JP7494418B2 - Composition for promoting collagen receptor production - Google Patents

Description

The present invention relates to a composition for promoting collagen receptor production.

The extracellular matrix fills the space around cells without leaving any gaps, giving biological tissues mechanical strength, flexibility, and plasticity, providing a foothold for surrounding cells to adhere to, and providing the external environment necessary for cells to assemble in an orderly manner to form tissues and organs. Furthermore, the extracellular matrix is known to control a variety of biological processes, such as development, differentiation, immune response, blood coagulation, and wound healing, by actively transmitting signals to cells. A typical molecule that constitutes the extracellular matrix is collagen. Collagen accounts for approximately 30% of the total protein content in the body. Collagen also forms tough fibers (collagenous fibers), giving tissues the mechanical strength to resist tension in the direction of the fibers.

There are various molecules in the body that interact with collagen. Collagen also binds to many molecules that make up the extracellular matrix, such as fibronectin and laminin, to form higher-order structures that provide a foothold for cell adhesion. Furthermore, collagen exists just beneath endothelial cells, so it acts as a sensor when the vascular wall is damaged. Receptors that bind to collagen are expressed on the surface of cells in the body.
Collagen is involved in intercellular signaling and cell-cell binding via this receptor. Representative collagen receptors expressed on the cell surface include integrin, discoidin domain receptor (DDR), glycoprotein IV, leukocyte-associated immunoglobulin-like receptor-1, and Endo180.

Integrins are the receptors that have been studied for the longest time, and four collagen-binding integrins (α1β1, α2β1, α10β1, and α11β1) have been reported to date, with the integrin β1 subunit (ITGB1) being a common component molecule (Non-Patent Documents 1 to 3). Integrins act as adhesion molecules that connect cells to cells and cells to extracellular matrix, and are involved in cell proliferation and tissue formation and repair.
DDR has two isoforms, DDR1 and DDR2, which are mainly expressed in epithelial cells and mesenchymal cells, respectively. DDR is a receptor tyrosine kinase activated by collagen molecules that retain their native structure, and is involved in cell adhesion and migration (see Non-Patent Documents 1 to 4). DDR2 expressed in skin fibroblasts is a membrane protein that is important for development, differentiation, and wound healing via remodeling of the extracellular matrix, and it has been reported that knockout mice show dwarfism, with immature bone formation, and skin wound healing is inhibited (Non-Patent Document 5).
Endo180 is a membrane protein that recognizes fragmented or denatured collagen and takes them into cells, thereby controlling collagen remodeling by phagocytosis (Non-Patent Document 6). Both receptors are present on the membrane of fibroblasts and play a role in transmitting intracellular and extracellular information via collagen.
Increased expression of collagen receptors on the surface of these cells strengthens cell-cell junctions and activates information exchange between cells, promoting wound healing and repair.

Various collagen receptor production promoters have been proposed so far.
Patent Document 1 describes that evening primrose extract and fruit passionflower extract are useful as substances that promote the production of DDR2, which is a collagen receptor.
Patent Document 2 describes an integrin production promoter containing pelargonidin as an active ingredient. Patent Document 3 describes an integrin expression promoter containing extracts of Chinese quince, Japanese quince, Chinese angelica, Akebia moniliforme, Auricularia japonica, and pea pollen as active ingredients. Patent Document 4 describes an integrin production promoter containing apple extract as an active ingredient, and Patent Document 5 describes an integrin production promoter containing hexagonal bean extract as an active ingredient.

Patent Document 6 describes lotus germ extract, shell ginger leaf extract, and Lactobacillus plantarum, which promote the production of Endo180, a collagen receptor. Patent Document 7 describes rosmarinic acid, caffeic acid, and Melissa extract, and Patent Document 8 describes plant extracts selected from the group consisting of taiso, wisteria tea, gentian, prune, chinese ginger, star fruit, royal jelly, green tea, wild thyme, horsetail, Japanese pepper, sweet osmanthus, goldenrod, and yarrow, which promote the production of Endo180.

On the other hand, a compound called quercetin is a flavonoid compound contained in vegetables and fruits. Isoquercitrin, one of the glycosides of quercetin, is a flavonol glycoside in which one glucose is bound to the 3-position of quercetin, and is known to have strong antioxidant activity and to prevent the fading of pigments (Patent Document 9). It is also known to have effects on the body, such as preventing arteriosclerosis and improving blood flow.

JP 2019-199441 A JP 2011-20951 A JP 2016-65019 A JP 2011-219454 A JP 2010-24211 A JP 2019-182753 A JP 2019-108398 A JP 2019-55924 A JP 2008-131888 A

B. Leitinger, Annu. Rev. Cell Dev. Biol., 27, 265-290 (2011) J. Heinoa et al., Int. J. Biochem. Cell Biol., 41, 341-348, (2009) YC. Yeh et al., Am. J. Physiol. Cell Physiol., 303, C1207-C1217 (2012) B. Leitinger, Int. Rev. Cell Mol. Biol., 310, 39-87, (2014) J.P. Labrador, EMBO Rep., 2, 446-452, (2001) MC Melander et al., Int. J. Oncol., 47, 1177-1188, (2015)

The objective of the present invention is to provide a new composition for promoting collagen receptor production.

The present invention comprises the following configurations.
(1) A composition for promoting collagen receptor production, comprising isoquercitrin as an active ingredient.
(2) The composition according to (1), wherein the collagen receptor is one or more selected from integrin, DDR2, and Endo180.

The present invention provides a novel composition for promoting collagen receptor production that contains isoquercitrin as an active ingredient. The composition of the present invention has the effect of promoting the production of integrin, DDR2, and Endo180, which are representative collagen receptors expressed on the surface of cells.

1 is a graph showing test results confirming that isoquercitrin promoted the production of DDR2, one of the collagen receptors. 1 is a graph showing the test results confirming that isoquercitrin promoted the production of Endo180, which is one of the collagen receptors. This is a graph showing the test results confirming that isoquercitrin promoted the production of integrin β1 (ITGB1), which is a collagen receptor.

The present invention will now be described in detail.
The term "collagen receptor" as used herein refers to an extracellular protein having a site that specifically binds to collagen. In the present specification, the collagen receptor may be referred to as a collagen receptor. This extracellular protein having a site that specifically binds to collagen can selectively bind to extracellular collagen as a collagen receptor. Representative examples of such collagen-binding proteins include fibronectin, integrin, laminin, DDR, Endo180, CD49b, CD29, Glycoprotein IV, Leukocyte-associated immunoglobulin-like receptor-1, and Secreted protein acidic and rich in cysteine.

"Isoquercitrin" is a glycoside represented by the following chemical formula 1, in which a glucose residue is bound to quercetin.

There is no particular limitation on the method for producing isoquercitrin. For example, as described in Japanese Patent Publication No. 54-32073, a glycosyltransferase is allowed to act on a solution containing quercitrin and a glucose donor such as starch, so that an equimolar or greater amount of glucose residues are transferred from the glucose donor to the quercitrin, and then amylase is allowed to act to produce α-glucosylisoquercitrin. The α-glucosylisoquercitrin produced by this method is called enzyme-modified isoquercitrin (EMIQ). Furthermore, it is also possible to purify only α-glucosylisoquercitrin by contacting it with a porous synthetic adsorbent and utilizing the difference in its adsorption properties. In the present invention, both the purified α-glucosylisoquercitrin and EMIQ can be used. EMIQ is commercially available, and for example, Sanemic, Sanmelin AO-3000, Sanmelin Powder C-10 (Sanei Gen F.F.I.), etc. may be used.

The "composition" in the present invention refers to a substance that contains isoquercitrin and promotes the production of collagen receptors. The "composition" includes foods, medicines, health foods, and animal feeds.
The composition containing the isoquercitrin of the present invention can be used as a collagen receptor production promoter or various pharmaceuticals, or as food, health food, animal feed, etc. The dosage form is not particularly limited and can be in various forms, specifically, it can be in a form for oral administration such as tablets, capsules, granules, etc. There is no particular limit to the excipients added when preparing the formulation. In addition, other physiologically active ingredients, etc. can be contained within a range that does not inhibit the action and effect of the present invention.

The amount of isoquercitrin, which is the active ingredient in the collagen receptor production promoting composition according to the present invention, is not particularly limited, and differs depending on the purpose and the target disease, so cannot be generally prescribed. However, in general, collagen receptor production can be promoted by using 0.001 to 99% by mass of isoquercitrin per composition, preferably 0.1 to 99% by mass, and more preferably 1 to 99% by mass.

The present invention will be further described below with reference to a test example confirming the collagen receptor production promoting effect using isoquercitrin (hereinafter abbreviated as "IQC").
<Test Method>
1. Cell Culture As test cells, normal human fibroblasts (NHDF: Normal Human Dermal Fibroblasts, Neonatal skin, Cat.# CC-2509; Lot No. 0000490825; LONZA) were cultured in an incubator at 37°C in the presence of 5% _CO2 using DMEM (Dulbecco's Modified Eagle's Medium, GIBCO) medium containing 10% (v/v) fetal bovine serum (FBS) SA (Nichirei) and 1% (v/v) Penicillin-Streptomycin (Sigma-Aldrich).

2. Preparation of IQC IQC (isoquercitrin, Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted to the required concentration with DMEM medium (serum-free) and used in this test.

3. Test Method for Evaluation of Collagen Receptor Production-Promoting Activity The production-promoting effect on three types of receptors, DDR2, Endo180, and integrin β1 (ITGB1), was confirmed.
NHDF cells were seeded in a 12-well plate at 80,000 cells/well and cultured for 24 hours. After culture, the medium was removed and 1.5 ml of IQC prepared using the medium was added. The IQC was diluted with 0.05% DMSO-containing DMEM medium (serum-free) to prepare 100 and 400 μg/ml.
After the addition of IQC, the cells were cultured for 48 hours, and then the medium was removed and the cells were washed with 2 ml of PBS. The cells were dissolved in 100 μl of Laemmli buffer (0.09 M Tris-HCl (pH 6.8), 3% SDS, 10.3% glycerol), collected in an Eppendorf tube, and sonicated (10-second treatment, 5-second standing on ice, 3 times) to dissolve the cells.
The mixture was then centrifuged at 15,000×g for 5 minutes at 4° C. The supernatant was mixed with 4×SDS-loading buffer and heat-treated at 95° C. for 3 minutes. Proteins were then separated by SDS-PAGE. Proteins in the gel were electrically transferred to a PVDF membrane.
After the transfer, the membrane was immersed in StartingBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific) for about 15 minutes for blocking. Next, the PVDF membrane was immersed in a primary antibody, Anti-DDR2 (AF2538, R&D Systems), Anti-Endo180 (ab70132, abcam), Anti-Integrin β1 (MAB1965, Millipore), or Anti-βActin (sc-47778, Santa Cruz Biotechnology), diluted 1,000-fold with StartingBlock, and reacted overnight at 4°C.
After washing four times with PBST (PBS, 0.05% Tween 20), the cells were incubated with secondary antibodies diluted 10,000 times, such as Rabbit anti-Goat IgG (H+L) Secondary Antibody (HRP, Polyclonal, Invitrogen, 81-1620, Thermo Fisher Scientific), Goat Anti-Rabbit IgG, HRP-linked Antibody (7074S, CST), or Goat anti-Mouse IgG (H+L) Cross-Adsorbed (HRP, Polyclonal, Secondary Antibody). The plate was immersed in a 50-μm anti-body (Invitrogen, G21040, Thermo Fisher Scientific) and reacted at room temperature for 1 hour.
After the reaction was completed, the plate was washed four times with PBST, and then signals were detected using an ECL detection kit with a chemiluminescence detector (LAS-4000 mini, Fujifilm). The signal intensity of the detected band was analyzed (Multi Gauge, Fujifilm), standardized with the band signal intensity of βActin, and the relative value to the control to which only DMSO was added was calculated to evaluate the increase in each collagen receptor.

4. Results IQC promotes collagen receptor protein production IQC promoted the production of all three collagen receptors (DDR2, Endo180, ITGB1) expressed in NHDF, as shown in Figures 1 to 3. In particular, at a concentration of 400 μg/ml IQC, the production was increased by 2.98, 1.65, and 1.73 times, respectively, compared to the control (DMSO) to which only DMSO was added.
It was revealed that IQC promotes the production of collagen receptors.

Claims (1)

A composition for promoting collagen receptor DDR2 production, comprising isoquercitrin as an active ingredient.