Phloroglucinol derivatives

Abstract

To provide a novel compound derived from an Agrimonia pilosa extract.SOLUTION: As novel phloroglucinol derivatives with an anti-aging action, five compounds have been discovered. The compounds represented by chemical formulae in this invention have been confirmed to have an action of decreasing a fluorescence value of SPiDER-βGal, that is, decreasing senescent cells. As one example of the novel phloroglucinol derivatives, the compound represented by a chemical formula 1 has been found.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a novel phloroglucinol derivative having anti-aging properties. [Background technology]

[0002] Aging, associated with the aging process, causes a decline in the function and metabolism of various organs and is involved in many diseases. In recent years, senescent cells that accumulate with age have attracted attention. It is thought that as senescent cells increase in the body with age, chronic inflammation and cancer are triggered in surrounding tissues through the senescence-associated secretory phenotype (SASP) of senescent cells. Chronic inflammation induced by senescent cells is known to cause systemic metabolic failure and abnormalities. Furthermore, it is known that senescent cells in the body are caused not only by aging but also by lifestyle irregularities such as high-fat diet intake, excessive stress, and ultraviolet radiation. Against this backdrop, there is a search for substances that selectively remove senescent cells accumulated in the body, and it has been reported that some substances improve the pathology of lifestyle-related diseases and age-related diseases (Non-patent Literature 1).

[0003] The present inventors have focused on and continuously explored the functionality of agrimol and the Kinmizuhiki extract containing it, and have already proposed compositions for anti-Helicobacter pylori (Patent Document 1), nerve activation (Patent Document 2), and immunoaging improvement functions (Patent Document 3).

[0004] [Patent Document 1] Japanese Patent Publication No. 2003-342190 [Patent Document 2] Japanese Patent Publication No. 2018-008888 [Patent Document 3] Japanese Patent Publication No. 2022-006259 [Non-Patent Document 1] Ther Adv Chronic Dis. 2020 Oct 13;11:2040622320964125.

Summary of the Invention

Problems to be Solved by the Invention

[0005] As described above, since improvements and therapeutic effects can be obtained against various diseases and aging by removing senescent cells, a novel substance that promotes the removal of senescent cells is required.

[0006] An object of the present invention is to provide a novel anti-aging substance that promotes the removal of senescent cells.

[0007] As a result of intensive studies on the above problems, the present inventors have found a novel compound having a senescent cell removal effect from the extract of Kinmizuhiki.

Means for Solving the Problems

[0008] The main configuration of the present invention is as follows. (1) A compound represented by Chemical Formula 1.

[0009]

Chem.

[0010] (2) A compound represented by Chemical Formula 2.

[0011]

Chem.

[0012] (3) A compound represented by Chemical Formula 3.

[0013]

Chem.

[0014] (4) A compound represented by Chemical Formula 4.

[0015] [ka] (Chemical formula 4)

[0016] (5) Compounds represented by chemical formula 5.

[0017] [ka] (Chemical formula 5) [Effects of the Invention]

[0018] The present invention provides novel phloroglucinol derivatives having anti-aging effects. The compounds of the present invention have been confirmed to have the effect of reducing the fluorescence value of SPiDER-βGal, i.e., reducing senescent cells. Therefore, the compounds of the present invention are useful as anti-aging substances and can also be used in foods, beverages, and pharmaceuticals containing the compounds of the present invention. [Brief explanation of the drawing]

[0019] [Figure 1] Isolation flow of compounds 1-5 from Agrimonia pilosa [Figure 2] Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) data of compound 1 [Figure 3] Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) data of compound 2 [Figure 4] Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) data of compound 3 [Figure 5] Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) data for compound 4 [Figure 6] Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) data for compound 5 [Figure 7] Figure showing the fluorescence values ​​of SPiDER-βGal upon addition of compound 1. [Figure 8]Figure showing the fluorescence values ​​of SPiDER-βGal upon addition of compound 2. [Figure 9] Figure showing the fluorescence values ​​of SPiDER-βGal upon addition of compound 3. [Figure 10] Figure showing the fluorescence values ​​of SPiDER-βGal upon addition of compound 4. [Figure 11] Figure showing the fluorescence values ​​of SPiDER-βGal upon addition of compound 5. [Modes for carrying out the invention]

[0020] Each of the novel compounds of the present invention can be obtained by extraction and purification processes using Agrimonia pilosa (Kinmizuhiki) as a raw material. It is also possible to obtain them by chemical synthesis. Furthermore, even if the plant is not Agrimonia pilosa, the compounds of the invention can be isolated and purified using extracts, crude products, dried plant materials, or plant pastes from other plants that have been confirmed to contain the compounds of the present invention.

[0021] When extracting and purifying the compounds of the present invention from Agrimonia pilosa, any extraction and purification process commonly used in industry can be used in appropriate combination. The leaves, stems, roots, flowers, etc. of the plant, which are the raw materials, are collected at an appropriate time and then either used as is or subjected to a drying process such as conventional aerosol drying to obtain the extraction raw materials. When extracting from the above-mentioned dried plant material, known extraction methods can be employed.

[0022] In other words, after crushing or finely chopping the raw materials, extraction is carried out using a solvent. As the extraction solvent, water, alcohols such as ethanol, methanol, and isopropyl alcohol, ketones such as acetone and methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, and lipophilic solvents such as hexane and chloroform can be used individually or in mixtures. Preferably, a mixture of ethanol and water is used. The extraction temperature is usually 0 to 100°C, preferably 5 to 50°C. The extraction time is about 1 hour to 10 days, and the amount of solvent is usually 1 to 30 times the weight of the dry raw materials, preferably 5 to 10 times the weight. The extraction operation may be carried out by stirring or by immersion. The extraction process may be repeated two to three times as needed. Alternatively, commercially available extracts of Agrimonia pilosa may be used as raw materials for the isolation and purification of compounds. The extraction obtained by removing insoluble residue from the crude extract obtained in the above procedure by filtration or centrifugation, or the purification of each compound from the plant sap, can be carried out by any known method for separating and purifying crude drugs. Generally, it is preferable to use methods such as two-phase solvent partitioning, countercurrent partitioning, column chromatography, or preparative high-performance liquid chromatography, either alone or in combination. For example, a two-phase solvent partitioning method may involve recovering the target compound into the solvent phase by partitioning the extract with a solvent such as n-hexane, chloroform, methyl ethyl ketone, ethyl acetate, or methyl acetate, and water. Column chromatography methods may include ion exchange column chromatography, methods using normal-phase or reversed-phase silica gel as the support, adsorption column chromatography using DIAION-HP20, etc., and gel filtration using modified dextran gel such as Sephadex-LH20 as the support. These methods may be performed individually, in combination, or repeatedly. Preparative high-performance liquid chromatography methods may include methods using reversed-phase columns with octadecyl silica, etc., and methods using normal-phase columns with silica gel, etc.

[0023] Examples of food and beverage forms containing the compounds of the present invention include tea made from dried Agrimonia pilosa, pure products of each compound, partially refined products of the novel compounds, and foods containing crude extracts from Agrimonia pilosa.

[0024] As a tea ingredient, it can be used alone or mixed with other tea ingredients. Other tea ingredients can include any tea commonly consumed as tea, such as green tea, oolong tea, pu-erh tea, black tea, roasted green tea, brown rice tea, Eucommia tea, persimmon leaf tea, mulberry leaf tea, etc.

[0025] The compound of the present invention can be used in any form of food or beverage that is normally available as a food product, including tea, drinks, jellies, biscuits, tablets, pills, soft capsules, hard capsules, powders, granules, and more. Additives such as excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, and antibacterial agents may also be used as auxiliary ingredients.

[0026] The effective intake amount of each novel compound from a food containing the compounds of the present invention varies depending on the form of intake, the health condition of the subject, the age of the subject, etc., but is usually 0.001 to 100 mg per day for adults, preferably 0.01 to 10 mg, and more preferably 0.1 to 1 mg.

[0027] The route of administration of pharmaceuticals containing the compounds of the present invention is not particularly limited. Examples include enteral administration such as oral or rectal administration, mucosal administration such as nasal administration, and injection administration such as intravenous or subcutaneous administration. The dosage form of the pharmaceuticals of the present invention can be a formulation suitable for the method of administration. Examples include solid preparations such as tablets, powders, fine granules, granules, capsules, powders, pills, and lozenges, liquid preparations such as solutions, suspensions, emulsions, syrups, and injections, and gel-like preparations. Pure products, purified products, or crude products of each compound may be administered as is, or they may be administered with pharmacologically acceptable excipients. Furthermore, the compounds of the present invention may be included alone as active ingredients related to the activation of nerves involved in memory, etc. In addition, other active ingredients related to the activation of the nervous system may be used in combination. As excipients, any substances that are generally available for use in formulations, such as monosaccharides, disaccharides, polysaccharides, inorganic salts, oils and fats, and distilled water, can be used. When formulating, additives such as binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, and antioxidants may also be used.

[0028] The dosage of each compound of the present invention as a pharmaceutical product varies depending on the route of administration, dosage form, symptoms of the disease, age of the subject, etc. Generally, for adults, it is 0.1 to 1000 mg per day, preferably 0.5 to 300 mg, and more preferably 1 to 100 mg. [Examples]

[0029] The following examples show how each compound of the present invention was extracted and purified from Agrimonia pilosa, and how the isolated compounds were identified. Furthermore, the present invention will be explained in more detail by providing examples of SPiDER-βGal tests using each purified compound.

[0030] [Extraction / separation / purification] Eight kg of pulverized dried Agrimonia pilosa was reflux-extracted with 10 times the volume of 90% ethanol for 1 hour, and concentrated under reduced pressure to obtain 630 g of Agrimonia pilosa extract. The Agrimonia pilosa extract was passed through a column packed with synthetic adsorbent, and water → 80% methanol → methanol → acetone was passed through in that order. The acetone eluate portion was concentrated under reduced pressure to obtain 66.5 g of acetone fraction. The acetone fraction was subjected to a column packed with octylsilylated silica gel (C8), and eluted in the following order: 0.1% formic acid / 80% methanol → 0.1% formic acid / 90% methanol → 0.1% formic acid / methanol → 0.1% formic acid / acetone. Each eluate was concentrated under reduced pressure to obtain the 0.1% formic acid / 80% methanol fraction (13.0 g), the 0.1% formic acid / 90% methanol fraction (16.2 g), the 0.1% formic acid / methanol fraction (23.2 g), and the 0.1% formic acid / acetone fraction (14.1 g). The 0.1% formic acid / acetone fraction was passed through a silica gel-packed column, and then through a mixture of n-hexane and ethyl acetate (6:1 → 5:1) followed by 0.1% formic acid / methanol. Each eluent was concentrated under reduced pressure to obtain fractions Fr.1 (4.3 g), 2 (4.1 g), 3 (730 mg), and 4 (4.7 g). Fr.3 was further subjected to a silica gel-packed column, and n-hexane-ethyl acetate (6:1) followed by methanol was passed through it. Each eluent was then concentrated under reduced pressure to obtain Fr.3-1 (122 mg), 3-2 (158 mg), and 3-3 (450 mg). Fr.3-1 was subjected to preparative HPLC using an octadecylated silica gel (C18) column, fractionated with acetonitrile-water-0.05% trifluoroacetic acid solvent, and each fraction was concentrated under reduced pressure to obtain compounds 1 (1 mg), 2 (2 mg), 3 (2 mg), and 5 (9 mg). Fr.2 was subjected to a C18-packed column, and eluted in the following order: 0.1% formic acid / 95% methanol → 0.1% formic acid / methanol → 0.1% formic acid / acetone. Each eluent was concentrated under reduced pressure to obtain Fr.2-1 (183 mg), Fr.2-2 (350 mg), and Fr.2-3 (2.1 g). Compounds Fr.2-2 were subjected to preparative HPLC using a C18 column, fractionated with acetonitrile-water / 0.05% trifluoroacetic acid solvent, and each fraction was concentrated under reduced pressure to obtain compounds 2 (16 mg), 3 (6 mg), and 4 (5 mg). Fr.2-3 was subjected to preparative HPLC using a C8 column, fractionated with acetonitrile-water-0.05% formic acid-based solvent, and each fraction was concentrated under reduced pressure to obtain Fr.2-3-1 (105 mg), 2-3-2 (159 mg), 2-3-3 (305 mg), and Fr.2-3-4 (60 mg). Fr.2-3-4 was subjected to preparative HPLC using a phenylated silica gel (Ph) column and fractionated with methanol-water-0.05% formic acid-based solvent to obtain a purified product (21 mg). The purified product was subjected to preparative HPLC using a C18 column and fractionated with acetonitrile-water / 0.05% trifluoroacetic acid-based solvent to obtain compound 4 (10 mg). Figure 1 shows the fractionation flow of compounds 1-5.

[0031] [Determination of the chemical structure of isolated compounds] As shown in Figure 7-11, the chemical structures of compounds 1-5 were determined by comparing data obtained by liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) and nuclear magnetic resonance (NMR) with data from Agrimol B described in Non-Patent Literature 1. Non-patent document 1: Chin. Tradit. Herb. Drugs. 2011; 42(2): 255-256.

[0032] [LC conditions] TIFF0007869058000006.tif36148

[0033] [TOFMS conditions] TIFF0007869058000007.tif67138

[0034] [NMR conditions] TIFF0007869058000008.tif15123

[0035] [Compound 1] Molecular formula: C 34 H 40 O 12 HR negative-ion ESI-MS: calculated for C 34 H 39 O 12 m / z 639.2442 [MH] - Found: 639.2463

[0036]

change

[0037] [Compound 2] Molecular formula: C 35 H 42 O 12 HR negative-ion ESI-MS: calculated for C 35 H 41 O 12 m / z 653.2598 [MH] - Found: 653.2595

[0038]

change

[0039] [Compound 3] Molecular formula: C 38 H 48 O 12 HR negative-ion ESI-MS: calculated for C 38 H 47 O 12 m / z 695.3068 [MH] -, found: 695.3074

[0040] [ka]

[0041] [Compound 4] Molecular formula: C 38 H 48 O 12 HR negative-ion ESI-MS: calculated for C 38 H 47 O 12 : m / z 695.3068 [MH] - , found: 695.3065

[0042] [ka]

[0043] [Compound 5] Molecular formula: C 37 H 46 O 12 HR negative-ion ESI-MS: calculated for C 37 H 45 O 12 : m / z 681.2911 [MH] - , found: 681.2917

[0044] [ka]

[0045] Evaluation of the effect of removing senescent cells [Culture and treatment methods] Human fetal lung fibroblasts WI38, 5x10 4Cells were suspended in MEM medium (Sigma-Aldrich) containing 10% FBS, 1% penicillin-streptomycin (Sigma-Aldrich), and 1% non-essential amino acid solution (Sigma-Aldrich) to a concentration of cells / mL. 100 μL of each suspension was seeded into black clear-bottom 96-well plates (Greiner) and incubated at 37°C under 5% CO2 for 24 hours. After 24 hours of incubation, doxorubicin was added to a final concentration of 62.5 nM and incubated for another 24 hours. After 24 hours of incubation with doxorubicin, the medium was changed to doxorubicin-free medium and incubated for 2 days. After 2 days of incubation, the medium was changed and compounds 1-5 were added at concentrations of 0 (no addition: Control), 0.1, 0.3, and 1.0 μg / mL and incubated for 3 days.

[0046] [Evaluation of SA β-Gal activity] Cell counts were measured using a fluorescence plate reader according to the instructions for the Cell Count Normalization Kit (DOJINDO) (Ex: 350 nm / Em: 461 nm). After measuring the cell count, the reaction solution was removed and the plates were washed with PBS. Subsequently, the Senescence-associated β-galactosidase (SA β-Gal) activity of the cells was measured using a fluorescence plate reader according to the instructions for the Cellular Senescence Plate Assay Kit - SPiDER-βGal (DOJINDO) (Ex: 500 nm / Em: 550 nm). The obtained fluorescence values ​​were corrected using the fluorescence values ​​previously measured with the Cell Count Normalization Kit.

[0047] [result] As shown in Figure 7-11, the addition of compounds 1-5 resulted in a decrease in the fluorescence value of SPiDER-βGal, indicating a decrease in the number of senescent cells. These results revealed that compounds 1-5 have the effect of removing senescent cells.

Claims

1. A compound represented by chemical formula 1. 【Chemistry 1】 (Chemical formula 1)

2. A compound represented by chemical formula 2. 【Chemistry 2】 (Chemical formula 2)

3. A compound represented by chemical formula 3. 【Transformation 3】 (Chemical formula 3)

4. A compound represented by chemical formula 4. 【Chemistry 4】 (Chemical formula 4)

5. A compound represented by chemical formula 5. 【Transformation 5】 (Chemical formula 5)

Images