JP2012031102A - Mmp-1 activity inhibitor and fibroblast proliferation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a matrix metalloprotease-1 (MMP-1) activity inhibitor and a fibroblast proliferation promoter containing a highly safe natural extract and/or a component derived from the natural extract as an active ingredient.SOLUTION: The matrix metalloprotease-1 (MMP-1) activity inhibitor and the fibroblast proliferation promoter contain the extract from a flower part and/or a bud part of Gallica Rose (botanical name; Rosa gallica) as the active ingredient.

Description

  The present invention relates to an MMP-1 activity inhibitor and a fibroblast proliferation promoter.

  The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as elastin and collagen that are outside these cells and support the skin structure. In young skin, fibroblasts are actively proliferating, and the interaction between skin tissues such as fibroblasts and collagen keeps their homeostasis, ensuring moisture retention, flexibility, elasticity, etc. It is also kept fresh with tension and gloss.

  However, elastin, which is a major component of the extracellular matrix, degrades and deteriorates when affected by certain external factors such as UV irradiation, drastic air drying, excessive skin washing, and so on. In addition, the production amount of collagen is reduced, and the elasticity is lowered due to crosslinking. As a result, the moisturizing function and elasticity of the skin are lowered and abnormal exfoliation of the keratin occurs, so that the skin loses its tension and gloss, and exhibits aging symptoms such as rough skin and wrinkles. As described above, changes accompanying aging of the skin, that is, wrinkles, dullness, disappearance of texture, decrease in elasticity, and the like are associated with reduction and degeneration of dermal matrix components such as collagen and elastin.

  In recent years, the involvement of matrix metalloproteinases MMPs (Matrix metalloproteinases) has been pointed out as factors that induce changes associated with skin aging. Among these MMPs, matrix metalloproteinase-1 (MMP-1) is known as an enzyme that degrades collagen, which is a major component of the dermal extracellular matrix of skin, but its expression is greatly increased by irradiation with ultraviolet rays. However, it is thought to contribute to the decrease / denaturation of collagen, and to be a major factor such as the formation of wrinkles on the skin and the decrease in elasticity. Therefore, inhibiting the activity of MMP-1 is important in preventing and improving skin aging symptoms.

  As what has such MMP-1 activity inhibitory action, for example, an extract from Himalayan cherry (see Patent Document 1), an extract from Ginger family Ginkgo balssumner or Mulberry ficus nerifolia (see Patent Document 2) ) Etc. are known.

  In addition, a decrease in fibroblast proliferation is cited as a cause of skin aging accompanying aging. In other words, when there is an influence of certain external factors such as ultraviolet rays, significant drying of air, excessive skin washing, etc., or when aging progresses, the growth of fibroblasts slows down and the skin moisturizing function and elasticity are descend. And the skin loses its tension and gloss, and exhibits aging symptoms such as roughness and wrinkles. Therefore, it is considered that aging of the skin can be prevented or improved by promoting the proliferation of fibroblasts.

  Based on this idea, Kusunohagashi extract (see Patent Document 3) and the like have been proposed as having a fibroblast proliferation promoting action.

JP 2003-176232 A JP 2003-176230 A JP 2003-146837 A

  It is an object of the present invention to provide a matrix metalloproteinase-1 (MMP-1) activity inhibitor and a fibroblast proliferation promoter comprising a highly safe natural extract and / or a natural extract-derived component as an active ingredient. And

  In order to achieve the above object, the matrix metalloproteinase-1 (MMP-1) activity inhibitor or fibroblast proliferation promoter of the present invention comprises an extract from the flower part and / or the heel part of gallica rose as an active ingredient. It is characterized by containing.

  According to the present invention, a highly safe matrix metalloproteinase-1 (MMP-1) activity inhibitor and fibroblast, which contains an extract from the flower part and / or heel part of a natural product, gallica rose, as an active ingredient A cell growth promoter can be provided.

Hereinafter, embodiments of the present invention will be described.
The matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter according to the embodiment of the present invention contains an extract from the flower part and / or the heel part of gallica rose as an active ingredient.

  Here, the “extract from the flower part and / or the buttock of the gallica rose” includes an extract obtained from the flower part and / or the buttock of the gallica rose as an extraction raw material, a diluted or concentrated solution of the extract, A dry product obtained by drying the extract, or a crude product or a purified product thereof is included.

  The plant which is an extraction raw material used in this embodiment is gallica rose (scientific name: Rosa gallica). Galica rose is a deciduous shrub that has been cultivated in Europe and the Middle East for a long time, and can be easily obtained from these areas.

  The site | part used as an extraction raw material is a flower part and / or a buttock. Here, the "flower" generally refers to the whole organ involved in sexual reproduction of a seed plant, and is composed of a flower leaf that is a leaf deformation and a flower axis that is a stem deformation. This includes organs such as pupae, petals, stamens, and heart skin. “Acupuncture” is composed of the same organs as the “flowers”, but is in a so-called unflowered state in which the anthers or petals are not open. In the present embodiment, the “flower part” and the “buttock part” used as the raw material for extraction include, in addition to the total organs involved in sexual reproduction of seed plants, a part thereof, for example, flower leaves, flower coats (buds and corolla). ), Corolla, petals and the like.

  Details of substances having an inhibitory action on matrix metalloprotease-1 (MMP-1) activity or a fibroblast growth-promoting action contained in extracts from the flower part and / or the buttock part of gallica rose are unknown, but plant extraction In general, an extract having a matrix metalloproteinase-1 (MMP-1) activity inhibitory action or a fibroblast proliferation promoting action can be obtained from the flower part and / or the heel part of Galicalose.

  For example, it can be obtained by drying the extraction raw material, pulverizing the raw material as it is or using a crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, extraction with a polar solvent can be performed efficiently.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a mixed solution of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of a lower aliphatic alcohol with respect to 10 parts by volume of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by volume of a lower aliphatic ketone with 10 parts by volume of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by volume of a polyhydric alcohol with respect to the volume part.

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent in an amount 5 to 15 times (mass ratio) of the extraction raw material, the soluble component is extracted at room temperature or under reflux heating, and then filtered to remove the extraction residue. Can be obtained. The obtained extract is diluted, concentrated, dried, purified, etc. 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 crude purified product or a purified product thereof. Processing may be performed.

  Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like. The obtained extract can be used as an active ingredient of a matrix metalloproteinase-1 (MMP-1) activity inhibitor or a fibroblast proliferation promoter as it is, but a concentrated solution or a dried product is used. It's easy to do.

  In addition, the extract from the flower and / or cocoon of gallica rose has a unique odor, so it can be purified for the purpose of decolorization, deodorization, etc. within a range that does not reduce its physiological activity. However, the matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter according to this embodiment is not incorporated in a large amount when blended in skin cosmetics or foods and drinks. Therefore, there is no practical problem even if it is not purified.

  Since the extract from the flower part and / or the buttock of the gallica rose obtained as described above has a matrix metalloproteinase-1 (MMP-1) activity inhibitory action or a fibroblast proliferation promoting action, Can be used as an active ingredient of a matrix metalloproteinase-1 (MMP-1) activity inhibitor or a fibroblast proliferation promoter.

  The matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter of this embodiment may consist only of an extract from the flower part and / or the buttock of gallica rose, It may be a formulation of an extract from a flower part and / or a buttock of a gallica rose.

  The extract from the flower part and / or the buttock of gallica rose is in the form of powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and any other auxiliary agent. It can be formulated into 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. Examples of the form of matrix metalloproteinase-1 (MMP-1) activity inhibitor or fibroblast growth promoter formulated with an extract from the flower part and / or the buttock of gallica rose include, for example, an ointment, a liquid for external use, Examples include patches. Moreover, the extract from the flower part and / or the buttock part of a gallica rose can be mix | blended and used for other compositions (for example, skin cosmetics, food-drinks, etc.).

  In addition, the matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter of the present embodiment is, as necessary, a matrix metalloproteinase-1 (MMP-1) activity inhibitory action or a fibroblast. A natural extract or the like having a growth promoting action can be blended with an extract from the flower part and / or the cocoon part of the gallica rose and used as an active ingredient.

  Examples of the administration method of the matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter of this embodiment generally include transdermal administration, oral administration, etc., but depending on the type of disease, What is necessary is just to select a suitable method for the prevention, treatment, etc. suitably.

  In addition, the dose of the matrix metalloproteinase-1 (MMP-1) activity inhibitor or fibroblast proliferation promoter of this embodiment is also dependent on the type of disease, severity, individual differences among patients, administration method, administration period, etc. What is necessary is just to increase / decrease suitably.

  The matrix metalloproteinase-1 (MMP-1) activity inhibitor of the present embodiment can be used for anti-aging applications as described above. Furthermore, it can also be used for the treatment / prevention of diseases and the like that are known to be associated with pathological conditions of extracellular matrix destruction. Examples of such applications include cancer treatment by preventing or suppressing cancer invasion / metastasis, rheumatoid arthritis, osteoarthritis of the knee, periodontitis, diabetic retinopathy, age-related macular degeneration, and the like. . In addition, the use of the matrix metalloprotease-1 (MMP-1) activity inhibitor of this embodiment is not limited to these, and all that is meaningful in exhibiting the matrix metalloprotease-1 (MMP-1) activity inhibitory action. It can be used for

  The fibroblast proliferation promoter of this embodiment can be used for anti-aging applications as described above. Fibroblasts play an important role in the healing process of wounds such as trauma and burns, and are also important in the healing process of skin diseases (eg, skin ulcers such as pressure sores, burn ulcers, diabetic ulcers, etc.). is there. Furthermore, in the field of regenerative medicine, a method for culturing and proliferating skin cells from a patient's own skin fragment and transplanting it to a patient as a treatment method for a patient suffering from a wound that is difficult to self-heal (such as a severe burn) However, the use of a cell growth promoter is expected to shorten the culture period of skin cells. Therefore, the fibroblast proliferation promoter of this embodiment can also be used for uses such as wound treatment, skin disease treatment, and application to the field of regenerative medicine. In addition, the use of the fibroblast proliferation promoter of this embodiment is not limited to these, It can use for all the uses meaningful in exhibiting the fibroblast proliferation promoting effect.

  In addition, although the matrix metalloproteinase-1 (MMP-1) activity inhibitor or the fibroblast proliferation promoter of this embodiment is suitably applied to humans, the respective effects are exhibited. As long as it is applicable to animals other than humans.

Hereinafter, although a test example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.
In this test example, as a sample, rose butts extract powder (manufactured by Maruzen Pharmaceutical Co., Ltd., sample 1), which is an extract from the flower part and the buttocks part of Galica Rose, was used.

[Test Example 1] MMP-1 activity inhibitory action test MLP-1 activity inhibitory action was tested as follows for extracts (sample 1) from the flower part and the buttock part of Galicalose.

  Sample solution dissolved in 0.1 mol / L Tris-HCL buffer (pH 7.1) containing 20 mmol / L calcium chloride in a test tube with a lid (Sample 1, see Table 1 below for sample concentration) 50 μL 50 μL of MMP-1 solution (COLLAGENASE Type IV from Clostridium histolyticum, Sigma) and 400 μL of Pz-peptide solution (Pz-Pro-Leu-Gly-Pro-D-Arg-OH, BACHEM Feinchemikalien AG) are mixed. The mixture was reacted at 37 ° C. for 30 minutes, and then 1 mL of a 25 mmol / L citric acid solution was added to stop the reaction.

Thereafter, 5 mL of ethyl acetate was added and shaken vigorously. This was centrifuged (1600 × g, 10 minutes), and the absorbance of the ethyl acetate layer at a wavelength of 320 nm was measured. Similarly, a blank test was performed and corrected. From the obtained results, the MMP-1 activity inhibition rate (%) was calculated by the following formula, and the sample concentration IC ₅₀ (μg / mL) that inhibits the activity of MMP-1 by 50% was determined by interpolation.

MMP-1 activity inhibition rate (%) = {1− (C−D) / (A−B)} × 100
In the formula, A represents “absorbance at a wavelength of 320 nm when no sample is added and an enzyme is added”, B represents “absorbance at a wavelength of 320 nm when no sample is added and an enzyme is not added”, and C is “addition of sample and enzyme is added” And “D” represents “absorbance at a wavelength of 320 nm when a sample is added and no enzyme is added”.
The results are shown in Table 1.

  As shown in Table 1, it was confirmed that the flower part and the buttock extract of Galicalose have an excellent MMP-1 activity inhibitory action. In addition, it was confirmed that the degree of MMP-1 activity inhibitory action can be adjusted by the concentration of the flower part and the buttock extract of Galicalose.

[Test Example 2] Skin fibroblast proliferation promoting action test The skin fibroblast proliferation promoting action of Galica Rose flower part and buttock extract (sample 1) was tested as follows.

Human normal skin fibroblasts (NB1RGB) were cultured using α-MEM containing 10% FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with 5% FBS-containing α-MEM to a cell density of 7.0 × 10 ⁴ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured overnight.

  After completion of the culture, 100 μL of a sample solution dissolved in 5% FBS-containing α-MEM (sample 1, sample concentration is shown in Table 2 below) was added to each well and cultured for 3 days. Fibroblast proliferation was measured using the MTT assay.

  After completion of the culture, 100 μL of the medium was removed from each well, and 20 μL of MTT dissolved in PBS buffer at a final concentration of 5 mg / mL was added to each well. After culturing for 4.5 hours, 100 μL of 0.01 mol / L hydrochloric acid solution in which 10% SDS was dissolved was added to each well and incubated overnight, and then the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. In addition, a blank test was performed and corrected in the same manner. From the obtained results, the fibroblast proliferation promotion rate (%) was calculated by the following formula.

Fibroblast proliferation promotion rate (%) = (St−Sb) / (Ct−Cb) × 100
In the formula, St represents “absorbance in cells to which a sample was added”, Sb represents “absorbance in a blank test to which a sample was added”, Ct represents “absorbance in cells to which no sample was added”, and Cb It represents “absorbance of blank test without adding sample”.
The results are shown in Table 2.

  As shown in Table 2, it was confirmed that the flower part and the buttock extract of Galicalose have an excellent fibroblast proliferation promoting action.

  The matrix metalloproteinase-1 (MMP-1) activity inhibitor and the fibroblast proliferation promoter of the present invention can greatly contribute to the prevention and improvement of skin aging symptoms.

Claims (2)

  A matrix metalloproteinase-1 (MMP-1) activity inhibitor comprising an extract from a flower part and / or a cocoon part of a gallica rose (Rosa gallica) as an active ingredient.   A fibroblast growth promoter characterized by containing an extract from a flower part and / or a buttock part of gallica rose (Rosa gallica) as an active ingredient.