 to 1:30 (plant raw material: water), and in the range of 1: 3 to 1:20. Is preferable, and the range of 1: 5 to 1:10 is more preferable. The extraction time is suitably in the range of 1 hour to 15 days. The extraction temperature is suitably in the range of 5 to 100 ° C, and preferably in the range of 60 to 100 ° C. The extraction method is not particularly limited, and any method such as batch extraction or continuous extraction using a column can be applied.

The obtained extract can be used as it is, but if necessary, further purification treatment may be added within a range that does not affect its activity. Such purification treatment may be performed by a usual method, for example, by filtering the extract by a conventional method. Moreover, the obtained filtrate can also be used for vacuum concentration, freeze-drying, or a spray dryer.
McLullin or a glycoside thereof McLullin (Macrulin, generic name: (3,4-dihydroxyphenyl) (2,4,6-trihydroxyphenyl) ketone) according to the present invention is represented by the following chemical formula (I) A compound. In addition, the McLurin glycoside is a compound represented by the following chemical formula (II).

  As for the McLullin and its glycoside according to the present invention, commercially available products or those isolated from plant extracts can be used.

  Examples of the plant containing McLullin or its glycoside include, for example, Japanese white mulberry (Morus alba L.), American black-tailed whale (Maclura ponifera (Raf.) Schneid.), Odoris (Iris florentina L.), mango (Mangifera L.). ), Chlorophora tinctoria (L.) Gandich., Gentiana rhodantha Franch., Gentiana verna s. Garcinia mangostana L.). The part of the plant used for extraction is not particularly limited as long as it is a part containing makullin and / or its glycoside, but for example, flowers, ears, pericarp, fruits, pulp, stems, leaves, branches, Branches, leaves, stems, bark, rhizomes, root barks, roots, seeds, insect gills, heartwood, above-ground parts, underground parts or whole grasses can be used. Of these, mangosteen peel is preferred.

The plant extract can be produced by a method usually used from a plant containing mcurulin or a glycoside thereof. Specifically, it can be produced by cutting each part of the plant as it is or cutting it into an appropriate size, blanching or drying as necessary, and extracting with juice or a solvent. The blanching can be performed, for example, by immersing each part of the plant raw or frozen and then thawed in hot water at 60 ° C. or higher for 5 minutes or less. As the extraction solvent, for example, water can be used.
The amount of water used varies depending on the plant material used, extraction conditions, and the like, but is preferably within a range of 1: 2 to 1:30 (plant material: water) in a weight ratio of 1: 3 to 1:20. Within the range, the range of 1: 5 to 1:10 is more preferable. The extraction time is suitably in the range of 1 hour to 15 days. The extraction temperature is suitably in the range of 5 to 100 ° C. The extraction method is not particularly limited, and any method such as batch extraction or continuous extraction using a column can be applied.

  The obtained plant extract can be used as it is, but if necessary, further purification treatment may be added within a range that does not affect its activity. Such purification treatment may be performed by a normal method, for example, by filtering a plant extract by a conventional method. The obtained filtrate can also be concentrated under reduced pressure and lyophilized.

  The method for isolating the mcululin or the glycoside thereof according to the present invention from the plant extract is not particularly limited, and examples thereof include gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase chromatography and the like. be able to. Moreover, you may isolate combining them.

As the mullulin or glycoside thereof according to the present invention, an aqueous extract of mangosteen peel is preferable. In particular, those extracted with hot water at 60 to 100 ° C. are preferred. More preferably, it exists in the range of 70-100 degreeC.
Fibroblast growth promoter The growth promoter of the present invention can promote the growth of fibroblasts and promote the production of collagen and hyaluronic acid.
The growth promoter of the present invention contains an aqueous extract of mangosteen peel and hyaluronic acid as active ingredients. In addition, the growth promoter of the present invention also contains macululin or a glycoside thereof, polyphenol, and hyaluronic acid as active ingredients. Examples of such polyphenols include epicatechin, catechin, and procyanidin. As such polyphenol, a commercially available product can be used, or a product extracted from a plant by a usual method can be used.
The growth promoting agent of the present invention is prepared by mixing the components obtained as described above and mixing them with a raw material that can be used as it is or as a carrier, and then in various forms such as powder, lump, and liquid by conventional methods. It can manufacture by processing.
The content of the water extract of mangosteen peel in the growth promoter of the present invention is not particularly limited. For example, 0.01% by weight (hereinafter simply referred to as “%”) or more is suitable, and preferably 0.05 Within the range of ~ 50%. In addition, the content of makullin or a glycoside thereof in the growth promoter of the present invention is not particularly limited, but for example, it is suitably in the range of 0.0001 to 50%, preferably 0.001 to 10%. Within range. The blending ratio of muculin or its glycoside and polyphenol in the growth promoter of the present invention is not particularly limited, but is in the range of 1: 1 to 1: 100 as a weight ratio (macklin or its glycoside: polyphenol, the same shall apply hereinafter). The inside is preferable, and the range of 1: 5 to 1:20 is more preferable.
In addition, the mixing ratio of muculin or its glycoside and polyphenol or hyaluronic acid is not particularly limited, but the weight ratio is 1: 1: 10 to 1: 100: 10000 (macklin or its glycoside: polyphenol: hyaluronic acid. Within the range of 1: 5: 100 to 1: 20: 1000, more preferably.
The growth promoting agent of the present invention may optionally contain a pharmaceutically or food acceptable additive. Examples of such additives include binders, disintegrants, lubricants, dispersants, suspending agents, emulsifiers, buffers, antioxidants, excipients, surfactants, UV inhibitors, and sequestering agents. , Thickeners, preservatives, antibacterial agents, moisturizers, and pigments, and one or more of these can be used.
The growth-promoting agent of the present invention can be appropriately prepared in dosage forms such as tablets, powders, granules, capsules, solutions, syrups, drop tablets, tonics, lotions, ointments, creams and the like by conventional methods.
The intake of the growth promoter of the present invention varies depending on the symptoms, dosage form, age of the administration subject, body weight, etc., but is usually 0.01 per day per adult as the weight of the extract or the growth promoter of the present invention. It is appropriate to be within the range of ˜100 g, preferably within the range of 0.03 g to 60 g, and as the weight of muculin or its glycoside, it is appropriate to be within the range of 0.01 mg to 10 g. Although it is preferable to be within the range of 0.03 mg to 5 g, it is not necessarily limited to this range. Such daily intake may be taken once, or may be taken divided into 2 to 4 times.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples. However, it goes without saying that the present invention is not limited to the following examples.
Example 1 Isolation of McLurin Glycoside 1.5 kg of dried mangosteen peel was packed into a φ13.5 cm × 60 cm stainless steel column with a 100 mesh filter attached to the bottom, hot water was added from above, and from below The extract was collected at a rate of 3 liters per hour. A solution concentrated with a concentrator (RVT-T, manufactured by HISAKA WORKS LTD) until a solid content (Brix) of 25% is placed in a tray, and a freeze dryer (RLEII-103, Nissei ( What was dried in (made by Co., Ltd.) was pulverized with a blender to obtain a powdered mangosteen hot water extract.

  Liquid-liquid distribution was performed on 25 g of this powder according to the scheme shown in FIG. The HPLC chart obtained by liquid-liquid distribution is shown in FIGS. The water-saturated butanol (BuOH) layer was evaporated to dryness, redissolved with water, charged onto an ODS open column (YMC-gel ODSAQ12S50, φ2 × 17 cm, manufactured by YMC Co., Ltd.), eluted with water and fractions 1-70 Divided into. Of these, the 15-18th fraction was collected and further collected by HPLC (column: YMC-Pack ODS-A 250 × 10 mm, S-5 μm, 12 nm, manufactured by YMC Co., Ltd.) and isolated. 6.7 mg was obtained.

The obtained compound was analyzed by HPLC under the following conditions, confirmed to be a pure product, and measured for an absorption spectrum.
<HPLC conditions>
Column: YMC-pack ODS-A A302, S-5 μm, 12 nm (150 mm × 4.6 mm, manufactured by YMC Co., Ltd.)
Mobile phase: A; 0.1% aqueous formic acid, B; acetonitrile containing 0.1% formic acid

Furthermore, the isolated compound was identified by NMR. The assignment results of ¹³ C and ¹ H are shown in Table 2. Furthermore, from the results of DEPT, 1D-NOESY, HMQC, and HMBC (4 Hz, 12 Hz), it was identified as McLurin glycoside (II).

Test Example 1 Measurement of Fibroblast Growth Promoting Activity of the Growth Promoter of the Present Invention Human normal fibroblasts (manufactured by Takara Bio Inc .; hereinafter referred to as NHDF cells) were used as the cells. NHDF cells include DMEM medium (Gibco) (100 unit / ml Penicillin-100 mg / ml Streptomycin (Gibco)) and 0.1 mM NEAA (Gibco) containing 10% FBS (Fetal Bovine Serum, the same applies hereinafter) ), 7 ml seeded in a culture flask for adherent cells (manufactured by Nippon Biosciences; bottom area: 25 cm ² ) to a final concentration of 1 × 10 ⁴ cells / ml, and a CO ₂ incubator (Sanyo Biomedica) It was cultured for 4-5 days in C using CO ₂ concentration 5%, humidity 90%. After confirming with a microscope that it was 70-90% confluent, it was used in the experiments in sequence.
Hyaluronic acid 3000 (manufactured by Nippon Shinyaku Co., Ltd.) was used as the hyaluronic acid used as the specimen. In addition, Mangosteen Aqua (manufactured by Nippon Shinyaku Co., Ltd.) was used as the mangosteen extract. Each specimen was dissolved in PBS (Phosphate Buffered Saline, the same applies hereinafter). The sample solution was sterilized by using a 0.2 μm filter and used for the experiment.
Cell proliferation activity was measured using WST-1 reagent (Roche Diagnostics). First, NHDF cells which became 70-90% confluent were peeled off using Trypsin-EDTA (manufactured by Gibco) and collected. The NHDF cells were seeded on a 96-well plate at 1.0 × 10 ⁴ cells / well (100 μl) and then incubated for 24 hours. After incubation, the culture solution was removed and washed twice with PBS. Thereafter, a DMEM medium containing 0.5% FBS and a specimen (hyaluronic acid; final concentration 1.0 μg / ml, mangosteen extract; final concentration 1.0 μg / ml) were added singly or plurally, and cultured for 48 hours. After incubation, 5 μl of WST-1 reagent (Roche Diagnostics) was added to each well and incubated for 30 minutes. Thereafter, the absorbance value at 450 nm was measured with a microplate reader (CORONA ELECTRIC, MTP-300).
In the case where no sample was added (control), the same operation as described above was performed, and the relative value of the absorbance value when each sample was added to the absorbance value obtained here was determined to obtain the cell growth rate (%). The result is shown in FIG.

As shown in FIG. 8, the mangosteen extract (the extract of the present invention) alone promoted the proliferation of fibroblasts. Hyaluronic acid also promoted fibroblast proliferation. On the other hand, the combined use of mangosteen extract and hyaluronic acid synergistically enhanced the cell growth promoting activity of each component alone.

Test Example 2 Measurement of Fibroblast Growth Promoting Activity of the Growth Promoter of the Present Invention Human normal fibroblasts (manufactured by Takara Bio Inc .; hereinafter referred to as NHDF cells) were used as the cells. NHDF cells were dispersed in DMEM medium (containing 100 unit / ml Penicillin-100 mg / ml Streptomycin and 0.1 mM NEAA) containing 10% FBS, and a culture flask for adherent cells (manufactured by Nippon Biosciences; bottom area: 25 cm ^2). ) 7 ml so as to give a final concentration of 1 × 10 ⁴ cells / ml, and using a CO ₂ incubator (manufactured by Sanyo Biomedica Co., Ltd .; CO ₂ concentration 5%, humidity 90%), 37 (4-5 at C) The cells were cultured for a day and confirmed to be 70-90% confluent by microscopic examination, and then used for experiments.
Hyaluronic acid 3000 (manufactured by Nippon Shinyaku Co., Ltd.) was used as the hyaluronic acid used as the specimen. Reagents purchased from Funakoshi were used for catechin and epicatechin, and reagents purchased from Sigma were used for procyanidin B2. The McLurin glycoside used in Example 1 was used. Each specimen was dissolved in PBS. The sample solution was sterilized by using a 0.2 μm filter and used for the experiment. Cell proliferation activity was measured using WST-1 reagent (Roche Diagnostics).
First, NHDF cells which became 70-90% confluent were peeled off using Trypsin-EDTA (manufactured by Gibco) and collected. The NHDF cells were seeded on a 96-well plate at 1.0 × 10 ⁴ cells / well (100 μl) and then incubated for 24 hours. After incubation, the culture solution was removed and washed twice with PBS. Thereafter, DMEM medium containing 0.5% FBS and specimen (hyaluronic acid; final concentration 1.0 μg / ml, catechin; final concentration 0.01 μg / ml, epicatechin; final concentration 0.01 μg / ml, procyanidin B2; final A concentration of 0.01 μg / ml, McLullin glycoside; final concentration of 0.001 μg / ml) was added alone or in a plurality of types, and cultured for 48 hours. After incubation, 5 μl of WST-1 reagent (Roche Diagnostics) was added to each well and incubated for 30 minutes. Thereafter, the absorbance value at 450 nm was measured with a microplate reader (CORONA ELECTRIC, MTP-300).
In the case where no sample was added (control), the same operation as described above was performed, and the relative value of the absorbance value when each sample was added to the absorbance value obtained here was determined to obtain the cell growth rate (%). The results are shown in FIGS.
As shown in FIG. 9, the cell growth activity by each component was synergistically enhanced by using a combination of a macrulin glycoside, epicatechin and hyaluronic acid.

As shown in FIG. 10, the cell growth activity of each component was synergistically enhanced by using a combination of the muculin glycoside, procyanidin B ₂ and hyaluronic acid.

  As shown in FIG. 11, cell proliferation activity was synergistically enhanced by using a combination of the muculin glycoside, catechin and hyaluronic acid.

  Since the growth promoting agent of the present invention has an excellent fibroblast proliferation promoting action, for example, skin aging, which is a symptom caused by decrease or alteration of collagen (collagen causing skin elasticity, wrinkles and sagging) It is useful for the suppression of the formation of crosslinks, pigmentation that causes dull skin, and the like.

Claims (3)

A fibroblast proliferation promoter comprising a water extract of mangosteen peel and hyaluronic acid as active ingredients. A fibroblast proliferation promoter containing macullin or a glycoside thereof, polyphenol, and hyaluronic acid as active ingredients. The fibroblast proliferation promoter according to claim 2, wherein the muculin or a glycoside thereof and polyphenol are derived from an aqueous extract of mangosteen peel.

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