M2 macrophage inducer

Abstract

We provide M2 ​​macrophages that are effective in suppressing inflammation, repairing tissue, and preventing and improving photoaging. [Solution] The present invention provides an M2 macrophage inducer comprising rosehip extract.

Description

Technical Field

[0001] The present invention relates to an M2 macrophage inducer comprising rose extract.

Background Art

[0002] Macrophages are classified into two types, M1 and M2, according to their roles. M1 macrophages are differentiated from inflammatory monocytes stimulated by stimuli such as TNF-α and IFN-γ, and while they function in the defense against pathogen and parasite infections, they are also known to promote inflammation. M2 macrophages are differentiated from tissue-resident monocytes stimulated by Th2-type cytokines such as IL-4 and IL-13, and are said to be involved in suppressing and resolving inflammation and tissue repair. M2 macrophages induce the expression of proteins essential for the assembly of collagen fibers, such as SPARC, lysyl oxidase gene LOX, and ADAMTS2. SPARC is a matrix protein, and it has been reported that SPARC-deficient mice show a decrease in the diameter of collagen fibers and abnormalities in the dermis. Similar to SPARC, lysyl oxidase also plays an important role in the stabilization of collagen fibers by initiating the formation of covalent crosslinks. ADAMTS-2 is a major N-protease of procollagen, and ADAMTS2 knockout mice have fragile skin due to abnormal collagen fibers caused by the overaccumulation of procollagen 1. In fact, it has been reported that CD206-positive M2-like macrophages control the formation of collagen fibers during skin repair. Furthermore, in addition to supporting the assembly of collagen by these proteins, M2 macrophages also induce the expression of not only type I but also type III, V, and VI collagens. Type III, V, and VI collagens are the main collagens in the dermis, coexist with type I collagen in collagen fibers, and support the fiber structure and skin elasticity (Non-Patent Document 1).

[0003] Human skin exposed to sunlight and aged becomes fragile due to collagen fragmentation and loss. The inventors have reported that the balance between M1 macrophages and M2 macrophages is related to inflammatory changes associated with chronic inflammation in human skin exposed to sunlight. It has also been found that the number of M2 macrophages is positively correlated with the amount of type I collagen, while the M1 / M2 ratio is negatively correlated with the amount of type V and VI collagen, which are essential for collagen formation in the skin. Furthermore, since M2 macrophages induce the expression of proteins necessary for the assembly of collagen fibers, the M1 / M2 balance is important not only for ensuring the quantity of the collagen matrix but also for ensuring its quality. On the other hand, it has been shown that M1 macrophages induce abnormal collagen fibers composed of type I, V, and VI collagen. An imbalance in the balance between M1 and M2 macrophages is associated with photoaging, and adjusting the M1 / M2 balance is particularly important in preventing and improving photoaging (Patent Documents 1 and 2). Furthermore, pigmentation, including blemishes and dullness, is caused by the accumulation of melanin produced by melanocytes in the basal layer of the epidermis. Melanin is normally present in the epidermis and basal layer, but because the epidermis has a relatively fast turnover cycle, this melanin is easily excreted. On the other hand, melanin may be present in the dermis due to reasons such as melanin falling into the dermis through gaps in the basement membrane. Because the turnover cycle of dermal cells is much slower than that of the epidermis, this melanin is often not excreted and accumulates. For these reasons, improving pigmentation in the dermis is extremely difficult. The inventors have reported that M2 macrophages have a high ability to phagocytose melanin in the dermis, and M2 macrophages are important in preventing and improving blemishes by removing melanin in the dermis (Patent Documents 1 and 2).

[0004] Thus, M2 macrophages are deeply involved in inducing and normalizing collagen formation in the skin, and are known to be effective in suppressing inflammation, repairing tissue, preventing and improving photoaging, removing dermal melanin, and consequently preventing age spots. Therefore, the emergence of drugs that effectively induce M2 macrophages is desirable. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2020 / 213743 [Patent Document 2] WO2020 / 213745 [Non-patent literature]

[0006] [Non-Patent Document 1] Horiba et al.,JID Innovations(2023);3:100222 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This invention provides an M2 macrophage inducer. [Means for solving the problem]

[0008] As a result of diligent research by the inventors, it was discovered that rose extract has an M2 macrophage-inducing effect.

[0009] This application provides the following invention. (1) An M2 macrophage inducer consisting of rose extract. (2) An M2 macrophage inducer of (1), in which increased CD206 (MRC1) gene expression is an indicator of M2 macrophage induction. [Effects of the Invention]

[0010] Administration of the rose extract of the present invention can promote the induction of differentiation from monocytes to M2 macrophages. [Brief explanation of the drawing]

[0011] [Figure 1] The addition of rose extract significantly promoted the differentiation of monocyte cells into M2 macrophages compared to the control group (****P<0.0001 (unpaired t-test)). [Modes for carrying out the invention]

[0012] This invention is based on the discovery that rose extract has an M2 macrophage-inducing effect. In this invention, induction of M2 macrophages means enhancing the differentiation induction of monocytes into M2 macrophages. Since M2 macrophages are effective in suppressing inflammation, repairing tissue, and preventing and improving photoaging, the inducer of this invention is expected to enhance the differentiation induction of M2 macrophages, thereby producing a wide range of effects resulting from an increase in M2 macrophages, including not only suppression of inflammation, repairing tissue, preventing and improving photoaging, such as the removal of dermal melanin and the resulting prevention of age spots.

[0013] The European rose (also known as the Cabbage Rose or Provance Rose) is a cultivated variety that was mainly grown in southern Europe from the Greek and Roman eras to the Renaissance. As its English name suggests, this flower has double petals resembling a cabbage head and is of hybrid origin (R. bifera XR. alba).

[0014] European rose (R. centifolia L. or R. gallica L. var. centifolia (L.) Reg.) can be used fresh or dried, but it is preferable to use it as a dried powder or solvent extract from the standpoint of usability and formulation.

[0015] For use, it is preferable to use the flowers or the entire plant of the European rose (R. centifolia L. or R. gallica L. var. centifolia (L.) Reg.), but other parts can also be used.

[0016] European rose extract can be obtained by processing the plant body by known methods such as saping, drying, purification, and extraction, and commercially available products are readily available. The plant material used for European rose 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 sap. 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 in the full ingredient labeling of cosmetics as defined by the Japan Cosmetic Industry Association and the international display name according to the INCI name (International Nomenclature for Cosmetic Ingredients), it is expressed as: Display name / INCI name: Centifolia rose flower extract / ROSA CENTIFOLIA FLOWER EXTRACT.

[0017] For example, the method for extracting rose extract can be carried out by solvent extraction. In the case of solvent extraction, preferably, the flowers of rose, or other various parts (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 as a preferred solvent according to the nature of the raw materials and the use of the composition, etc., and is used at room temperature or heated for extraction. However, the extraction method is not limited to solvent extraction, and it may also be by common methods known in the art. 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 appropriately diluted or concentrated by common methods, and further, it may be a powdery or块状 solid obtained by drying the extract, or the squeezed juice may be appropriately diluted or concentrated by common methods. The extract may be subjected to fermentation or enzyme treatment such as protease or pectinase, or dextrin or gum arabic, etc. may be added and powdered.

[0018] As examples of the water-containing organic solvent, water-containing lower alcohols (e.g., 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 the flowers or various parts (bark, leaves, fruits, seeds, roots, etc.) of rose and then drying, or a method of drying the plant and then shredding or pulverizing to obtain a dry powder. Also, a method can be appropriately adopted such as 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. Dextrin or gum arabic, etc. may be added to the dried product and powdered.

[0020] In the present invention, the induction of M2 macrophages refers to increasing the mRNA amount of markers of M2 macrophages, such as CD206, CD163, Agr1, IL-10, etc. or the number of M2 macrophages. 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. A preferred marker for M2 macrophages is CD206. Therefore, in a preferred embodiment of the present invention, the enhancement of CD206 gene expression serves as an indicator for the induction of M2 macrophages.

[0021] As a result of inducing M2 macrophages, the M2 macrophage inducer of the present invention can adjust the M1 / M2 balance. The M1 / M2 balance may refer to the ratio of the number of M1 macrophages to the number of M2 macrophages, or may refer to the ratio of the number of M1 macrophages to the number of M2 macrophages, or may refer to the ratio of the mRNA amount of markers of M1 macrophages (e.g., CD86, CD80, iNOS, etc.) to the mRNA amount of markers of M2 macrophages (e.g., CD206, CD163, Agr1, IL-10, etc.). It is preferable to reduce the M1 / M2 balance, which is the ratio of M1 / M2 (the number of M1 / the number of M2, and / or the mRNA amount of M1 marker / the mRNA amount of M2 marker). The reduction may be, for example, a reduction having a statistically significant difference (e.g., Wilcoxon's rank sum test, Student's t-test, etc.) with a significance level of 5%, and / or, for example, a reduction 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%.

[0022] This application also provides compositions containing the agent of the present invention. The agent or composition of the present invention preferably contains rosehip extract in an amount that is sufficient to exert the effects of the present invention, and the amount can be appropriately determined according to their type, purpose, form, and method of use. For example, the active ingredient rosehip extract 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 rosehip extract.

[0023] All references made herein are incorporated herein by citation in their entirety.

[0024] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0025] Experimental method Culture of human monocyte-derived cell line THP-1 and differentiation stimulation of M2 macrophages Human monocyte-derived cell line THP-1 was cultured in a T175 flask in 10% FBS-RPMI (Nacalai Tesque) supplemented with 1 mM Na Pyruvate (Nacalai) and 2 mM L Glutamine (Nacalai). After confirming proliferation to subconflu, the entire cell suspension was collected, centrifuged, and the number of cells was counted. The cells were then used for differentiation stimulation experiments to M2 macrophages at an appropriate cell concentration. Specifically, 100 nM PMA (abcam) was added to THP-1 cells, and they were stimulated for 24 hours to differentiate into M0 macrophages. Subsequently, 20 ng / mL IL-4 (R&D) and 20 ng / mL IL-13 (R&D) were added, and they were stimulated for 72 hours to differentiate into M2 macrophages.

[0026] Verification of M2 macrophage induction effect (MRC1 (CD206) gene expression analysis) As a drug to evaluate the M2 macrophage induction effect, we investigated 0.1% rose extract (manufacturer: Maruzen Pharmaceutical Co., Ltd. (extracted with 1,3-butylene glycol aqueous solution)). We evaluated the M2 macrophage induction effect using MRC1 (CD206), a known surface marker of M2 macrophages, as an indicator. 100 nM PMA (abcam) was added to THP-1, and simultaneously 0.1% rose extract was added. The cells were stimulated for 24 hours to differentiate into M0 macrophages. Subsequently, 20 ng / mL IL-4 (R&D) and 20 ng / mL IL-13 (R&D) were added, along with 0.1% rose extract. The cells were stimulated for 72 hours to differentiate into M2 macrophages. In the control group, the cells were cultured in the same medium and under the same differentiation stimulation conditions, but without rose extract. After culturing under the above stimulation conditions, the entire volume was recovered using RLT buffer. Subsequently, mRNA was extracted using the RNeasy mini kit (QUIAGEN), and cDNA was synthesized using SuperScript VILO (invitrogen). The synthesized cDNA was then subjected to quantitative PCR analysis using platinum SYBER green (invitrogen). The primers used were PrimePCR PreAmp for SYBER Green Assay: MRC1, Human (BIO-RAD, 10041595), and the GAPDH primer set with the sequences shown in Table 1 below as an endogenous control.

[0027] [Table 1] statistical analysis Each group was tested with n=3. An independent t-test was performed to compare the control with the rose extract. (****P<0.0001)

[0028] The results are shown in Figure 1. The addition of rose extract significantly promoted the differentiation of monocyte cells into M2 macrophages compared to the control group.**** P<0.0001 (unpaired t-test). These experiments show that rose extract promotes the differentiation of human monocyte-derived cells into M2 macrophages. Therefore, if M2 macrophage induction is promoted by rose extract, it can be expected to produce a wide range of effects resulting from an increase in M2 macrophages, not only in the suppression of inflammation, tissue repair, and prevention and improvement of photoaging.

Claims

1. An M2 macrophage inducer derived from rose extract.

2. The M2 macrophage inducer according to claim 1, wherein increased CD206 gene expression is an indicator of M2 macrophage induction.

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