JP2017218412A - Fibroblast cell growth promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a fibroblast growth-promoting substance capable of acting on skin fibroblasts and promoting growth thereof.SOLUTION: According to the present invention, there is provided a method for producing a fibroblast growth-promoting substance. The method comprises a process of obtaining a parsley leaf extract using parsley leaves and a solvent; and a process of culturing precursor fat cells. The culturing process includes the steps of obtaining a culture solution using a differentiation induction medium containing DMEM, FBS, insulin, and a parsley leaf extract, and, subsequently, further culturing the culture solution obtained in the previous step to obtain a culture solution, and subsequently, collecting a supernatant from the resulting culture solution to obtain a fibroblast growth-promoting substance.SELECTED DRAWING: None

Description

  The present invention relates to a fibroblast growth promoter that is effective for growing fibroblasts present in the dermis of the skin.

It is known that fibroblasts existing in human dermis are cells that produce collagen, elastin, hyaluronic acid and the like.
It is known that when these fibroblasts are damaged or decreased due to aging or exposure to ultraviolet rays, skin aging such as wrinkles and sagging easily occurs.

Claim 5 of Patent Document 1 describes an invention of a cosmetically acceptable natural extract that converts TGFb1-L to active TGFb1 under conditions acceptable for cosmetics. Contains parsley root extract.
Claim 7 of Patent Document 2 discloses binding of TGFb1 and LAP protein (substance that is potentially bound to a protein) to cosmetics under dermatological and / or pharmaceutically acceptable conditions. The invention describes a natural extract that is converted into active TGFb1 by cutting, and is a cosmetic, dermatologically or pharmaceutically acceptable natural extract, and the natural extract contains an extract of parsley root It is included.
Patent Document 3 describes an invention of a cosmetic preparation containing an effective amount of a germinating plant extract, and describes parsley as the plant (Claims 1 and 2).

JP 2009-292847 A Japanese Patent Laid-Open No. 2005-34384 JP 2004-532269 A

  An object of the present invention is to provide a method for producing a fibroblast growth-promoting substance capable of promoting proliferation by acting on skin fibroblasts.

The present invention comprises a step of obtaining a parsley leaf extract using parsley leaves and a solvent and a step of culturing preadipocytes,
The culture step is a step of obtaining a culture solution using a differentiation induction medium containing DMEM, FBS, insulin and parsley leaf extract;
Thereafter, using a medium obtained by removing the parsley leaf extract from the differentiation induction medium, further comprising culturing the culture solution obtained in the previous step to obtain a culture solution,
Thereafter, a method for producing a fibroblast growth promoting substance is provided, which comprises the step of collecting the supernatant of the obtained culture broth to obtain a fibroblast growth promoting substance.

The present invention also includes a step of obtaining a parsley leaf extract using a parsley leaf and a solvent and a culture step,
The culturing step comprises:
Culturing preadipocytes in a differentiation-inducing medium 1 containing DMEM, FBS, insulin, IBMX (3-isobutyl-1-methylxanthine) to obtain a first culture solution;
A step of culturing the first culture medium using a differentiation induction medium 2 containing DMEM, FBS, insulin and parsley leaf extract instead of the differentiation induction medium 1 to obtain a second culture medium;
In place of the differentiation-inducing medium 2, the step of culturing the second culture solution using the differentiation-inducing medium 3 obtained by removing the parsley leaf extract from the differentiation-inducing medium 2 to obtain a third culture solution And
Furthermore, the present invention provides a method for producing a fibroblast growth promoting substance, comprising the step of collecting the supernatant of the third culture solution to obtain a fibroblast growth promoting substance.

Further, the present invention, after producing a fibroblast growth promoting substance by the above production method,
Provided is a method for producing a skin cosmetic, in which the obtained fibroblast growth promoting substance and a cosmetic ingredient are mixed to obtain a skin cosmetic.

  The fibroblast growth-promoting substance obtained by the production method of the present invention can produce collagen by acting on fibroblasts.

The figure which shows the amount of protein (collagen) production when the fibroblast growth promotion substance obtained in Example 1 is made to act on a fibroblast.

<Method for Producing Fibroblast Growth Promoter>
Hereafter, the manufacturing method of this invention is demonstrated for every process.
In the first step, a parsley leaf extract is obtained using parsley (Petroselinum Crispum or Petroselinum sativum) leaves and solvent.
Parsley (Petroselinum Crispum) is only leaves and does not contain other parts such as roots, flowers, and seeds, but because it is a natural product, it may contain a very small amount of roots in addition to leaves ( Preferably, it is about 5% by mass or less of the total mass, more preferably about 3% by mass or less, and further preferably about 1% by mass or less.
The parsley leaves may be raw leaves that have not been dried or may be dried, but it is preferable to use shredded leaves.

As the extraction solvent, polyhydric alcohol, methanol, ethanol, or a mixed solution (aqueous solution) thereof and the like can be used, but one selected from 1,3-butylene glycol, propylene glycol, hexylene glycol, or A mixed liquid of water selected from them and water (for example, alcohol concentration of 30 to 70% by mass) is preferable, and 1,3-butylene glycol or an aqueous solution of 1,3-butylene glycol is particularly preferable.
When the 1,3-butylene glycol aqueous solution is used, the concentration of the 1,3-butylene glycol aqueous solution is preferably 40 to 60% by mass.

Extraction conditions can be extracted at room temperature (10-30 ° C) using a solvent about 2 to 20 times the volume of parsley leaves (volume basis), but shortening extraction time and increasing extraction solids Therefore, it may be heated (for example, a temperature range of more than 30 ° C. to the boiling point of the extraction solvent).
As the parsley leaf extract, a commercially available product can be used, for example, trade name parsley extract BG-J (manufactured by Maruzen Pharmaceutical Co., Ltd.) or the like can be used.
In addition, it can also manufacture using a manufacturing method to a well-known plant extract. For example, a method in which the plant in Extraction Example 4 in paragraph No. 19 of JP 2000-239145 A is replaced with a parsley leaf, and the plant of Examples 1 and 2 in JP 2002-53427 A is used as a parsley leaf. A method to be carried out in place, a method to carry out by replacing the olive branch and leaf part of the olive branch and leaf part extraction example 4 in JP 2000-128765 A with a parsley leaf, and a parsley foliage in Table 2 in JP 2002-12516 A It can also be produced using a method that is carried out in place of parsley leaves.
The solid content concentration in the parsley leaf extract can be, for example, in the range of 1.0 to 0.1% by mass, but is not limited to the above range. Further, in any case outside the solid content concentration range or the solid content concentration range, it can be appropriately diluted with distilled water or the like and used in the next step.

The subsequent process is a culture process.
The culturing step is a step of culturing preadipocytes using a differentiation-inducing medium, and a medium (liquid) containing DMEM (Dulbecco's Modified Eagle's Medium) and FBS (fetal bovine serum) as a pretreatment step as necessary. The step of proliferating preadipocytes using a medium to obtain a culture solution can be performed.
Preadipocytes are cells that are the source of fat cells.
As the FBS, one dispersed in low glucose DMEM so as to have a concentration of 5 to 15% by mass can be used.
The culture condition is preferably 5 to 14 days at 37 ° C. If necessary, the old medium can be replaced with a new medium when half of the culture period has elapsed.

In the first step, preadipocytes are cultured in a differentiation induction medium 1 (liquid medium) containing DMEM, FBS, insulin, IBMX (3-isobutyl-1-methylxanthine) and dexamethasone to obtain a first culture solution.
When the pretreatment step is carried out, the culture solution (pretreatment step culture solution) in which the preadipocytes obtained in the pretreatment step are used is used to change the medium to obtain the first culture solution. Transition.
As the FBS, one dispersed in high glucose DMEM so as to have a concentration of 5 to 15% by mass can be used.
Insulin can be used at 5-15 μg / ml.
IBMX can use 0.1-1.0 mM.
Dexamethasone can be used at 0.05 to 0.2 μM.
The culture conditions are preferably 1 to 3 days at 37 ° C.

In the next second step, instead of the differentiation induction medium 1, the first culture medium is cultured using a differentiation induction medium 2 (liquid medium) containing DMEM, FBS, insulin and parsley leaf extract, A second culture is obtained.
As the FBS, one dispersed in high glucose DMEM so as to have a concentration of 5 to 15% by mass can be used.
Insulin can be used at 1-10 μg / ml.
The parsley leaf extract can be used in a form dispersed to a concentration of 1 to 0.01% by mass.
The culture conditions are preferably 2 to 6 days at 37 ° C. If necessary, the old differentiation-inducing medium 2 can be replaced with a new differentiation-inducing medium 2 when half of the culture period has elapsed.

In the next third step, the second culture medium is cultured using the differentiation induction medium 3 obtained by removing the parsley leaf extract from the differentiation induction medium 2 in place of the differentiation induction medium 2, obtain.
The culture conditions are preferably 1 to 3 days at 37 ° C.

In the subsequent step (fourth step), the supernatant of the third culture solution is recovered to obtain a fibroblast growth promoting substance.
The fibroblast growth-promoting substance contained in the supernatant can be used as it is, or can be used as a powder by a method such as freeze-drying.
The manufacturing method of the present invention can carry out the pretreatment step, the first step to the fourth step, but the second step to the fourth step are essential steps, and the pretreatment step and the first step are carried out. It is a preferable process.

<First skin cosmetic production method>
The first skin cosmetic production method of the present invention is a method in which a fibroblast growth promoting substance is produced by the production method described above, and then the obtained fibroblast growth promoting substance and a cosmetic ingredient are mixed. is there.

<Second skin cosmetic production method>
According to the second method for producing a skin cosmetic of the present invention, after a fibroblast growth promoting substance is produced by the production method described above, the obtained fibroblast growth promoting substance is allowed to act on the fibroblasts. This is a method for producing cosmetics by producing collagen and further using the collagen produced by the above method as a cosmetic ingredient.

Cosmetic ingredients that can be used in the first and second skin cosmetic production methods of the present invention include parsley leaf extract.
Other cosmetic ingredients that can be used in the first and second skin cosmetic production methods of the present invention include known cosmetic ingredients (for example, skin external preparations disclosed in JP-A-10-36279). Component) can be used.
Known cosmetic ingredients include fats, waxes, mineral oils, fatty acids, solvents (ethanol, polyhydric alcohol, water, etc.), surfactants, vitamins, amino acids, plant extracts (excluding parsley leaf extract), Selected from animal extracts, inorganic pigments, colorants (excluding inorganic pigments), UV absorbers, astringents, antioxidants, anti-inflammatory agents, bactericides, moisturizers, fragrances, nutrient enhancers, deodorants, etc. However, the present invention is not limited to these.

The content ratio of the fibroblast growth promoting substance (solid content in the supernatant) in the skin cosmetic obtained by the production method of the present invention is preferably in the range of 1 to 0.001% by mass.
When the parsley leaf extract is used as another cosmetic ingredient, the parsley leaf extract preferably has a concentration of 1 to 0.01% by mass. The concentration of the parsley leaf extract can be obtained by diluting the obtained parsley leaf extract with distilled water so that the concentration of the extract is in a concentration range of 1 to 0.01% by mass.

  The skin cosmetic dosage form obtained by the method for producing a skin cosmetic of the present invention can be in the form of powder, granules, liquid, solid, liquid, gel, emulsion, cream, ointment, sheet, etc. However, it is not limited to these dosage forms.

  Skin cosmetics to which the production method of the present invention can be applied include basic cosmetics such as lotions, emulsions, creams, ointments, lotions, oils, packs, shampoos, rinses, hair treatments, hair creams, pomades, hair sprays, hair styling agents. Hair cosmetics such as hair dyes, hair restorations (hair growth agents), makeup cosmetics such as foundation, white powder, lipstick, blusher, eye shadow, eyeliner, mascara, eyebrow, etc. Is not to be done.

In the following Examples and Reference Examples, the following materials were used.
-Parsley leaf extract: Trade name Parsley extract BG-J; a product (solid content concentration 0.3% by mass; Maruzen Pharmaceutical Co., Ltd.) extracted with a 50% by mass 1,3-butylene glycol aqueous solution was used.
・ Mouse-derived 3T3-L1 preadipocytes (DS Pharma Biomedical)
・ Normal human fibroblasts (Kurabo)
・ Dulbecco's Modified Eagle's Medium (DMEM)
・ FBS (fetal bovine serum)
・ IBMX (3-isobutyl-1-metylxanthine)
・ Dexamethasone

Reference Example 1 <Toxicity confirmation test method>
(1) Fibroblasts were seeded in 96-well plates.
(2) Incubated for 24 hours at 37 ° C.
(3) The medium was replaced with a fresh medium supplemented with parsley leaf extract.
Medium exchange was performed in a clean bench by sucking and removing the old medium (lower layer) with an aspirator and then adding a new medium with Pipetman. The method for exchanging the medium is the same in the following.
(4) Incubated for 24 hours at 37 ° C.
(5) MTT assay was performed.
As a result, it was considered non-toxic if the solids concentration of the parsley extract in the medium was 0.5% by mass or less.

Example 1 <Method for Inducing Differentiation of Preadipocytes>
(1) 3T3-L1 cells were seeded in dish (DMEM-low glucose / 10% FBS).
(2) The cells were cultured at 37 ° C. for 5 days.
(3) Seeded on a 24-well plate (DMEM-low glucose / 10% FBS).
(4) The cells were cultured at 37 ° C. for 5 days.
(5) Medium replacement (production of the first culture)
Differentiation induction medium 1 [DMEM-high glucose / 10% FBS + 10 μg / ml insulin, 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 0.1 μM dexamethasone (Dexamethasone)] was replaced.
(6) The cells were cultured at 37 ° C. for 2 days.
(7) Medium replacement (production of second culture)
Differentiation induction medium 2 [DMEM-high glucose / 10% FBS + 5 μg / ml insulin + parsley leaf extract 0.5-0.0625 mass%] was replaced. The parsley leaf extract was diluted with distilled water so that the concentration of the extract was in the concentration range of 0.5 to 0.0625% by mass.
(8) The cells were cultured at 37 ° C for 2 days.
(9) Medium replacement (production of second culture)
Differentiation induction medium 2 [DMEM-high glucose / 10% FBS + 5 μg / ml insulin + parsley leaf extract 0.5-0.0625 mass%] was replaced. The parsley leaf extract was diluted with distilled water so that the concentration of the extract was in the concentration range of 0.5 to 0.0625% by mass.
(10) The cells were cultured at 37 ° C. for 2 days.
(11) Medium replacement (production of third culture)
The medium was replaced with differentiation induction medium 3 (DMEM-high glucose / 10% FBS + 5 μg / ml insulin).
(12) The cells were cultured at 37 ° C. for 2 days.
(13) Culture supernatant (supernatant fluid containing a fibroblast growth promoting substance) was collected and stored at -80 ° C.

Test Example 1 <ELISA Method for Collagen Production of Fibroblasts>
Collagen production ability was confirmed when a fibroblast growth promoting substance obtained by the production method of Example 1 was allowed to act on fibroblasts.
(1) Fibroblasts were seeded in 96-well plates.
(2) The cells were cultured at 37 ° C for 1 day.
(3) Medium replacement Replace with adipocyte culture supernatant (supernatant fluid containing fibroblast growth-promoting substance), L-ascorbic acid-2-magnesium phosphate 100 μM as positive control and human plate I on ELISA plate 100 μl / well of collagen (Human type I collagen; Daiichi Fine Chemical Co.) was added and coated on the plate.
(4) The cells were cultured at 37 ° C. for 2 days. The ELISA plate was kept in the refrigerator for 2 days.
(5) The ELISA plate was washed with 200 μl of 0.05% PBS-T (phosphate buffer).
(6-1) After washing the ELISA plate, the plate was blocked with 150 μl of Block Ace, a blocking agent for immunity experiments. Then, it incubated at 37 degreeC for 1 hour.
(6-2) Simultaneously, another 96-well plate was diluted with 0.5 μg / ml of human type I collagen as a standard, and 60 μl of fibroblast culture supernatant and 60 μl were added. Further, 60 μl each of 10,000-fold diluted anti-collagen type I (ROCKLAND) was added. This plate was also incubated at 37 ° C. for 1 hour.
(7) Only the ELISA plate was washed with 200 μl of 0.05% PBS-T.
(8) 100 μl of the mixed solution prepared in another 96-well plate was added to the washed ELISA plate and incubated at 37 ° C. for 1 hour.
(9) Washed with 0.05 μl PBS-T 200 μl.
(10) 100 μl of 500-fold diluted Streptavidin-HRP (R & D Systems) was added and incubated at 37 ° C. for 1 hour.
(11) Washed with 200 μl of 0.05% PBS-T.
(12) ABTS 150 μl as a coloring reagent was added and incubated at room temperature in the dark for 20-30 minutes.
(13) Absorbance at 405 nm was measured.
(14) The amount of protein in each well of the fibroblast plate was measured.
The results are shown in FIG. As is clear from FIG. 1, it was confirmed that the fibroblast growth-promoting substance obtained by the production method of the present invention was enhanced in collagen production ability by acting on fibroblasts.

Example 2 (Manufacture of skin cream)
The following components (1) to (5) were heated and dissolved after mixing to 75 ° C., and mixed, heated and melted to 75 ° C., and (6) to (9) and (12) were added and emulsified. . After stirring and cooling, (10) and (11) were added to obtain a skin cream.
(Composition) (mass%)
(1) Stearic acid 1.00
(2) Batyl alcohol 1.00
(3) Glycerol monostearate 0.50
(4) Squalane 15.00
(5) Sorbitan monostearate 2.00
(6) Polyoxyethylene (20) sorbitan monostearate 2.00
(7) Potassium hydroxide 0.05
(8) Carboxyvinyl polymer 0.10
(9) Methyl parahydroxybenzoate 0.10
(10) Fibroblast growth promoting substance (Example 1) 0.10
(11) Fragrance 0.10
(12) Purified water balance ratio Total 100.00

Example 3 (Manufacture of skin cream)
The following components (1) to (5) were heated and dissolved after mixing to 75 ° C., and mixed, heated and melted to 75 ° C., and (6) to (9) and (12) were added and emulsified. . After stirring and cooling, (10) to (12) were added to obtain a skin cream.
(Composition) (mass%)
(1) Stearic acid 1.00
(2) Batyl alcohol 1.00
(3) Glycerol monostearate 0.50
(4) Squalane 15.00
(5) Sorbitan monostearate 2.00
(6) Polyoxyethylene (20) sorbitan monostearate 2.00
(7) Potassium hydroxide 0.05
(8) Carboxyvinyl polymer 0.10
(9) Methyl parahydroxybenzoate 0.10
(10) Fibroblast growth promoting substance (Example 1) 0.10
(11) Parsley leaf extract (trade name Parsley extract BG-J) 0.10
(12) Fragrance 0.10
(13) Purified water balance ratio Total 100.00

  The fibroblast growth promoting substance obtained by the production method of the present invention acts on skin fibroblasts to promote the production of collagen and the like, and is therefore used as an active ingredient in skin cosmetics. can do.

Claims (8)

It has a process of obtaining a parsley leaf extract using parsley leaves and a solvent and a step of culturing preadipocytes,
The culture step is a step of obtaining a culture solution using a differentiation induction medium containing DMEM, FBS, insulin and parsley leaf extract;
Thereafter, using a medium obtained by removing the parsley leaf extract from the differentiation induction medium, further comprising culturing the culture solution obtained in the previous step to obtain a culture solution,
Thereafter, a method for producing a fibroblast growth promoting substance, comprising a step of collecting the culture supernatant obtained to obtain a fibroblast growth promoting substance. It has a process of obtaining a parsley leaf extract using parsley leaves and a solvent and a culture process,
The culturing step comprises:
Culturing preadipocytes in a differentiation-inducing medium 1 containing DMEM, FBS, insulin, IBMX (3-isobutyl-1-methylxanthine) and dexamethasone to obtain a first culture solution;
A step of culturing the first culture medium using a differentiation induction medium 2 containing DMEM, FBS, insulin and parsley leaf extract instead of the differentiation induction medium 1 to obtain a second culture medium;
In place of the differentiation-inducing medium 2, the step of culturing the second culture solution using the differentiation-inducing medium 3 obtained by removing the parsley leaf extract from the differentiation-inducing medium 2 to obtain a third culture solution And
A method for producing a fibroblast growth promoting substance, further comprising the step of collecting the supernatant of the third culture solution to obtain a fibroblast growth promoting substance.   The fibroblast according to claim 2, wherein the culturing step includes a step of obtaining a culture solution in which preadipocytes are grown in a medium containing DMEM and FBS as a pretreatment step of the step of obtaining the first culture solution. A method for producing a cell growth promoting substance.   4. The solvent according to claim 1, wherein the solvent is a polyhydric alcohol selected from 1,3-butylene glycol, propylene glycol, and hexylene glycol, or a mixture of water selected from the polyhydric alcohol and water. 2. A method for producing a fibroblast growth promoting substance according to item 1. After producing a growth promoting substance for fibroblasts by the production method according to any one of claims 1 to 4,
A method for producing a skin cosmetic, wherein a skin cosmetic is obtained by mixing the obtained fibroblast growth promoting substance and a cosmetic ingredient. After producing a growth promoting substance for fibroblasts by the production method according to any one of claims 1 to 4,
A step of producing collagen by allowing the obtained fibroblast growth promoting substance to act on fibroblasts;
The manufacturing method of skin cosmetics which has the process of mixing the obtained collagen and cosmetics ingredients and obtaining skin cosmetics.   The method for producing a skin cosmetic according to claim 5 or 6, wherein the cosmetic ingredient is a parsley leaf extract.   The cosmetic ingredients are oil, wax, mineral oil, fatty acid, solvent, surfactant, vitamin, amino acid, plant extract (excluding parsley leaf extract), animal extract, inorganic pigment, colorant (inorganic pigment) Skin cosmetics according to claim 5 or 6, which are selected from those containing ultraviolet absorbers, astringents, antioxidants, anti-inflammatory agents, bactericides, moisturizers, perfumes and deodorants. Manufacturing method.