JP2023075657A - COL1A1 mRNA expression promoter, MMP-1 mRNA expression inhibitor and CYR61 mRNA expression inhibitor - Google Patents

Abstract

To discover, from among natural products, a substance having a COL1A1 mRNA expression promoting action, a MMP-1 mRNA expression inhibitory action and a CYR61 mRNA expression inhibitory action, and provide a COL1A1 mRNA expression promoter, a MMP-1 mRNA expression inhibitor and a CYR61 mRNA expression inhibitor each comprising the discovered substance as an active ingredient.SOLUTION: A COL1A1 mRNA expression promoter, a MMP-1 mRNA expression inhibitor and a CYR61 mRNA expression inhibitor each comprise a marsh mallow extract as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a COL1A1 mRNA expression promoter, an MMP-1 mRNA expression inhibitor and a CYR61 mRNA expression inhibitor containing an extract of Velvet mallow.

Skin is composed of epidermis, basement membrane and dermis. The dermis is composed of fibroblasts and extracellular matrices such as collagen, elastin and hyaluronic acid secreted from these cells. The basement membrane is present at the boundary between the epidermis and the dermis and not only holds the epidermis and dermis together but also plays an important role in maintaining skin functions (see Non-Patent Document 1).

In young skin, the proliferation of fibroblasts is active, and the interactions between the epidermis and the dermis are maintained by maintaining the homeostasis of interactions between fibroblasts and skin tissues such as collagen, and by the action of the basement membrane. By maintaining constancy, moisture retention, flexibility, elasticity, etc. are secured, and the appearance is maintained in a fresh state with tension and luster.

However, when the skin is affected by external factors such as ultraviolet rays, extreme dryness of the air, excessive skin washing, stress, smoking, etc., or when aging progresses, collagen, which is the main component of the extracellular matrix, decomposes. Degeneration occurs and fibroblast proliferation slows down. As a result, the moisturizing function and elasticity of the skin are reduced, abnormal exfoliation of keratin occurs, and changes associated with aging of the skin, such as wrinkles, dullness, loss of texture, and the like, occur.

Among collagens, type I collagen is the most abundant collagen in the body, and is also abundantly contained in the dermis of the skin, and is known to play a role in creating strength of the skin. Type I collagen is composed of α1 chain and α2 chain of peptide chains, and each peptide chain is synthesized based on mRNA transcribed from COL1A1 gene and COL1A2 gene, respectively.

Therefore, promoting the expression of COL1A1 mRNA is thought to increase type I collagen synthesis and prevent or improve skin aging.

It is desired to obtain a substance having a collagen production-promoting effect from a highly safe natural product. So far, substances having a collagen production-promoting effect include, for example, star fruit leaf extract (Patent Document 1). , a camphor tree extract (Patent Document 2), and the like are known. As substances that promote the expression of COL1A1 mRNA, trehangerin (Patent Document 3), apple polyphenol that suppresses UVB-induced decrease in COL1A1 mRNA expression (Patent Document 4), and the like are known.

In recent years, degradation and reconstruction of a group of proteolytic enzymes called matrix metalloproteases (hereinafter sometimes referred to as "MMPs") have been known as factors that induce reduction or degeneration of dermal matrix components.
The MMPs are classified into (1) collagenase group (MMP-1, MMP-8 and MMP-13), (2) gelatinase group (MMP-2 and MMP-9), ( 3) stromelysin group (MMP-3 and MMP-10), (4) membrane-bound matrix metalloprotease group (MMP-14, MMP-15, MMP-16, and MMP-17), (5) others ( MMP-7, MMP-11, and MMP-12) (see, for example, Patent Document 5).
Among the MMPs, MMP-1 is known as an enzyme that degrades type I collagen, type II collagen, and type III collagen, which are the main constituents of the dermal matrix of skin. Moreover, its expression is greatly increased by irradiation with ultraviolet rays, and it is considered to be one of the causes of reduction or denaturation of collagen due to ultraviolet rays, which is a major factor in the formation of skin wrinkles and the like.

Therefore, suppressing the mRNA expression of matrix metalloprotease-1 (MMP-1) reduces the production of MMP-1 and prevents or improves skin aging such as skin wrinkle formation and skin elasticity loss. is considered possible.

So far, for example, extracts of Ginkgo biloba (Patent Document 6), silk extracts (Patent Document 7), etc. have been known to have an activity-inhibiting effect on MMP-1.

In recent years, it has been reported that cysteine-rich protein 61 (CYR61), which was found to be a factor involved in the regulation of collagen production, increases with aging and photoaging (see Non-Patent Document 2). In addition, in fibroblasts, it has been confirmed that by overexpressing CYR61, type I collagen precursors decrease and MMP-1, a collagenase, increases. CYR61 is a collagen production inhibitor and It is believed to have a function as a collagen degradation promoting factor. For this reason, CYR61 is considered to be a factor in the reduction or degeneration of collagen due to aging or ultraviolet rays, and to be a factor in the formation of wrinkles in the skin.

Therefore, it is thought that suppression of CYR61 mRNA expression can reduce CYR61 production and prevent or improve skin aging such as skin wrinkle formation and skin elasticity loss.

So far, for example, rambutan extract (Patent Document 8), apple polyphenol (Patent Document 4) that suppresses the enhancement of CYR61 protein expression by UVB, and the like have been known as substances having an effect of suppressing the production of CYR61 protein.

The extract of velvet mallow (Althaea Officinalis) has elastin production promoting action (Patent Document 9), suppressing action on MMP-1 expression enhancement by near infrared rays (Patent Document 10), AGEs degradation promoting action and desaccharification enzyme gene production promoting action. (Patent Document 11), mitochondrial transfer promoting effect (Patent Document 12) and other anti-aging effects. It is not known to have an MMP-1 mRNA expression-suppressing effect and a CYR61 mRNA expression-suppressing effect.

As described above, substances having COL1A1 mRNA expression-promoting action, MMP-1 mRNA expression-suppressing action, and CYR61 mRNA expression-suppressing action all contribute to promotion of collagen production, and prevention of skin wrinkle formation, elasticity loss, etc. It is considered to have a high effect on There is still a strong demand for formulations derived from natural products that have at least one of these actions, are highly safe and highly productive, are inexpensive, and can be used on a daily basis, and their prompt development is required. is.

Japanese Patent Application Laid-Open No. 2002-226323 JP-A-2003-146837 JP 2015-024985 A JP 2016-053007 A JP-A-2000-344672 JP 2013-177439 A JP 2010-235548 A Japanese Patent Publication No. 2020-518593 JP 2012-056933 A JP 2015-086183 A JP 2016-138148 A JP 2018-123130 A

Marinkovich MP et al. , J. Cell. Biol. , 1992, Vol. 199, p.695-703 Quan T et al. , Am J Pathol. , 2006, Aug; 169(2): 482-490

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, it is an object of the present invention to provide a COL1A1 mRNA expression-enhancing agent that is safe, highly productive, inexpensive, and routinely usable, and that has excellent COL1A1 mRNA expression-enhancing activity.
A second object of the present invention is to provide an MMP-1 mRNA expression inhibitor having an excellent MMP-1 mRNA expression inhibitory effect.
A third object of the present invention is to provide a CYR61 mRNA expression inhibitor having an excellent CYR61 mRNA expression inhibitory effect.

In order to solve the above problems, the present inventors conducted extensive studies and found that extracts of velvet mallow are useful as excellent COL1A1 mRNA expression promoters, MMP-1 mRNA expression inhibitors, and CYR61 mRNA expression inhibitors. I found out something.

The present invention is based on the above findings of the present inventors, and includes a COL1A1 mRNA expression promoter, an MMP-1 mRNA expression suppressor, and a CYR61 mRNA expression comprising an extract of Velvet mallow as an active ingredient. Provides an inhibitor.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a COL1A1 mRNA expression-enhancing agent that is safe, highly productive, inexpensive, and routinely usable, and that has excellent COL1A1 mRNA expression-enhancing activity.
Secondly, according to the present invention, it is possible to provide an MMP-1 mRNA expression inhibitor having an excellent MMP-1 mRNA expression inhibitory effect.
Thirdly, according to the present invention, it is possible to provide a CYR61 mRNA expression inhibitor having an excellent CYR61 mRNA expression inhibitory effect.
In addition, these COL1A1 mRNA expression promoters, MMP-1 mRNA expression inhibitors, and CYR61 mRNA expression inhibitors prevent, treat, or improve skin aging, that is, wrinkles, dullness, loss of texture, loss of elasticity, and the like. can do.

The present invention will be described in detail below.
The COL1A1 mRNA expression promoter, MMP-1 mRNA expression inhibitor and CYR61 mRNA expression inhibitor of the present invention contain an extract of Velvet mallow as an active ingredient.

Here, in the present invention, the "extract" includes an extract obtained by using velvet mallow as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude Both purified and purified products are included.

The velvet mallow (scientific name: Althaea Officinalis) is a perennial herb that grows naturally in wetlands of Europe from Norway to Spain, warm regions of West Asia and North Asia, Asia Minor, Australia, eastern North America, etc., and these regions can be easily obtained from The parts that can be used as raw materials for extraction are not particularly limited, and can be appropriately selected according to the purpose. is preferred.

The details of the substances contained in the velvet mallow extract that promote COL1A1 mRNA expression, suppress MMP-1 mRNA expression, and suppress CYR61 mRNA expression are unknown, but extraction methods commonly used to extract natural products An extract having COL1A1 mRNA expression-promoting action, MMP-1 mRNA expression-suppressing action and CYR61 mRNA expression-suppressing action can be obtained from Velvet mallow.

For example, after drying the raw material for extraction, it is crushed as it is or using a crusher and subjected to extraction with an extraction solvent, so that it has a COL1A1 mRNA expression promoting effect, an MMP-1 mRNA expression suppressing effect, and a CYR61 mRNA expression suppressing effect. An extract can be obtained. Drying may be performed in the sun or using a commonly used dryer. In addition, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment with a polar solvent can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent, etc., which are used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent. is preferred.

Water that can be used as an 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. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic adjustment, buffering, and the like. Therefore, water that can be used as an extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered physiological saline, and the like.

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 include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

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

The extraction treatment is not particularly limited as long as the soluble components contained in the raw material for extraction can be eluted into the extraction solvent, and can be carried out according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent of 5 to 15 times the amount (mass ratio) of the extraction raw material, and the soluble components are eluted at room temperature or under reflux heating, and then filtered to remove the extraction residue. You can get the liquid. The obtained extract is subjected to dilution, concentration, drying, purification, 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 or purified product thereof. may be treated.

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 it is as an active ingredient for promoting COL1A1 mRNA expression, suppressing MMP-1 mRNA expression and suppressing CYR61 mRNA expression. Cheap.

Since the above extract has a characteristic odor and taste, it is possible to purify the extract for the purpose of decolorization, deodorization, etc., as long as it does not reduce its physiological activity. Since it is not used in a large amount when it is blended into , there is no practical problem even if it is unrefined.

The extract of Velvet mallow obtained as described above has COL1A1 mRNA expression promoting action, MMP-1 mRNA expression suppressing action and CYR61 mRNA expression suppressing action. agent, MMP-1 mRNA expression inhibitor and CYR61 mRNA expression inhibitor.

The COL1A1 mRNA expression-enhancing agent, MMP-1 mRNA expression-suppressing agent, and CYR61 mRNA expression-suppressing agent of the present invention may consist of only the extract of Velvet mallow, or the extract of Velvet mallow may be formulated.

The extract of Velvet mallow can be made into any dosage form such as powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin, or any other adjuvant. It can be formulated and provided, and can be used by blending with other compositions (for example, skin cosmetics, etc.), and can also be used as ointments, external liquids, patches, and the like. At this time, as auxiliary agents, for example, excipients, binders, disintegrants, lubricants, stabilizers, corrigents and the like can be used.

The COL1A1 mRNA expression-enhancing agent, MMP-1 mRNA expression-suppressing agent, and CYR61 mRNA expression-suppressing agent of the present invention can exhibit COL1A1 mRNA expression-enhancing action, MMP-1 mRNA expression-suppressing action, and CYR61 mRNA expression-suppressing action, if necessary. Other natural extracts, etc. possessed by velvet mallow can be blended together with the velvet mallow extract and used as an active ingredient.

The COL1A1 mRNA expression promoter of the present invention can promote the production of collagen through the COL1A1 mRNA expression-promoting action possessed by the extract of Velvet mallow, thereby reducing skin aging-related changes such as wrinkles, dullness, and texture. disappearance, loss of elasticity, etc. can be prevented or improved. However, the COL1A1 mRNA expression-enhancing agent of the present invention can be used for all other applications in which exhibiting the COL1A1 mRNA expression-enhancing effect is significant in addition to these applications.

The MMP-1 mRNA expression suppressing agent of the present invention can suppress the degradation of collagen through the MMP-1 mRNA expression suppressing action of the extract of Velvet mallow, thereby suppressing changes associated with skin aging, that is, wrinkles. , dullness, loss of texture, loss of elasticity, etc. can be prevented or improved. However, the MMP-1 mRNA expression-suppressing agent of the present invention can be used for all other uses that are significant in exhibiting the MMP-1 mRNA expression-suppressing action.

The CYR61 mRNA expression inhibitor of the present invention can contribute to the promotion of collagen production and the suppression of collagen degradation through the CYR61 mRNA expression inhibitory action possessed by the velvet mallow extract. , wrinkles, dullness, loss of texture, loss of elasticity, etc. can be prevented or improved. However, the CYR61 mRNA expression-suppressing agent of the present invention can be used for all uses other than these uses that are significant in exhibiting the CYR61 mRNA expression-suppressing action.

The COL1A1 mRNA expression promoter, MMP-1 mRNA expression inhibitor, and CYR61 mRNA expression inhibitor of the present invention are preferably applied to humans. It can also be applied to animals other than humans.

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

[Production Example 1] Production of velvet mallow root extract 100 g of coarsely pulverized velvet mallow root was added to 1500 mL of extraction solvent, and the mixture was heated at 80 to 90°C for about 2 hours using a reflux extractor while gently stirring. kept to After performing the above extraction treatment using three types of extraction solvents: 30% 1,3-butylene glycol, 50% 1,3-butylene glycol, or 80% 1,3-butylene glycol, filter and dry with a dryer. to obtain an extract of Table 1 shows the extraction rate when 30% 1,3-butylene glycol, 50% 1,3-butylene glycol or 80% 1,3-butylene glycol is used as the extraction solvent. When the extraction solvent is a mixture, the mixing ratio is based on volume.

[Test Example 1] COL1A1 mRNA expression promoting effect test Using each extract of Production Example 1 as a sample, the COL1A1 mRNA expression promoting effect was tested as follows.

Normal human dermal fibroblasts (NB1RGB) were cultured in α-MEM containing 10% FBS and then harvested by trypsinization. The collected cells were diluted with the above medium, seeded at 35×10 ⁴ cells/2 mL per 35 mm petri dish, and cultured for 24 hours.

After culturing, the culture medium was discarded, replaced with 2 mL of serum-free α-MEM, and cultured for 24 hours.

After culturing, 2 mL of the sample solution dissolved to the required concentration in serum-free α-MEM was added to each petri dish and cultured for 24 hours.

After culturing, the culture medium was discarded, total RNA was extracted with ISOGEN (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to 200 ng/μL. Using this total RNA as a template, the expression levels of COL1A1 and internal standard GAPDH mRNA were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (R) Real Time System III (manufactured by Takara Bio Inc.) using PrimeScript (TM) RT Master Mix (Perfect Real Time) and TB Green (R) Fast qPCR Mix (manufactured by Takara Bio Inc. ) by a two-step real-time RT-PCR reaction. The expression level of COL1A1 mRNA was calculated after correcting it with the expression level of GAPDH mRNA.
From the obtained results, the COL1A1 mRNA expression promotion rate was calculated according to the following formula. Table 2 shows the results.
COL1A1 mRNA expression promotion rate (%) = A/B x 100
A to B in the above formula each represent the following.
A: Correction value when sample solution is added B: Correction value when sample solution is not added

As shown in Table 2, each extract of the root of Velvet mallow was confirmed to have an excellent effect of promoting COL1A1 mRNA expression.

[Test Example 2] MMP-1 mRNA expression inhibitory activity test Using each extract of Production Example 1 as a sample, the MMP-1 mRNA expression inhibitory activity was tested as follows.

Normal human dermal fibroblasts (NB1RGB) were cultured in α-MEM containing 10% FBS and then harvested by trypsinization. The collected cells were diluted with the above medium, seeded at 35×10 ⁴ cells/2 mL per 35 mm petri dish, and cultured for 24 hours.

After culturing, the culture medium was discarded, replaced with 2 mL of serum-free α-MEM, and cultured for 24 hours.

After culturing, 2 mL of the sample solution dissolved to the required concentration in serum-free α-MEM was added to each petri dish and cultured for 24 hours.

After culturing, the culture medium was discarded, total RNA was extracted with ISOGEN (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to 200 ng/μL. Using this total RNA as a template, the expression levels of MMP-1 and internal standard GAPDH mRNA were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (R) Real Time System III (manufactured by Takara Bio Inc.) using PrimeScript (TM) RT Master Mix (Perfect Real Time) and TB Green (R) Fast qPCR Mix (manufactured by Takara Bio Inc. ) by a two-step real-time RT-PCR reaction. The expression level of MMP-1 mRNA was calculated after correcting it with the expression level of GAPDH mRNA.
From the obtained results, the MMP-1 mRNA expression suppression rate was calculated by the following formula. Table 3 shows the results.
MMP-1 mRNA expression suppression rate (%) = (1-A / B) × 100
A to B in the above formula each represent the following.
A: Correction value when sample solution is added B: Correction value when sample solution is not added

As shown in Table 3, each extract of the root of Velvet mallow was confirmed to have an excellent MMP-1 mRNA expression inhibitory effect.
[Test Example 3] CYR61 mRNA expression inhibitory action test Each extract of Production Example 1 was used as a sample to test the CYR61 mRNA expression inhibitory action as follows.

Normal human dermal fibroblasts (NB1RGB) were cultured in α-MEM containing 10% FBS and then harvested by trypsinization. The collected cells were diluted with the above medium, seeded at 35×10 ⁴ cells/2 mL per 35 mm petri dish, and cultured for 24 hours.

After culturing, the culture medium was discarded, replaced with 2 mL of serum-free α-MEM, and cultured for 24 hours.

After culturing, 2 mL of the sample solution dissolved to the required concentration in serum-free α-MEM was added to each petri dish and cultured for 24 hours.

After culturing, the culture medium was discarded, total RNA was extracted with ISOGEN (manufactured by NIPPON GENE), the amount of each RNA was measured with a spectrophotometer, and total RNA was prepared to 200 ng/μL. Using this total RNA as a template, the expression levels of MMP-1 and internal standard GAPDH mRNA were measured. Detection was performed using a real-time PCR device Thermal Cycler Dice (R) Real Time System III (manufactured by Takara Bio Inc.) using PrimeScript (TM) RT Master Mix (Perfect Real Time) and TB Green (R) Fast qPCR Mix (manufactured by Takara Bio Inc. ) by a two-step real-time RT-PCR reaction. The expression level of CYR61 mRNA was calculated after correcting it with the expression level of GAPDH mRNA.
From the obtained results, the CYR61 mRNA expression suppression rate was calculated according to the following formula. Table 4 shows the results.
CYR61 mRNA expression suppression rate (%) = (1-A / B) × 100
A to B in the above formula each represent the following.
A: Correction value when sample solution is added B: Correction value when sample solution is not added

As shown in Table 4, each extract of the root of Velvet mallow was confirmed to have an excellent CYR61 mRNA expression inhibitory effect.

The COL1A1 mRNA expression promoter, MMP-1 mRNA expression inhibitor, and CYR61 mRNA expression inhibitor of the present invention prevent or improve skin aging-related changes, such as wrinkles, dullness, loss of texture, and loss of elasticity. can make a significant contribution to

Claims (3)

A COL1A1 mRNA expression promoting agent comprising an extract of Velvet mallow as an active ingredient. An MMP-1 mRNA expression inhibitor characterized by containing an extract of Velvet mallow as an active ingredient. A CYR61 mRNA expression inhibitor comprising an extract of Velvet mallow as an active ingredient.