TGF-β signaling inhibitors

Abstract

To provide a novel TGF-β signaling inhibitor. [Solution] An extract of Oenothera biennis is used as an active ingredient. Since the extract of Oenothera biennis has an inhibitory effect on TGF-β signal transduction, it is expected to have preventive and therapeutic effects on fibrotic diseases and preventive and ameliorative effects on cellulite by suppressing the promotion of fibrosis in tissues by TGF-β.

Description

[Technical field]

[0001] The present invention relates to a TGF-β signaling inhibitor. [Background technology]

[0002] TGF-β (transforming growth factor β) is a multifunctional cytokine that controls cell proliferation and differentiation. TGF-β is also involved in various pathological diseases, for example, it has the effect of promoting fibrosis in tissues, and is closely related to the onset and progression of fibrotic diseases such as pulmonary fibrosis. In addition, although it is not a disease, it has been suggested that TGF-β is involved in the uneven skin condition (cellulite) that occurs when adipose tissue degenerates due to fibrosis.

[0003] It is known that the signal of TGF-β is transmitted by the TGF-β / Smad pathway, and when TGF-β binds to a receptor present on the surface of a cell, Smad, a signal transduction molecule present in the cell, is phosphorylated, and the phosphorylated Smad migrates to the nucleus to regulate the transcription of target genes. In recent years, a method of preventing or treating fibrotic diseases or preventing or improving cellulite by inhibiting the signal transduction of TGF-β, which inhibits the promotion of fibrosis in tissues by TGF-β, has attracted attention. For example, Patent Document 1 introduces that a compound having a specific chemical structure can be used as an active ingredient of a TGF-β signal transduction inhibitor. However, the search for an ingredient that has a TGF-β signal transduction inhibitory effect is still in a significant state. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5846527 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a novel TGF-β signaling inhibitor. [Means for solving the problem]

[0006] In view of the above, the present inventors have conducted extensive research and have found that an extract of evening primrose (Oenothera biennis L.) has an inhibitory effect on TGF-β signaling.

[0007] The TGF-β signaling inhibitor of the present invention, which has been made based on the above findings, may contain water as described in claim 1. 1,3-Butylene glycol of Oenothera biennis using Seed The extract is the active ingredient. Effect of the Invention

[0008] According to the present invention, a novel TGF-β signaling inhibitor can be provided. [Brief description of the drawings]

[0009] [Figure 1] 1 is a graph showing the TGF-β signaling inhibitory effect of an evening primrose extract in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The TGF-β signaling inhibitor of the present invention contains an extract of Oenothera biennis as an active ingredient.

[0011] Oenothera biennis used in the present invention is a biennial plant of the genus Oenothera and the family Onagraceae, also known as evening primrose. In the present invention, either naturally growing Oenothera biennis or cultivated Oenothera biennis may be used.

[0012] In the present invention, the extract of Oenothera biennis can be obtained, for example, by adding water or an organic solvent (such as monoalcohols exemplified by methanol, ethanol, and isopropanol, and polyhydric alcohols exemplified by 1,3-butylene glycol, ether, hexane, ethyl acetate, acetonitrile, etc., which may contain water) as an extraction solvent to the seeds themselves (which may be dried or crushed) or to the pressed seeds, leaving the mixture at room temperature for 1 hour to 1 week to extract the components, or by refluxing the mixture to extract the components, and then filtering the mixture to obtain a filtrate. The extraction solvent may be a single solvent or a mixture of multiple solvents. The obtained filtrate may be used as it is as a liquid extract, or may be used as a solid or powder by vacuum concentration or freeze-drying. The filtrate may be further purified by ammonium sulfate fractionation, gel filtration, ion chromatography, etc., and the obtained fraction may be used as a liquid extract. Alternatively, for example, an extraction operation may be performed first using hexane, and then extraction operations may be performed successively using solvents with gradually increasing polarity, such as ethyl acetate, acetone, and water, and the filtrate obtained by using any of the solvents may be used as the liquid extract.

[0013] The TGF-β signaling inhibitor of the present invention can be formulated into various formulations and can be taken or ingested as a medicine or health food by itself, and can also be incorporated into various foods and drinks and ingested. The TGF-β signaling inhibitor of the present invention can also be applied to the skin in the form of an external preparation such as an ointment, cream, or lotion. The dosage, intake, or application amount can be appropriately determined based on the age, sex, weight, and severity of symptoms of the subject, and by taking, ingesting, or applying an appropriate amount, it is possible to expect preventive and therapeutic effects on fibrosis disease and preventive and ameliorative effects on cellulite based on its TGF-β signaling inhibitory action. EXAMPLES

[0014] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to the following description.

[0015] Experimental Example 1: Inhibitory effect of Oenothera biennis extract on TGF-β signal transduction (Experimental Method) The inhibitory effect of Oenothera biennis extract on TGF-β signaling was evaluated by the SBE (Smad binding element)-Luciferase reporter assay, which is known as a monitoring system for the TGF-β / Smad pathway. Specifically, pNL[NlucP / SBE / Hygro]Vector (Promega, USA) was introduced into mouse embryonic fibroblasts NIH3T3 (BRC, Japan) by electroporation using Cell Line Nucleofector Kit V (Lonza, Switzerland). After 24 hours, drug-resistant cells were selected by adding hygromycin at a final concentration of 200 μg / mL, and a hygromycin-resistant NIH3T3 cell line stably expressing the SBE-Luciferase reporter was established. Next, about 10,000 SBE-Luciferase reporter stably expressing NIH3T3 cell lines were seeded in 100 μL of medium (low glucose DMEM (Sigma, USA) supplemented with 10% FBS (Gibco, USA) and 200 mmol / L L-glutamine solution (×100) (Wako, Japan)) in a 96-well plate per well, and cultured at 37 °C for 16 to 24 hours. After that, 1 μL of evening primrose extract (final concentration 1%) was added and cultured for another hour. After 1 hour, human TGF-β1 (Wako, Japan) at a final concentration of 20 ng / mL was added and cultured at 37 °C for 4 hours. After 4 hours, the TGF-β signaling inhibitory effect of the evening primrose extract was evaluated by measuring luciferase activity using the Nano-Glo Luciferase Assay System (Promega, USA) according to the protocol. The evening primrose extract used as the sample was a commercially available product, and its composition (weight %) was evening primrose seed extract (extraction solvent was 1,3-butylene glycol containing 50% water): 1.0, 1,3-butylene glycol: 49.5, water: 49.5.

[0016] (Experimental Results) As a control, 1,3-butylene glycol was added at a final concentration of 0.5% instead of the evening primrose extract, and the luciferase activity without human TGF-β1 was set to 1. Figure 1 shows the relative values. As is clear from Figure 1, the increase in luciferase activity, which indicates activation of the TGF-β / Smad pathway caused by the addition of human TGF-β1, was greatly reduced by adding the evening primrose extract before adding human TGF-β1. From these results, the inhibitory effect of evening primrose extract on TGF-β signal transduction was confirmed.

[0017] Formulation Example 1: Tablets Tablets having the following composition and TGF-β signal transduction inhibitory activity were produced by a method known per se (unit: weight %). Powder of evening primrose extract used in Experimental Example 1 1 lactose 80 Magnesium stearate 19

[0018] Formulation example 2: Biscuits Biscuits having the following component composition and TGF-β signaling inhibitory activity were produced by a method known per se (unit: weight %). Evening primrose extract used in Experimental Example 1 1 32g plain flour Whole eggs 16 Butter 16 Sugar 24 water 10 1 teaspoon baking powder

[0019] Formulation example 3: Jelly A jelly having the following component composition and TGF-β signaling inhibitory activity was produced by a method known per se (unit: weight %). Evening primrose extract used in Experimental Example 1 0.01 Gelling agent 1.3 Sugar 20 pH adjuster 2.5 Wednesday 76.19

[0020] Formulation Example 4: Ointment An ointment having the following component composition and having a TGF-β signaling inhibitory effect was produced by a method known per se (unit: weight %). Powder of evening primrose extract used in Experimental Example 1 5 White Vaseline 95

[0021] Formulation Example 5: Cream A cream having the following component composition and having a TGF-β signaling inhibitory effect was produced by a method known per se (unit: weight %). Powder of evening primrose extract used in Experimental Example 1 5 White Vaseline 30 Cetanol 15 Sodium Lauryl Sulfate 1 Ethyl parahydroxybenzoate 0.1 Butyl paraoxybenzoate 0.1 Water 48.8

[0022] Formulation Example 6: Lotion A lotion having a TGF-β signaling inhibitory effect and consisting of the following component composition was produced by a method known per se (unit: weight %). Evening primrose extract used in Experimental Example 1 10 Polyoxyethylene (40) hydrogenated castor oil 0.5 Ethanol 5 Paraben: 0.2 Water 84.3 [Industrial Applicability]

[0023] INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a novel TGF-β signaling inhibitor.

Claims

[Claim 1] A TGF-β signaling inhibitor comprising, as an active ingredient, an extract from seeds of Oenothera biennis using 1,3-butylene glycol which may contain water as an extraction solvent.

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