JP5774839B2 - Macrophage foaming inhibitor in arteriosclerotic tissue - Google Patents

Description

  The present invention relates to an inhibitor of macrophage foaming in arteriosclerotic tissue, which contains ε-viniferin as an active ingredient. The present invention is suitably applied to foods, pharmaceutical preparations, cosmetics, feeds and the like.

Oxygen is an essential substance for life support of many living organisms, and plays various roles such as energy production in the living body. In this energy production system and other reaction systems, oxygen becomes active oxygen such as superoxide anion, singlet oxygen, and hydroxy anion by the action of enzymes, ultraviolet rays, and other radiations.
Active oxygen plays an extremely important role for a living body in terms of a bactericidal action by leukocytes, that is, an action of decomposing foreign substances taken by phagocytes. On the other hand, if the active oxygen is excessively produced, it causes damage to living tissue itself, promotes peroxidation of unsaturated fatty acids that form phospholipids in biological membranes, and produces inflammatory factors such as lipid peroxides. Trigger. In addition, there is a risk that, together with the lipid peroxide, an alkoxy radical or a hydroxy radical is generated to attack a biological membrane, induce membrane damage, and deactivate various useful enzymes.

  On the other hand, in vivo, examples of enzymes that deactivate active oxygen include superoxide dismutase (SOD), catalase, peroxidase, glutathione peroxidase, and the like. Examples of various vitamins having an antioxidant action include α-tocopherol (vitamin E). A normal living body is maintained by the action of these enzymes and vitamins. However, a large amount of various chemical substances that promote in vivo production of active oxygen, such as herbicides, insecticides, detergents, pharmaceuticals, and various food additives, are present in the environment surrounding modern living bodies. Due to the influence of these chemical substances, generation of active oxygen exceeding the ability of an appropriate defense mechanism by the enzymes and vitamins, and generation and accumulation of lipid peroxides occur. When the active oxygen is abnormally increased in the body, for example, various inflammations, liver disorders, kidney disorders, gastric disorders, arteriosclerosis, hemolysis, senile dementia are associated with the progression of chain reaction of peroxidation. Serious disorders such as retinopathy, pulmonary disorders, and ischemic vascular diseases may occur. In particular, it has recently become clear that arteriosclerosis occurs due to active oxygen. In particular, LDL cholesterol oxidized by reactive oxygen has various actions including the expression of cytokines and adhesion molecules that cause inflammation of vascular endothelial cells to promote atherosclerosis, and also a monocyte migration factor But there is. In addition, it shows a cytotoxic effect on endothelial cells. Monocytes in the arteries gather in the intima via adhesion molecules, differentiate into macrophages and express several scavenger receptors, which promote uptake of oxidized LDL into the cells. Uptake of oxidized LDL by macrophages is irreversible, and since there is no mechanism for controlling excessive uptake, it is known that foaming occurs and deposits in the arterial intima (Non-Patent Documents 1-3). It has been clarified that accumulation of such macrophage foaming enlarges part of the artery, resulting in atherosclerosis.

Thus, it is widely recognized that excessive active oxygen is a primary factor of arteriosclerosis and has an adverse effect on the living body. For this reason, it has been studied to positively impart an action of removing active oxygen to foods and drinks used daily, and for example, a food and drink having an action of removing active oxygen has been proposed (Patent Document 1).
Alternatively, in recent years, interest has been concentrated on natural substances present in plants and exhibiting physiological activity, in particular, compounds of polyphenols. Polyphenols are generally contained in tea, vegetables, fruits, herbs and the like, and can be expected as a therapeutic / preventive agent having no side effects and having long-term ingestion as a food or a favorite product.
Polyphenolic compounds are secondary metabolites of plants. They are universally present in the plant kingdom and are known to exhibit a variety of physiological activities. They used to be used in the fields of pharmacy, phytochemistry, etc. Attracted attention. For example, tea polyphenols, especially catechins, have a wide range of physiology, including antibacterial, antiviral, antimutation, antioxidant, antihypertensive, hypocholesterolemic, anticariogenic, antiallergic, intestinal flora improvement, and deodorization. It is known to have activity.

Among polyphenols, stilbenoids called resveratrols have attracted attention. Stilbenoids are a group of compounds composed of a C ₆ -C ₂ -C ₆ basic skeleton having 14 carbon atoms. Although stilbenoids have a limited distribution in plants, these are included in the vine family, and the representative compounds are trans-resveratrol ((E) -3,4 ', 5-trihydoroxy- stilbene).

  Resveratrol, a representative stilbene compound, is active through the inhibitory activity of the active oxygen generator NADPH oxidase or xanthine oxidase, the superoxide dismutase of the active oxygen scavenging system, the increase of catalase activity, and the increase of glutathione. It is known to exert an antioxidant effect, and has other excellent physiological activities such as a vasorelaxant action, an anti-inflammatory action, a platelet aggregation inhibitory action and the like (Non-patent Document 4). Furthermore, a life extension effect similar to calorie restriction has been reported even under high-fat diet conditions (Non-Patent Document 5), and is applied as a naturally derived substance having a health maintenance effect for anti-aging.

Stilbene compounds typified by resveratrol are naturally found in plants of the seed plant family, and are especially found in wines made by fermenting weedbirds, grape shoots, young leaves, vines, grape skins, and grapes. It is a compound. Using these as materials, stilbene compounds have been separated as a mixture and have been marketed by being blended in beverages, confectionery, health foods, cosmetics, hair restorers and the like.

  Plants containing stilbene compounds contain mixtures of various degrees of polymerization, from those having a high degree of polymerization to those having a low degree of polymerization. For example, resveratrol is a monomer, but from butter seedlings, Besides veratrol, peceatannol, piceid (glycoside), astringin (glycoside), dimer, epsilon-viniferin, delta-viniferin, iso-epsilon-viniferin, ampelopsin A, pallidol, trimmer, miyabenol C, Hopephenol, R-viniferin, R-2-viniferin, etc. have been found as tetramers.

High fat and high calorie are known as factors that promote arteriosclerosis, but in France, the incidence of coronary artery disease is low despite the high fat and high calorie diet, and it is known as the so-called French paradox. This phenomenon is related to the high consumption of red wine in France, and the polyphenols derived from grapes have been highlighted. Among them, a substance called resveratrol has attracted attention. However, resveratrol needs to be absorbed and used by the living body in order to exhibit a useful bioregulatory effect, but it is known that bioavailability in humans is low because it is excreted immediately after ingestion. (Non-Patent Document 6). Therefore, it has been found that it is difficult to completely prevent or treat arteriosclerosis even if it depends only on such an antioxidant effect. The present inventor conducted research on ε-viniferin which is a dimer of resveratrol in a so-called narrow sense, and found a strong anti-atherosclerosis action and an active oxygen scavenging activity in living cells which were not known at all. It was.
Upon further investigation, ε-viniferin, a dimer of resveratrol, is a member of the CD36 protein, a family of scavenger receptors on the surface of monocyte cells that are invaded and immobilized in macrophages and atherosclerotic layers. It was found that expression can be suppressed, excessive phagocytosis of oxidized LDL-cholesterol can be suppressed, and foaming of macrophages can be suppressed. CD36 is a collective term for a plurality of glycoprotein molecules having a molecular weight of 78-88 kDa, and is expressed in many cell types such as monocytes, macrophages, platelets and adipocytes. In macrophages, CD36 functions as a scavenger receptor that recognizes specific oxidized phospholipids and oxidized lipoproteins and is involved in the intracellular translocation of apoptotic cells, certain bacterial and fungal pathogens, and modified low density lipoproteins, It contributes to inflammatory response and atherothrombosis. Actually, by suppressing the overexpression of CD36 on the surface of macrophages, it is shown that macrophages retain the mobility without foaming from the vascular intima and regress arteriosclerotic lesions (Non-patent Document 7). ing.

We also found that ε-viniferin suppresses the expression of CD11a, an adhesion molecule expressed on the monocyte surface. Thereby, it is possible to suppress peripheral monocytes from adhering to the arteriosclerotic lesion and invading into the artery, and to suppress the acceleration of arteriosclerosis. LFA-1 (a heterodimer of CD11a and CD18) is involved in allogeneic adhesion between lymphocytes and heterogeneous adhesion of lymphocytes to vascular endothelium. As ligands of LFA-1, intercellular adhesion molecules (ICAMs), that is, ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50) and the like are known. Here, CD11a is expressed in leukocytes such as monocytes, macrophages, granulocytes, and T cells, and CD11a and CD18 are also expressed in activated platelets. The CD11a (LFA-1α) antigen is an integrin αL chain with a molecular weight of 180 kDa and non-covalently associates with a 95 kDa CD18 antigen (integrin β2).

Furthermore, we have also found a phenomenon in which the superoxide anion produced by macrophages is strongly eliminated. Macrophages produce superoxide anions among reactive oxygen species when they exert phagocytosis. Since this promotes the inflammation reaction of the arteriosclerotic lesion and accelerates the foaming of macrophages, the approach to prevent the progression of the arteriosclerotic lesion from the viewpoint of inhibiting NADPH oxidase activity is also studied to suppress superoxide anion production (Non-Patent Document 7). Therefore, defoaming of macrophages can be suppressed by detoxifying superoxide anions produced by macrophages themselves.

As mentioned above, substances that suppress the expression of CD36 on the macrophage surface and the adhesion molecule CD11a, and further suppress the harmful effects of superoxide anion, prevent excessive aggregation of macrophages in the arteriosclerotic lesion, The progression of the lesion can be suppressed. In particular, by preventing foaming of macrophages, macrophages that have taken up oxidized LDL can not only be deposited on the intima of the arteries, but also maintain normal cell function, so that the cholesterol that is taken in is secreted out of the cells and is released by HDL. The secretory cholesterol can be extracted from the arteriosclerotic lesion via the ABCA1 transporter and recovered, and the arteriosclerotic lesion can be regressed and improved.

As a result of diligent investigations, the present inventors have investigated the scavenger receptor expression inhibitor of macrophages in the arteriosclerotic layer characterized by containing ε-viniferin as an active ingredient, and the adhesion molecule expression inhibitory action and superoxide anion scavenging activity In addition, a macrophage foaming inhibitor in arteriosclerotic tissue based on these actions has been completed.

JP-A-11-346715 JP 2001-122723 A JP-A-6-49053 International Publication No. 00/64883 Pamphlet International Publication No. 03/091237 Pamphlet International Publication No. 2004/103988 Pamphlet

Gerrity RG. The American Journal of Pathology, The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. 103 (2): 181-190, 1981. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL.New England Journal of Medicine, Beyond cholesterol.Modifications of low-density lipoprotein that increase its atherogenicity, 320 (17): 915-924, 1989. Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ. Circulation, Atherosclerosis: Basic mechanisms. Oxidation, inflammation, and genetics, 91 (9): 2488-2496, 1995. Toshihiro Ichiki. Cardiology, Resveratrol in wine and its mechanism, 67 (2): 216-222, 2010. Baur JA, Pearson KJ, Sinclair DA, et al. Resveratrol improves health and survival of mice on a high-calorie diet.Nature, 444 (7117): 337-342, 2006. Walle T, Hsieh F, Walle K, et al.High absorption but very low bioavailability of oral resveratrol in humans.Drug Metabolism and Dispositions, 32 (12): 1377-1382, 2004. Curtiss LK. Reversing atherosclerosis? New England Journal of Medicine, 360 (11): 1144-1146, 2009.

  The present invention relates to a pharmaceutical or health food having an excellent arterial cholesterol deposition inhibitory effect.

    In order to solve the above-mentioned problems, the present inventors diligently searched for various compounds and natural substances. As described above, among the group of compounds collectively called resveratrol, ε-viniferin is particularly known. Specifically, it was found that it has a function of specifically suppressing the expression of CD36 on the surface of macrophages and suppressing the foaming of macrophages in the arteriosclerotic layer as compared with resveratrol. Furthermore, the present inventors have completed a method for suppressing adhesion of cholesterol in an artery based on these actions, having both an adhesion molecule expression suppressing action and a superoxide anion scavenging activity.

That is, the present invention
CD36 protein expression inhibitor containing ε-viniferin as an active ingredient (excluding food forms and arteriosclerosis prevention / treatment applications).
About.

  The present invention has a high effect of preventing foaming by suppressing the expression of CD36, which is one of the macrophage scavenger receptor family, and further suppresses the expression of CD11a which is a constituent protein of LFA-1 which is an adhesion molecule of macrophages. It can suppress the atherogenesis of the inflammatory site of the artery. In addition, since macrophages themselves have high scavenging activity for superoxide anions, they can provide various disease treatments and health foods using them, and metabolic syndrome, diabetes and its complications that are thought to involve active oxygen. It is useful for the treatment and prevention of various pathological conditions such as arteriosclerosis and gastrointestinal diseases.

The figure which shows the expression of CD36 by a flow cytometry method. Control group (shadow area), 24 hours after addition of TNFα (solid line). The figure which shows the expression of the flow cytometry method CD36. 24 hours after addition of TNFα (shaded area), resveratrol (upper solid line) and biniferin (lower solid line). The figure which shows the expression of CD11a by the flow cytometry method. 24 hours after the addition of the control group (shadow area), resveratrol (upper solid line) and biniferin (lower solid line).

  Hereinafter, the present invention will be described in detail.

  The ε-viniferin used in the present invention is a stilbene compound and is known as a kind of grape phytoalexin. Resveratrol in the present invention refers to a polyphenol monomer having a stilbene skeleton as a basic structure. ε-viniferin is this dimer. Ε-viniferin can be extracted from a plant body containing a stilbene compound, preferably from a grape side branch, using various extraction solvents, preferably 70% methanol. The extracted ε-viniferin can be separated and purified by separation and purification means such as preparative high performance liquid chromatography.

  Although ε-viniferin is generically called resveratle, it is used separately in the present invention. As described above, resveratrol is a kind of polyphenol present in grape leaves, shoots, peels, etc., and is a stilbene compound, and trans-resveratrol is a typical compound. In the past, so-called sirtuin activity was measured as a complex or mixture, and the ability to remove active oxygen has been measured and evaluated as part of its action process. The present inventors have further studied and confirmed the strong effect of suppressing the expression of CD36 on the surface of macrophages by ε-viniferin alone, and confirmed the effect.

  ε-Viniferin can be extracted from a plant such as the above-mentioned grapes in addition to a high-purity product synthesized using organic chemistry or microorganisms, in which case an extract may be used, or Resveratrol isolated from the extract may be used. The natural product may be any as long as the content of ε-viniferin is controlled, and examples thereof include grapes, peanuts, and teraceae plants such as Japanese knotweed and clover.

Grape is particularly preferred as a natural product. The type of grape is not particularly limited, but Delaware, Kyoho, Koshu, Pione, Muscat, Chenin Blanc, Grenache, Mataro, Muller Thurgau, Trebbiano, Berry A, Cabernet Sauvignon, Merlot, Pinot Noir, Cabernet Franc, Schiller, Chardonnay, Sauvignon Blanc, Semillon, Schiller, Gamay, Riesling, Alligote and the like are preferred. A grape extract refers to an extract from a product derived from grape berries, grape leaves or grapes. The thing derived from grape means grape juice, wine, the residue at the time of wine manufacture, wine concentrate, etc. Extraction, for example, resveratrols are known to be abundant in grape skins, shoots, leaves, vines, so after drying them as necessary, soak in a solvent for a certain period of time, Alternatively, it can be brought into contact with a heating refluxing extraction solvent, then filtered, concentrated, and further collected by the above-described preparative chromatography. As the extraction solvent, any solvent that is usually used for extraction can be used. For example, water, methanol, ethanol, propylene glycol, 1,3-butylene glycol, glycerol and other alcohols, chloroform, dichloroethane, four Organic solvents such as carbon chloride, acetone and ethyl acetate can be used alone or in combination. The extract obtained by extraction with the above solvent is used as it is, or concentrated extracts are used, or these extracts are removed by using an adsorption method, for example, ion exchange resin, or a porous polymer (for example, Amberlite XAD- After adsorbing on the column of 2), elution with methanol or ethanol and concentration can also be used. Further, a partitioning method, for example, an extract extracted with water / ethyl acetate can be used. Moreover, since it is contained also in wine, the concentrate of wine, or the solid substance concentrated and dried, they can also be used. The grape residue generated during wine production can be used as it is, or can be extracted in the same manner as the above extraction method. As the organic solvent, alcohol such as ethanol is preferable, and ethanol is particularly preferable.

The active oxygen scavenger of the present invention can be administered to humans and animals as a pharmaceutical preparation, and can also be ingested even if blended with various foods and drinks and feed (such as pet food).

  The pharmaceutical preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.). The dosage form is also arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as internal liquids and syrups, and parenteral liquid preparations such as injections. These forms can also be appropriately prepared by known methods. For these pharmaceutical preparations, commonly used binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonicity Excipients such as agents, stabilizers and pH adjusters may be used as appropriate.

  In the pharmaceutical preparation of the present invention, the dose of ε-viniferin, which is an active ingredient, varies depending on the type, dosage form, patient age, body weight, indication symptoms, etc. It is administered once to several times, and about 1 mg to 200 mg, preferably about 3 mg to 20 mg / person is administered once a day.

  When the active oxygen scavenger of the present invention is used as food or drink, feed, etc., in addition to general food and drink, prevention and treatment of metabolic syndrome, prevention and treatment of diabetes and its complications, cancer (endometrial cancer, menopause Post-breast cancer, leukemia, colorectal cancer, gastric cancer, prostate cancer, etc.), non-alcoholic steatohepatitis (NASH), and the physiological function such as prevention and treatment of liver disease derived from it as a concept It can be applied to sex foods and drinks, foods for the sick, foods for specified insurance, and the like.

  As a form of food-drinks, it can shape | mold arbitrarily, for example in the shape of a granule, a granular form, a paste form, a gel form, solid form, or liquid. These include various known substances that are recognized to be contained in foods, such as binders, disintegrants, thickeners, dispersants, reabsorption accelerators, flavoring agents, buffers, surfactants, Excipients such as solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers and pH adjusters can be contained as appropriate.

  The content of ε-viniferin, which is an active ingredient contained in the food and drink of the present invention, can be determined as appropriate according to the type, purpose, form, usage method, and the like, for example, about 1 to 10% by mass. It can be. In particular, when used as a health food or drink, it is preferable to contain the active ingredient of the present invention in such an amount that a predetermined effect is sufficiently exhibited. Therefore, in such a case, the food / beverage product of the present invention contains ε-viniferin and is used for the prevention or improvement of various diseases such as reduction of oxidized LDL or metabolic syndrome. The attached food and drink can be used.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to a following example. In addition, all compounding quantities are shown by the mass%.

CD36の場合は、ヒトTNＦα（最終濃度：20 ng/mL）の添加により、有意ではないものの、発現が増強したため、このTNＦα添加の条件で、trans-resveratrol（レスベラトロール）、epsilon-viniferin（ビニフェリン）による、このマーカーの発現に対する影響を検討した。図1には、対照群の発現とTNＦα添加時のCD36発現の典型例を示す（図1）。図1に示すように、24hr後の対照群の細胞におけるＭ２％は、６３．９９ ± ６．５３０％（ｎ＝３）である。ＴＮＦα20 ng/mＬ添加群のＭ２％は、６６．０７ ± 2.944%（n=3）であり、実験により、M2画分の細胞集団の割合には、TNＦα添加により、U937細胞におけるCD36の発現は、僅かながら増強した。このTNＦα添加の条件で、レスベラトロール １００ μＭ添加、及び、ビニフェリン １００ μＭ添加による、CD36発現への影響を検討した。

図2には、レスベラトロール １００ μＭ添加、及び、ビニフェリン １００ μＭ添加時の典型例を示す（図2）。3回の独立した実験結果を表１に示すが、TNＦαによって惹起されたCD36の発現は対照に比較してレスベラトロールによって抑制傾向を示した。一方、ビニフェリンは、対照に比較して有意にCD36の発現が抑制されることを確認した。

Example 1
Confirmation test of CD36 expression induction on macrophage surface

<Method> The U937 (human histiocytic lymphoma derived, macrophage-like) cell line was used as a cell for flow cytometry (FCM) examination.
U937 cells (DS Pharma Biomedical Co., Ltd., Suita, Osaka) were purchased in flasks (> 10 ⁶ ) and designated as the _F0 generation. As long as the cells were subcultured, the generation number was not changed, but cells within 10 passages were subjected to the experiment. Cells that had reached submaximal confluent were subcultured at a ratio of 1:10, and cells cultured for 24 hours were used. U937 cells are suspension cells, and the medium is sodium bicarbonate (Sodium Bicarbonate, Life Technologies Japan, Hatchobori, Tokyo) and antibacterial-antimycotic (Antibiotic-Antimycotic, 10 mg / L). HyClone ^R fetal calf serum (Lot No .: 10%) in a modified RPMI-1640 liquid medium (R7388, containing 20 mM HEPES, Sigma-Aldrich Japan, Shinagawa, Tokyo) with Life Technologies Japan) ATD31995, Fetal Bovine Serum Defined; FBS, HyClone Laboratories, Inc., Logan, UT, USA) was used.
At 0 hr, the cell suspension was used as an expression stimulant, with human TNFα (Recombinant Human Tumor Necrosis Factor α, Lot No .: AA3309072, R & D Systems, Inv., Mineapolis, MN, USA) at a final concentration of 20 ng / mL. At the same time, trans-resveratrol (resveratrol,> 98%, 185-01721, C ₁₄ H ₁₂ O ₃ = 228.24, Wako Pure Chemical Industries, Doshumachi, Osaka) and epsilon with a final concentration of 100 μM -viniferin (viniferin, 228-01741, C ₂₈ H ₂₂ O ₆ = 454.47, Wako Pure Chemical Industries, Ltd.) was added. Human TNFα is not added to the cell suspension of the control group, but the same amount of ethanol (EtOH, 99.5v / v%, 053-06531, Wako Pure Chemical Industries, Ltd.) is used as the solvent for the test drug (20 μL) Added. After the start of the test, the cell suspension after 6, 12, 24, 36, and 48 hr is dispensed into a 1.5 mL Eppendorf tube (eppendorf AG, Hamburg, Germany) and stored in Staining Buffer (SB) [2% Bovine Serum Albumin (BSA, > 95%, 014-15134, Lot No .: CDK4319, Wako Pure Chemical Industries, Ltd.) and 0.5% NaN ₃ (Sodium Azide, NaN ₃ = 65.01, Lot No .: PER1519,> 98%, 190-14901, Japanese Wash cells with Dulbecco's Phosphate Buffered Saline (DPBS, (-) CaCl ₂ (-) MgCl ₂ , 14190-144, 500 mL, Gibco-Invitrogen, Hatchobori, Tokyo) Subsequently, specific binding to cell surface antigen was performed using an anti-human CD36 mouse monoclonal antibody (4 ° C, 30 min), and then cells were fixed with CellFIX ^™ (340181, BD Bioscience, San Jose, CA, USA) After fixation, it was subjected to FCM analysis (FCM measurement was possible for about one week after cell fixation).
Regarding human-specific antibodies, CD36 used Allophycocyanin (APC) -conjugated antibody (Isotype: Mouse IgM) and, as a negative control, Isotype Control, which is an antibody against mouse immunoglobulin to which each dye was bound (PE-). IgG _2a , APC-IgM). PE is excited with an argon ion (Ar) laser at an excitation wavelength of 564 nm and a fluorescence wavelength of 585 nm, and APC is a helium-neon laser with an excitation wavelength of 650 nm and a fluorescence wavelength of 660 nm. Excited by. FCM used BD's Fluorescence Activated Cell Sorter (FACS; FACSCalibur ^™ , BD Bioscience) to acquire data and analyzed the data using CellQuestPro ^™ software (MacOS, BD Bioscience). Fluorescence intensity data in isotype control was used as a reference, and fluorescence intensity data stronger than this was defined as positive (M2%).
Data were subjected to one-way analysis of variance, and Scheffe's multiple comparison test was performed for comparison between groups. The significance level was 5% or less.

<result>

In the case of CD36, expression was enhanced by the addition of human TNFα (final concentration: 20 ng / mL), but the expression was enhanced. Therefore, under the conditions of addition of TNFα, trans-resveratrol (resveratrol), epsilon-viniferin ( The effect of biniferin on the expression of this marker was examined. FIG. 1 shows typical examples of expression in the control group and CD36 expression upon addition of TNFα (FIG. 1). As shown in FIG. 1, M2% in the cells of the control group after 24 hr is 63.99 ± 6.530% (n = 3). The M2% of the TNFα20 ng / mL addition group is 66.07 ± 2.944% (n = 3). According to the experiment, the proportion of the cell population of the M2 fraction was found to be increased in the expression of CD36 in U937 cells by the addition of TNFα. Slightly enhanced. Under the conditions of this TNFα addition, the effect of 100 μM resveratrol and 100 μM biniferin on CD36 expression was examined.

FIG. 2 shows typical examples when resveratrol is added at 100 μM and vinyliferin is added at 100 μM (FIG. 2). The results of three independent experiments are shown in Table 1, and the expression of CD36 induced by TNFα tended to be suppressed by resveratrol compared to the control. On the other hand, it was confirmed that the expression of CD36 was significantly suppressed for biniferin compared to the control.

図3に示すように、対照群の細胞におけるCD11aのM2%で示されるように高度にCD11aの発現が認められた。レスベラトロール １００ μＭ添加群及びビニフェリン１００ μＭ添加群のM2%は、いずれも、CD11aの発現を抑制していたが、その作用は、ビニフェリンの方が強かった。なお、3回の独立した実験結果を表2に示すが、ほぼ、同様の成績であった。

Example 2
Confirmation test of CD11a expression induction on macrophage surface

<Method> As in Example 1, U937 (human histiocytic lymphoma derived, macrophage-like) cell line was used as a cell for flow cytometry (FCM) examination. Detailed culture conditions and procedures were also performed according to Example 1. However, in Example 1, human TNFα was added as an expression stimulator to the cell suspension at a final concentration of 20 ng / mL at 0 hr, but CD11a was preliminarily examined in cells without human TNFα addition. High expression was observed, and conversely, the expression was reduced by the addition of human TNFα. Therefore, the expression level of this marker was evaluated under culture conditions in which no TNFa was added. Resveratrol and vinyliferin are trans-resveratrol (resveratrol,> 98%, 185-01721, C ₁₄ H ₁₂ O ₃ = 228.24, Wako Pure Chemical Industries, Doshumachi, Osaka) and epsilon at a final concentration of 100 μM -viniferin (viniferin, 228-01741, C ₂₈ H ₂₂ O ₆ = 454.47, Wako Pure Chemical Industries, Ltd.) was added. Human TNFα is not added to the cell suspension of the control group, but the same amount of ethanol (EtOH, 99.5v / v%, 053-06531, Wako Pure Chemical Industries, Ltd.) is used as the solvent for the test drug (20 μL) Added. After the start of the test, 24 hr later, the cell suspension is aliquoted into 1.5 mL Eppendorf tubes (eppendorfAG, Hamburg, Germany) and stored in Staining Buffer (SB) [2% Bovine Serum Albumin (BSA,> 95%, 014-15134, Lot No .: CDK4319, Wako Pure Chemical Industries, Ltd.) and 0.5% NaN ₃ (Sodium Azide, NaN ₃ = 65.01, Lot No .: PER1519,> 98%, 190-14901, Wako Pure Chemical Industries, Ltd.) were added. , Dulbecco1's Phosphate Buffered Saline (DPBS, (-) CaCl ₂ (-) MgCl ₂ , 14190-144, 500 mL, Gibco-Invitrogen, Hatchobori, Tokyo)] and then anti-human CD11a mouse monoclonal antibody After specific binding to a cell surface antigen (4 ° C., 30 min), the cells were fixed with CellFIX ^™ (340181, BD Bioscience, San Jose, Calif., USA) and subjected to FCM analysis. Regarding human-specific antibodies, data is obtained using CD11a using Phycoerythrin (PE) -conjugated antibody (Isotype: Mouse IgG _2a ), and FCM using BD's Fluorescence Activated Cell Sorter (FACS; FACSCalibur ^™ , BD Bioscience). Data analysis was performed using CellQuestPro ^™ software (MacOS, BD Bioscience). Fluorescence intensity data in isotype control was used as a reference, and fluorescence intensity data stronger than this was defined as positive (M2%).

<Result> In the case of CD11a, a typical example of FCS is shown (Fig. 3).

As shown in FIG. 3, CD11a expression was highly observed as shown by M2% of CD11a in the cells of the control group. M2% in the resveratrol 100 μM addition group and the biniferin 100 μM addition group both suppressed the expression of CD11a, but the action of biniferin was stronger. The results of three independent experiments are shown in Table 2, and the results were almost the same.

Example 3
Evaluation test of superoxide anion scavenging activity

The ability to erase various active oxygen species of resveratrol and biniferin was evaluated by electron spin resonance.

<Method>
(1). Singlet oxygen scavenging action Evaluation method of singlet oxygen scavenging action is ESR (Electron Spin Resonance) spin trap method using 2,2,6,6-tetramethyl-4-poperidinol (4-OH TEMP) as a spin trap agent. It was measured by. 0.1 mL of a 99.5% (v / v) methanol aqueous solution of the test drug prepared to an arbitrary concentration was dropped into a test tube. Subsequently, 0.02 mL of milliQ water, 0.04 mL of 100 mM 4-OH TEMP aqueous solution, and 0.04 mL of 0.1 mM Rose Bengal aqueous solution were mixed in this order to obtain a mixed solution having a total amount of 0.2 mL. This was irradiated with a light emitting diode (green LED) having a maximum wavelength of 530 nm from the back of the test tube as a light source for the photosensitization reaction for 20 minutes to generate singlet oxygen. This mixed solution was transferred to a flat aqueous solution cell for ESR, an ESR spectrum was measured, and a singlet oxygen elimination rate was obtained from the ESR spectrum intensity.
Equipment used: JEOL JES RE1X, X-band ESR equipment (100 kHz, JEOL Ltd., Akishima, Tokyo)
Condition setting: Center field: 335.0 ± 5.0 mT
Microwave: 4 mW
Modulation amplitude: 0.1 mT
Gain: 200
Time constant: 0.1 second Scanning time: 1 minute Note that the preparation method of the test drug and the equipment used and the condition setting of the equipment are the same for other activated radical elimination methods described below.

(2). Nitric oxide scavenging action
200 μM sodium 2- (4-carboxyphenyl) -4,4,5,5-tetraimidazolin-1-oxy-3-oxide (carboxy-PTIO) 20 μL and 10 mM 1-hydroxy-2-oxo-3- The NO radicals generated from the reaction mixture of (N-methyl-3-aminopropyl) -3-methyl-1-triazen (NOC-7) were converted to 0.1 M phosphate buffer (pH7.) In the presence of 30% dimethyl sulfoxide (DMSO). 4) Measured in. NOC-7, developed as a NO radical donor, is an unstable compound that is stable in alkaline solution but generates NO radicals under neutral and acidic conditions (half-life at 37 ° C, pH 7.4). 5 minutes). The ESR signal of carboxy-PTIO was composed of five peak components with an intensity ratio = 1: 2: 3: 2: 1.

(3). Hydroxyl radical scavenging action The hydroxy radical scavenging action was evaluated by the ESR spin trap method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. First, 200 μM diethylenetriamine-N, N, N ′, N ″, N ′ ″-pentaacetic acid (DTPA) 0.1 mL was added to the test tube, followed by 500 μM iron (II) sulfate aqueous solution 0.02 mL, 10 Immediately add 0.02 mL of mM 5,5-demethyl-1-pyrroline-N-oxide (DMPO) aqueous solution, and then immediately after adding 0.02 mL of 10 mM aqueous hydrogen peroxide, transfer the mixture to a flat cell for ESR measurement. ESR spectrum was measured. Hydroxyl radical scavenging rate was determined from ESR spectrum intensity, using as a control a specimen to which 0.04 mL of an aqueous solution containing the same concentration of ethanol as each test drug was added.

(4). Superoxide anion scavenging action Superoxide anion scavenging action was evaluated by the ESR spin trapping method using DMPO as a spin trapping agent. First, 0.05 mL of 2 mM hypoxanthine 0.01 mM phosphate buffer (pH 7.4) was added to the test tube. Next, 0.013 mL of 1.33 mM DTPA, 0.015 mL of 1.33 M (stock solution), and 0.1 mL of aqueous test drug solution were added, and finally 0.02 mL of 200 mU / mL xanthine oxidase 0.1 mM phosphate buffer (pH 7.4) was added. Immediately after this, the mixture was stirred for 5 seconds using a test tube mixer. This mixed solution was transferred to a flat cell for ESR, and an ESR spectrum was measured 2 minutes after addition of xanthine oxidase.

<result>
Tables 3 to 6 show the concentration-dependent erasing activity (%) and 50% erasing concentration (IC ₅₀ ) of resveratrol and vinyliferin for each reactive oxygen species. Resveratrol and vinyliferin showed similar activities in terms of singlet oxygen scavenging ability, nitric oxide scavenging ability, and hydroxy radical scavenging ability. On the other hand, the superoxide anion scavenging activity was shown to be 20 times greater than that of resveratrol.

Example 4
Preparation of ε-viniferin ε-viniferin was extracted from about 5 kg of grape side branch (variety Delaware) with 10 l of 70% methanol, and separated and purified by preparative HPLC to obtain about 200 mg of ε-biniferin. Acetonitrile / water (7:13) is used as a mobile phase during HPLC fractionation, and it is detected after about 8 minutes at a flow rate of 1 ml / min. Detection was performed with an ultraviolet absorption of 288 nm. The vinyliferin thus obtained was used in the test example of Example 1. Moreover, the residue obtained by distilling off the solvent after methanol extraction was used as biniferin in the following formulation examples as roughly purified ε-viniferin.

Formulation Example 1: Supplement (Compound Component) (mass%)
Lactose 54.0
Crystalline cellulose 35.0
Starch degradation product 10.0
ε-vineferin 1.0

Formulation Example 2: Soft capsule (combination component) (mass%)
Edible soybean oil 50.0
ε-vinephrine 1.0
Glycerin fatty acid ester 12.0
Beeswax 5.0
Gelatin 12.0
Water 20.0

Formulation Example 3: Gummy (combined component) (mass%)
Reduced water tank 44.0
Granulated sugar 20.0
Glucose 20.0
Gelatin 4.7
Water 9.6
ε-vinephrine 1.0
Flavor 0.6
Dye 0.1

Formulation example 3: Soft drink (combined component) (mass%)
Fructose dextrose liquid sugar 30.0
Grape juice 20.0
Emulsifier 0.5
ε-vinephrine 1.0
Perfume Appropriate amount Purified water Residual

Formulation example 4: Tablet confectionery (mixed component) (mass%)
76.0 sugar
Glucose 19.0
ε-viniferin 1.0
Sucrose fatty acid ester 0.2
Purified water 3.8

Formulation Example 5: Candy (combination component) (mass%)
Sugar 50.0
Minamata 33.0
Water 14.0
Organic acid 2.0
ε-viniferin 0.5
Fragrance 0.5

Claims (1)

CD36 protein expression inhibitor containing ε-viniferin as an active ingredient (excluding food forms and arteriosclerosis prevention / treatment applications).

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