JP2009084210A - Cell activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance which activates the metabolism of fibroblast and/or promotes the growth of fibroblast and has reduced side-effects, particularly a cell activator comprising kale, an extract thereof, a kale sprout or an extract thereof which has an excellent cell activation effect on fibroblast and excels in the aspect of incorporation into an internal preparation or an external preparation. <P>SOLUTION: The cell activator comprises kale, an extract thereof, a kale sprout or an extract thereof. Furthermore, an internal preparation, an external preparation, a wound healing therapeutic agent or a cosmetic comprises the cell activator. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an agent that activates cell metabolism and / or promotes proliferation (hereinafter, this action is also referred to as “cell activation”, and an agent having the same action is also referred to as “cell activation agent”). In addition, the present invention relates to an internal preparation or an external preparation containing the cell activator, a wound healing promoter or a cosmetic.

  The skin forms three layers in order from the surface: epidermis, dermis and subcutaneous tissue. Among them, the cell component of the dermis is composed of fibroblasts, histiosphere / macrophages, mast cells and plasma cells.

  Fibroblasts are cells that produce collagen, elastin, hyaluronic acid, and the like, and have a function of forming a dermal structure by using collagen as a fiber bundle.

  Fibroblasts are also cells that migrate to the damaged area when skin tissue is damaged and begin to produce extracellular matrix such as collagen, interact with cells and extracellular matrix that renew extracellular matrix It plays an important role in the wound healing process, such as causing contraction of the wound.

  It is known that these fibroblasts are slowed in proliferation due to ultraviolet rays, drying, stress and aging, and do not divide regularly. Therefore, when abnormalities occur in the fibroblasts, problems such as the generation of wrinkles and spots, the progress of skin aging such as a decrease in skin elasticity, and the speed of wound healing are caused.

Thus, in order to activate the proliferation of fibroblasts, search for various physiologically active substances has been made (Patent Document 1, Patent Document 2). Moreover, the search of the plant-derived cell activator from viewpoints of safety etc. is also made | formed (patent document 3, patent document 4). However, there has been no report that kale or its extract activates fibroblast metabolism or / and promotes proliferation.
JP-A-4-305528 JP 2004-331543 A Japanese Patent Laid-Open No. 10-36279 JP 2002-3390 A

Therefore, the present inventor has studied a substance that activates metabolism and / or promotes proliferation of fibroblasts and has few side effects. In the present invention, attention has been paid to plants considered to have low side effects as a material of the substance.
An object of this invention is to provide the plant which has a cell activation effect, or its extract. In particular, a cell activator comprising a kale or extract thereof or kale sprout or extract thereof that has an excellent cell activation effect on fibroblasts and is excellent in terms of blending with an internal preparation or an external preparation. The purpose is to provide.

  As a result of intensive studies on these problems, the present inventors have paid attention to kale or its extract or kale sprout or its extract, and recognized that it has an excellent cell activation effect, thereby completing the present invention. It was.

  That is, the present invention relates to a cell activator characterized by containing kale or an extract thereof or kale sprout or an extract thereof.

  Preferably, the present invention relates to a cell activator wherein the cells are fibroblasts.

  The present invention also relates to an internal preparation containing the cell activator.

  Furthermore, this invention relates to the external preparation containing the said cell activator.

  Preferably, the present invention relates to a wound healing promoter comprising the cell activator or an external preparation containing an internal preparation or an external preparation.

  Also preferably, the present invention relates to a cosmetic comprising the cell activator, an internal preparation, or an external preparation containing an external preparation.

  ADVANTAGE OF THE INVENTION According to this invention, the cell activator which can activate the metabolism of a fibroblast or / and can accelerate | stimulate proliferation can be provided. Furthermore, it is possible to provide an internal preparation or an external preparation, a wound healing promoter or a cosmetic that can promote wound healing and prevent skin aging through activation of fibroblasts.

  Hereinafter, the present invention will be described in detail.

  The cell activator of the present invention is characterized by containing kale or its extract or kale sprout or its extract.

  The kind of kale as a raw material of the present invention is not particularly limited, and various kinds of kale such as kitchen kale, tree kale, bush kale, mallow kale, siberian kale, scotch kale, collard, and green leaf kanran can be used.

  In this invention, it can apply to any site | part of the leaf part and stem part of a kale.

  In the present invention, kale sprout can also be applied. Here, kale sprout refers to the above-mentioned various kale shoots, and specifically refers to seedlings harvested after germination and before true leaf development.

  The kale or kale sprout is preferably processed immediately after harvesting, and if it takes time to process, it can be stored by means commonly used by those skilled in the art, such as cold storage, to prevent its alteration.

  According to the present invention, the kale or the kale sprout is stripped to an appropriate size as needed after washing the deposits with water or the like.

  The fragmentation is performed by a method of fragmenting a plant body such as slicing, crushing, and chopping, which is usually used by those skilled in the art.

  The extract that can be used for the cell activator of the present invention includes an extract obtained from an extraction raw material by an extraction process, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these All of crude or purified products are included.

  An example of a suitable extraction solvent is water.

  Water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments.

  Examples of the treatment applied to the water used for extraction include heating, sterilization, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Accordingly, water that can be used as the extraction solvent includes hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  The extraction treatment is not particularly limited as long as the soluble component contained in the kale can be eluted in the extraction solvent, and can be performed according to a conventional method. In the extraction process, it is not necessary to adopt a special extraction method, and any apparatus can be used at room temperature or under reflux heating.

  An eluate can be obtained by eluting soluble components by extraction and then filtering to remove the extraction residue.

  The obtained extract is subjected to treatments such as dilution, concentration, drying and purification according to conventional methods in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a purified product thereof. Also good.

  In obtaining a dried extract, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

  The kale or extract thereof or kale sprout or extract thereof obtained as described above has a cell activation effect.

  The mechanism of cell activation of kale or its extract or kale sprout or its extract has not been clarified so far, but it is clear from the examples described later that this extract has a cell activation. The cell activator of the present invention utilizes the cell activation action of kale or its extract or kale sprout or its extract.

  Kale or its extract or kale sprout or its extract can be used as it is as a cell activator, and can be further incorporated into an internal preparation, an external preparation, a wound healing promoter, or a cosmetic. Not what you want.

  In addition, the cell activator, the internal preparation, the external preparation, the wound healing promoter, or the cosmetics of the present invention, in addition to the above essential components, if necessary, within the range not impairing the effects of the present invention, Ingredients and additives used in preparations such as quasi-drugs, cosmetics, and foods and drinks can be selected and used optionally.

  As internal preparations, ampoules, capsules, powders, granules, pills, tablets, solids, liquids, sheets, etc., or foods and drinks, pharmaceuticals and quasi drugs Etc. can also be provided.

  As an external preparation, it can be provided in various forms such as lotions, emulsions, gels, creams, ointments, powders, granules, etc., or as pharmaceuticals / quasi drugs, skin / hair cosmetics, bath preparations, etc. it can.

  As a wound healing promoter, it can be applied not only to human skin but also to animal skin, and it can be applied to healthy skin to contribute to prevention of skin aging, as well as cuts. By promoting cell migration against wounds such as cracks, scratches, scratches, and sores of burns, the cells are quickly concentrated in the wound area, and further, cell activation effects such as collagen production, cell proliferation, and differentiation are achieved. Can be increased.

  Cosmetics include soft lotions, astringent lotions, skin lotions such as cleaning lotions, emollient creams, moisture creams, massage creams, cleansing creams, creams such as makeup creams, emollient emulsions, and moisturizing emulsions. , Emulsions such as nourishing milk, cleansing milk, jelly-like pack, peel-off pack, wash-out pack, powder pack, etc. It can be provided in various dosage forms such as rinses, treatments, and bath preparations.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these specific examples.

(Example 1) Fibroblast metabolic activity promotion test The present inventors used Cell Counting Kit-8 (Dojindo Laboratories, Inc.) based on human normal fibroblast cell line (HFSKF-II). The fibroblast metabolic activity promoting action was tested and evaluated. The test method is as follows.

(1) was seeded in 96 well plates at 1 × 10 ⁴ cells / well of HFSKF-II and pre-cultured at 37 ° C. under 5% carbon dioxide for 24 hours.

  (2) Kale strips were suspended in a medium (Ham's F12 medium containing 15% FBS (fetal bovine serum)) to a concentration of 1 mg / ml and 10 mg / ml, and allowed to stand at room temperature for 1 hour. Centrifugation was performed and the supernatant was filtered and sterilized to obtain a Kale extract-containing medium. As negative control medium, medium without added kale strips was used.

  (3) After removing the medium, each kale extract-containing medium described in (2) and a negative control medium were added and cultured for 24, 48, and 72 hours.

  (4) Cell Counting Kit-8 using color development by reduction of tetrazolium salt (WST-8) that produces highly sensitive water-soluble formazan was used for calculation of metabolic activity of cells. WST-8 is reduced by intracellular dehydrogenase to produce water-soluble formazan. Since the number of cells and the amount of formazan produced are in a linear proportional relationship, the number of living cells can be easily measured by directly measuring the absorbance of this formazan at 450 nm.

  (5) In the same manner as in (3) above, cells cultured for 24, 48, and 72 hours were each washed once with a serum-free medium (Ham's F12 medium), and then the Cell Counting Kit-8 solution diluted with the medium was added. The mixture was added at 100 μl / well, and further cultured at 37 ° C. under 5% carbon dioxide for 1 hour. Thereafter, the absorbance at 450 nm was measured.

  (6) Metabolic activity was calculated as a relative value with the absorbance value of the negative control medium minus the blank as 100%. That is, the relative value (%) of each sample was obtained based on the following equation.

Relative value (%) = {(A−B) / (C−B)} × 100
A: Absorbance in cells with each sample added B: Absorbance in Cell Counting Kit-8 solution diluted with medium (Ham's F12 medium), Absorbance in wells without cells C: Negative control medium added Absorbance in cells (Results) The results are shown in FIG.

  As shown in FIG. 1, it was confirmed that the kale extract of the present invention promotes the metabolic activity of human normal fibroblasts in a concentration-dependent manner.

(Example 2) Wound healing acceleration test The present inventors tested and evaluated the wound healing acceleration effect using HFSKF-II in the same manner as in Example 1. The test method is as follows.

(1) 8 × 10 ⁴ cells / well of HFSKF-II cells were seeded in a 24-well plate and pre-cultured at 37 ° C. under 5% carbon dioxide for 24 hours until almost confluent. Wounds were made using the tip of the chip on the cells that were almost confluent in the monolayer, and this was designated as day 0 (FIG. 2A).

  (2) Kale strips were suspended in a medium (Ham's F12 medium containing 15% FBS) to a concentration of 1 mg / ml, 3 mg / ml, 10 mg / ml, and allowed to stand at room temperature for 1 hour. Centrifugation was performed and the supernatant was filtered and sterilized to obtain a Kale extract-containing medium. As negative control medium, medium without added kale strips was used.

  (3) The medium used for the pre-culture was removed, each kale extract-containing medium described in (2) and a negative control medium were added, and further cultured for 24 hours, and then each was observed.

  (Results) The results are shown in FIGS. 2B to 2E.

  Compared to the void on day 0 (FIG. 2A), the fibroblasts are only slightly observed in the void in the negative control medium (FIG. 2E). On the other hand, in each kale extract-containing medium, cell growth was observed in a concentration-dependent manner (FIGS. 2B and 2C). In particular, in the 10 mg / ml kale extract-containing medium, the voids were caused by almost proliferated fibroblasts. It was repaired (FIG. 2D). From this, it was confirmed that kale has an effect of promoting wound healing of fibroblasts in a concentration-dependent manner.

(Example 3) Fibroblast metabolic activity promotion test The present inventors tested and evaluated the fibroblast metabolic activity promoting effect of kale and kale sprout using HFSKF-II in the same manner as in Example 1. The test method is as follows.

(1) Preparation of kale extract and kale sprout extract Kale extract and kale sprout extract were prepared by extracting 50 g of kale sprout and kale strips with 50% ethanol and then concentrating them.

(2) 1 × 10 ⁴ cells / well of HFSKF-II cells were seeded in a 96-well plate and pre-cultured for 24 hours.

  (3) Ham's F12 medium containing 15% FBS containing Kale Sprout extract and kale extract extracted at the concentration of 1 mg / ml extracted by the method described in (1) above, and Ham 'containing 15% FBS as a control s F12 medium was added and cultured for 72 hours.

  (4) After washing twice with serum-free Ham's F12 medium, WST-8 solution diluted with serum-free Ham's F12 medium was added at 100 μl / well.

  (5) The cells were further cultured at 37 ° C. and 5% carbon dioxide for 1 hour. Thereafter, the absorbance at 450 nm was measured.

  (6) Metabolic activity was calculated as a relative value with the absorbance value of the negative control medium minus the blank as 100%. That is, the relative value (%) of each sample was obtained based on the following equation.

Relative value (%) = {(A−B) / (C−B)} × 100
A: Absorbance in cells with each sample added B: Absorbance in Cell Counting Kit-8 solution diluted with medium (Ham's F12 medium), Absorbance in wells without cells C: Negative control medium added Absorbance in cells (Results) The results are shown in FIG.

  As shown in FIG. 3, both the kale extract and the kale sprout extract showed a fibroblast metabolic activity promoting effect as compared with the negative control. In addition, kale sprout extract showed higher fibroblast metabolic activity promoting action than kale extract.

Effect of kale on the metabolic activity of normal human fibroblasts Photo immediately after scratching (Day 0) Photograph after 24 hours of culture (Kale extract-containing medium; 1 mg / ml) Photograph after 24 hours of culture (Kale extract-containing medium; 3 mg / ml) Photograph after 24 hours of culture (Kale extract-containing medium; 10 mg / ml) Photo after 24 hours of culture (negative control medium) Effects of kale sprout extract and kale extract on the metabolic activity of normal human fibroblasts

Claims (6)

  A cell activator comprising kale or an extract thereof or kale sprout or an extract thereof.   The cell activator according to claim 1, wherein the cells are fibroblasts.   An internal preparation containing the cell activator according to claim 1.   An external preparation containing the cell activator according to claim 1.   A wound healing promoter comprising the cell activator according to any one of claims 1 to 4, an internal preparation, or an external preparation.   A cosmetic comprising the cell activator according to any one of claims 1 to 4, an internal preparation, or an external preparation.