HSP47 gene expression enhancer

Abstract

To provide an HSP47 gene expression enhancer that is effective in collagen formation and normalization of the collagen environment. [Solution] The present invention provides an HSP47 gene expression enhancer comprising a Beni Shizuka extract.

Description

Technical Field

[0001] The present invention relates to an Hsp47 gene expression enhancer comprising gardenia extract.

Background Art

[0002] When a living body is subjected to stress caused by high temperatures exceeding a certain normal physiological temperature, chemical agents, heavy metals, radiation, ultraviolet rays, starvation, oxygen deficiency, etc., one of the defense mechanisms of the living body induces the expression (production) of specific proteins called heat shock proteins (hereinafter also referred to as "Hsp") to protect cells from the stress. There are several types of Hsp, which are classified into various families according to their molecular weights. For example, the Hsp90 family (molecular weight of 90 kDa or more and 110 kDa or less), the Hsp70 family (molecular weight of 70 kDa or more and less than 80 kDa), the Hsp60 family (molecular weight of 60 kDa or more and less than 70 kDa), and the low molecular weight Hsp family (molecular weight of less than 60 kDa) are known.

[0003] Hsp is useful for promoting the normal folding of proteins when the proteins in cells are denatured due to various stress loads and for restoring physiological functions. Also, a certain amount exists in the living body even under non-stress conditions, contributing to biological defense and maintaining the homeostasis of the living body, such as assisting proteins with folding problems. If the expression of Hsp having such functions is enhanced, effects such as protecting cells from various stresses, promoting the recovery of physiological functions of cells that have declined or malfunctioned, preventing and treating various diseases and symptoms caused by abnormal protein folding are expected. Also, it is known that inducing the expression of Hsp has effects on, for example, preventing or treating cancer, gastric ulcers, brain diseases such as cerebral infarction and Alzheimer's disease, ulcerative colitis, pulmonary fibrosis, promoting wound healing, anti-inflammation, anti-oxidation, suppressing melanin production, and anti-wrinkle.

[0004] The 47kDa heat shock protein (Hsp47) is a molecular chaperone that recognizes the triple helix of collagen within the endoplasmic reticulum. Mouse embryos with Hsp47 knockout lack maturation of type I and type IV collagen, and it is known that the triple helix of collagen formed in the absence of Hsp47 is more sensitive to protease digestion. It has also been shown that the fibrils of type I collagen produced by Hsp47- / - cells are abnormally thin and highly branched. Type I collagen accumulates highly within the endoplasmic reticulum of Hsp47+ / - cells, and its secretion rate is much slower than that of Hsp47+ / + cells. It is also known that insoluble aggregates of type I collagen accumulate within the cells. There are also findings that transient expression of Hsp47 in Hsp47- / - cells restores normal extracellular fiber formation and intracellular localization of type I collagen. Furthermore, it has been found that type I collagen with an unprocessed N-terminal propeptide (N-propeptide) is secreted from Hsp47- / - cells and accumulates in the extracellular matrix. In other words, Hsp47 is necessary for the correct folding and prevention of aggregation of type I collagen within the endoplasmic reticulum, and this function has been shown to be essential for the efficient secretion, processing, and fibrosis of collagen (Non-Patent Literature 1).

[0005] Thus, Hsp, particularly Hsp47, is deeply involved in the folding of proteins in the body, especially collagen, and the normalization of the collagen environment. For example, it supports the formation of the triple-strand helix structure of collagen within the endoplasmic reticulum, thereby preventing abnormalities in collagen triple-strand formation. fiber It is expected to be effective against malformations and basement membrane formation defects. Therefore, the emergence of drugs that effectively induce Hsp47 expression is desirable. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 09-012459 [Non-patent literature]

[0007] [Non-Patent Document 1] Molecular Biology of the Cell Vol.17,No.5(2006) [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention provides an agent that enhances HSP47 gene expression. [Means for solving the problem]

[0009] As a result of diligent research by the inventors, it was discovered that Beni Shizuka extract has the effect of enhancing HSP47 gene expression.

[0010] This application provides the following invention. (1) Consists of Beni Shizuka extract, fiber An agent that enhances Hsp47 gene expression in blast cells. (2) An Hsp47 gene expression enhancer as described in (1), which promotes the formation of collagen triple chains. [Effects of the Invention]

[0011] Administration of the Benishizuka extract of the present invention can promote the expression of the Hsp47 gene in fibroblasts. [Brief explanation of the drawing]

[0012] [Figure 1] The addition of Beni Shizuka extract significantly promoted the expression of the Hsp47 gene in fibroblasts compared to the control group (*p<0.05 (unpaired t-test)). [Modes for carrying out the invention]

[0013] This invention is based on the discovery that Benishizuka extract has an effect of enhancing Hsp47 gene expression. In this invention, enhancing Hsp47 gene expression means increasing the expression of the Hsp47 gene in fibroblasts. The enhancing agent of this invention normalizes the folding of collagen, such as type I collagen, and the collagen environment by Hsp47, and supports the formation of the triple helix structure of collagen within the endoplasmic reticulum, thereby preventing abnormalities in collagen triple-strand formation. fiber It is expected to be effective against malformations and basement membrane malformations.

[0014] Beni Shizuka is a medicinal variety of peony (scientific name: Paeonia lactiflora (broad sense) or Paeonia lactiflora var. trichocarpa (standard)) and is known to contain a high amount of paeoniflorin, which has been shown to have sedative, analgesic, anti-inflammatory, hypotensive, vasodilatory, and smooth muscle relaxant effects. Paeoniflorin is disclosed as an Hsp47 synthesis inhibitor in Japanese Patent Publication No. 9-12459 (Patent Document 1), and thus exhibits the opposite effect to the Hsp47 gene expression enhancement effect of the present invention.

[0015] While it is preferable to use the roots of the Japanese quince (Benishizuka), other parts can also be used.

[0016] Beni Shizuka extract can be obtained by processing the plant body using known methods such as juicing, drying, purification, and extraction, and commercially available products are readily available. The plant material used for Beni Shizuka extract can be used in its raw or dried form, but from the viewpoint of usability and formulation, it can also be used as a dried product, dried powder, powdered raw material, or juice. The form to be used can be appropriately selected depending on the raw material, and sterilization or other treatments may be applied as necessary. Furthermore, in the display name used for the full ingredient labeling of cosmetics as defined by the Japan Cosmetic Industry Association and the international display name according to the INCI (International Nomenclature for Cosmetic Ingredients), it is expressed as Display Name / INCI Name: Peony Root Extract / PAEONIA LACTIFLORA ROOT EXTRACT

[0017] For example, the method for extracting safflower extract can be carried out by solvent extraction. In the case of solvent extraction, preferably, various parts of safflower (bark, leaves, fruits, seeds, roots, etc.) are dried as necessary, and further shredded or pulverized as necessary, and then an aqueous extractant, water, such as cold water, warm water, or hot water at or below the boiling point, or a water-containing organic solvent, an organic solvent, such as ethanol, methanol, ether, 1,3-butylene glycol, etc., is appropriately selected according to the properties of the raw material and the use of the composition, etc., and used at room temperature or heated for extraction.S However, the extraction method is not limited to solvent extraction, and it may also be by common methods known in the industry. The extraction method and the form of the extract used in the present invention are arbitrary as long as the effects of the present invention are not impaired. The form of the above extract may be not only the extract itself, but also one appropriately diluted or concentrated by common methods. Furthermore, it may be a powdery or块状 solid obtained by drying the extract, or a squeezed juice appropriately diluted or concentrated by common methods. The extract may be subjected to fermentation or enzyme treatment such as protease or pectinase, or dextrin, gum arabic, etc. may be added and powdered.

[0018] As an example of the water-containing organic solvent, water-containing lower alcohols (for example, C1 to C4) such as water-containing ethanol, water-containing methanol, water-containing ether, water-containing 1,3-butylene glycol, etc. may be used. In that case, the water content rate may be, for example, 0 to 10 v / v%, 10 to 40 v / v%, 20 to 30 v / v%, 30 to 40 v / v%, 30 to 50 v / v%, 60 to 70 v / v%, 50 to 80 v / v%, 80 to 99.5 v / v%, etc.

[0019] As a method for obtaining a dry powder, there are a method of shredding or pulverizing various parts of safflower (bark, leaves, fruits, seeds, roots, etc.) and then drying, and a method of drying the plant and then shredding or pulverizing to obtain a dry powder. Also, a method of shredding or pulverizing the plant, subjecting it to fermentation or enzyme treatment such as protease or pectinase, then drying, and further pulverizing to a predetermined particle size as necessary can be appropriately adopted. Dextrin, gum arabic, etc. may be added to the dried product and powdered.

[0020] In the present invention, the promotion of the Hsp47 gene means increasing the amount of Hsp47 mRNA or the expression level of Hsp47 protein. For example, it may be an increase having a statistically significant difference (e.g., Student's t-test) with a significance level of 5%, and / or, for example, an increase of 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more. In a preferred embodiment of the present invention, the enhancement of the expression of Hsp47 mRNA serves as an indicator of the enhanced expression of the Hsp47 gene.

[0021] The present application also provides a composition containing the agent of the present invention. The agent or composition of the present invention preferably contains safflower extract in an amount such that the effects of the present invention can be fully exerted, and the blending amount can be appropriately determined according to their types, purposes, forms, usage methods, etc. For example, the safflower extract as an active ingredient can be 0.0001 to 100% by weight, 0.0001 to 90% by weight, 0.001 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.01 to 0.5% by weight, 0.05 to 0.2% by weight, 0.1% by weight, etc. based on the total weight, but is not limited as long as the effects of the present invention are exerted. In one embodiment, the agent of the present invention consists of safflower extract.

[0022] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0023] The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description in the claims. Changes to the present invention, for example, addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Examples

[0024] Experimental method method Culture of human neonatal-derived fibroblasts Human neonatal fibroblasts were cultured in 10% FBS-DMEM (Thermo Fisher Science) in T175 flasks. After confirming proliferation to subconfluence, 0.025% trypsin was added, and the cells were incubated at 37°C for 3 minutes. The cells were then detached, the reaction was stopped with 10% FBS-DMEM, and the cells were collected. After centrifugation, the number of cells was counted, and the cells were used in various experiments at appropriate concentrations.

[0025] Gene expression analysis of Hsp47 As a drug to be investigated for its effect on inducing Hsp47 expression, we examined Beni Shizuka (medicinal peony, Maruzen Pharmaceutical). Fibroblasts were treated with 10% FBS-DMEM 1 ml / well, 1 × 10⁶ cells. 5 Cells were seeded in a 24-well plate at a concentration of cells / well and cultured for 24 hours, after which *Hibiscus mutabilis* was added. *Hibiscus mutabilis* was added to 10% FBS-DMEM at a final concentration of 0.1% and cultured. For the control, cells were cultured in the same medium but without *Hibiscus mutabilis*. After 24 hours, the entire volume was collected using RLT buffer. mRNA was then extracted using the RNeasy mini kit (QUIAGEN), and cDNA was synthesized using SuperScript VILO (invitrogen). The synthesized cDNA was then used for quantitative PCR analysis using platinum SYBER green (invitrogen). The primers used were PrimePCR PreAmp for SYBER Green Assay: SERPINH1 (Hsp47), Human (BIO-RAD, 10041595), and the GAPDH primer set with the sequences shown in Table 1 below as an endogenous control. [Table 1]

[0026] statistical analysis Each group was tested with n=4. An independent t-test was performed to compare the control and *Hibiscus erythrosora* (*p<0.05*).

[0027] The results are shown in Figure 1. The addition of *Hibiscus erythrorhizon* extract significantly promoted the expression of the Hsp47 gene in fibroblasts compared to the control (*p<0.05 (unpaired t-test)). It should be noted that *Hibiscus erythrorhizon* is known to contain a high amount of paeoniflorin, and as mentioned above, Japanese Patent Publication No. 9-12459 discloses that paeoniflorin has an inhibitory effect on Hsp47 synthesis. The cells used in the experiment described in that publication were HeLa cells, not fibroblasts. These experiments show that Beni Shizuka extract promotes the expression of the Hsp47 gene in fibroblasts. Therefore, if the enhancement of HSP47 gene expression by Beni Shizuka extract is promoted, it may prevent abnormalities in collagen triple helix formation, for example, by supporting the formation of the triple helix structure of collagen in the endoplasmic reticulum. fiber It is expected to be effective against malformations and basement membrane malformations.

Claims

1. A fibroblast Hsp47 gene expression enhancer derived from Beni Shizuka extract.

2. An Hsp47 gene expression enhancer according to claim 1, which promotes the formation of collagen triple chains.

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