JP6628071B2 - Low-density lipoprotein oxidation inhibitor - Google Patents

Description

  The present invention relates to a low-density lipoprotein (LDL) oxidation inhibitor comprising a water-soluble nucleoprotein. The water-soluble nucleoprotein has an LDL antioxidant effect and can be used as an agent for preventing / ameliorating anti-atherosclerosis.

  In recent years, there has been a consensus that arteriosclerosis is a symptom that is located downstream of metabolic syndrome, which is mainly caused by a high-fat diet and lack of exercise. It forms and causes heart disease and cerebrovascular disease.

  According to recent demographic statistics of the Ministry of Health, Labor and Welfare of Japan, malignant neoplasms (tumors and cancers) rank first in disease-related deaths in Japan, heart disease in third, pneumonia in third, and cerebrovascular disease in fourth. However, the total number of deaths due to heart disease and cerebrovascular disease is about 25% of the total, which is almost the same as the death rate of malignant neoplasms of about 29% (2013 (2013)). Regarding the method of coping with malignant neoplasms, early detection and early treatment are important in view of the onset process, and a clinical approach is considered desirable. However, heart disease and cerebrovascular disease located downstream of atherosclerosis, which causes the above-mentioned metabolic syndrome, can be prevented by daily intake of food, and a method of coping with such food intake is desirable. It is believed that.

  As mentioned above, in atherosclerosis, a food-based approach including diet is important, but it is important to determine which mechanism of action is targeted in complex and diverse processes. Atherosclerosis is triggered by the onset of LDL oxidation due to oxidative stress and vascular endothelial dysfunction due to the incorporation of oxidized LDL into vascular endothelial cells, followed by foaming due to differentiation and dedifferentiation of macrophages and vascular smooth muscle. Exhibit a phenomenon. Eventually, thrombus is formed through a stenotic lesion.

  It is known that the onset and progression of arteriosclerosis is closely related to oxidative degeneration of LDL. Dysfunction of vascular endothelial cells, which is considered to be one of the early causes of atherosclerosis, is due to LOX-1 (Lectin-like oxidized low lipoprotein receptor-1), which is an oxidized LDL receptor. There is a report that it depends on being taken up into vascular endothelial cells via -density lipoprotein receptor-1) (see Non-Patent Document 1).

  From a preventive point of view, it is important to suppress LDL oxidation due to oxidative stress or the like, which is an initial lesion, or to suppress dysfunction due to incorporation of oxidized LDL into vascular endothelial cells.

  Materials having an LDL oxidation inhibitory effect include dihydrolipoic acid (see Non-Patent Document 2), proanthocyanidin (see Non-Patent Document 3) which is a main component of grape seed extract, vitamin E (see Non-Patent Document 4), lycopene and Vitamin E (see Patent Document 1), astaxanthin (see Patent Document 2), 2 ″ -O-glucosylisovitexin (see Patent Document 3), lactic acid bacteria or fermented milk obtained by fermenting a milk medium with the lactic acid bacteria (Patent Document 4) ), An algal body of a single-cell green algae or an extract thereof (see Patent Document 5), a compound having a chemical structure similar to shogaol and gingerol (see Patent Documents 6 and 7), and vitamin C and cocoa extract components (Patent Document 8) Combined use of Rabo-hima extract and vitamin C (see Patent Document 9), myricytosine (see Patent Document 10), α-li Acid, vitamin E, grape seed extract-containing material (Patent Document 11 reference) are known.

  On the other hand, regarding water-soluble nucleoprotein obtained from milt of fish, it has been reported that the water-soluble nucleoprotein has an effect of significantly reducing oxidative damage of genes (see Patent Document 12). There is no known effect of suppressing the effect.

JP-T-2002-504928 JP-A-10-155449 JP-A-9-67255 JP 2001-302523 A JP-A-2002-114703 JP 2005-272384A JP 2005-306829 A JP-A-2006-87352 JP 2006-160667 A JP 2006-219389 A JP 2006-306820 A JP 2003-325149 A

Sawamura, T. et al., Nature, 6; 386 (6620), 73-7, 1997. Lodge, J.K. et al., Free Radic. Biol. Med., 25 (3), 287-97 1998. Yamakoshi, Y. et.al., Atherosclerosis, 142 (1), 139-49, 1999. Toshima, A. et al., Arterioscler. Thromb. Vasc. Biol., 20 (10), 2243-7, 2000.

  In order to prevent arteriosclerosis, it is considered that a sufficient effect can be exerted by daily and continuous ingestion of a material having an LDL oxidation inhibitory effect. Therefore, the problem to be solved by the present invention is to provide a medicine or a drug which is safe even if taken for a long period of time, can effectively prevent the oxidation of LDL, and has an effect of preventing and improving anti-atherosclerosis. It is to provide an LDL oxidation inhibitor in the form of a functional food.

As a result of intensive studies on the above problems, the present inventors have found that a water-soluble nucleoprotein which is safe even if taken for a long period of time has an effect of suppressing the oxidation of LDL, and completed the present invention. That is, the present invention is as follows.
(1) A low-density lipoprotein (LDL) oxidation inhibitor comprising a water-soluble nucleoprotein as an active ingredient.
(2) The LDL oxidation inhibitor according to the above (1), wherein the water-soluble nucleoprotein is a water-soluble nucleoprotein derived from milt of fish.
(3) The LDL oxidation according to (1) or (2), wherein the water-soluble nucleoprotein is a low-molecular-weight water-soluble nucleoprotein containing 50 to 100% of a fraction having a molecular weight of 3000 or less. Inhibitors.
(4) The LDL oxidation inhibitor according to any one of the above (1) to (3), which is in the form of a functional food.

  ADVANTAGE OF THE INVENTION According to this invention, oxidation suppression of LDL in a living body, and prevention and treatment of atherosclerosis can be implemented efficiently, without accompanying a side effect. Since the water-soluble nucleoprotein used in the present invention can be freely prepared in the form of a functional food or a medicament without any problem in safety, it can be prepared not only from healthy subjects, but also from the elderly, the sick, and post-sick. Etc. can be taken over a long period of time.

It is a figure which shows the oxidation state (potential change) of LDL measured by the carbon nanotube (CNT) electrode method. (A) shows the potential change of native-LDL (unoxidized LDL) before oxidation, (B) shows the potential change when oxidation treatment is performed in the presence of native-LDL and water-soluble nucleoprotein, (C) Indicates potential changes when native-LDL is oxidized in the absence of a water-soluble nucleoprotein. It is a figure which shows the result of having measured the progress state of the oxidation of native-LDL when the addition density | concentration of the water-soluble nucleoprotein was changed and coexisted with native-LDL by the TBARS method and the CNT electrode method. The oxidation state when no water-soluble nucleoprotein was added is shown as 100%. Open circles show the results of the TBARS method, and black circles show the results of the CNT electrode method.

  Hereinafter, embodiments of the present invention will be described in detail. However, the following description is an example of the embodiments of the present invention, and the present invention is not limited to the following description. It should be noted that, when the expression “to” is used in this specification, it is used as an expression including numerical values or property values before and after the expression.

  The LDL oxidation inhibitor of the present invention is not particularly limited as long as it is a preparation in the form of a medicament or a functional food containing a water-soluble nucleoprotein as an active ingredient. The LDL oxidation inhibitor of the present invention includes atherosclerosis. It is useful as a preventive / ameliorating agent. Here, the formulation refers to tablets, capsules, granules, drinks, and other forms different from ordinary foods, and functional foods refer to foods that emphasize the function of regulating body tone. In addition, foods contain a small amount of various ingredients that help prevent disease and prevent aging, and have a function to condition the body that has been developed so that they can be extracted and consumed effectively. Foods that emphasize nutrition, nutritional foods, health foods, and supplements are also included in functional foods.

  The water-soluble nucleoprotein used in the present invention can surely suppress the oxidation of LDL as specifically shown in Examples described later. Therefore, by using this water-soluble nucleoprotein, it is an early lesion of atherosclerosis, which is caused by oxidization of LDL by oxidative stress or the like, and vascular endothelial dysfunction due to incorporation of oxidized LDL into vascular endothelial cells. Oxidation of LDL can be suppressed. Further, the present invention can be applied to prevention and treatment of atherosclerosis and other diseases caused by oxidation of LDL. In addition, the present invention can be applied to a method of suppressing oxidation of LDL by ingesting a water-soluble nucleoprotein (however, excluding medical practice for humans).

  In the present invention, the water-soluble nucleoprotein is preferably prepared from fish milt (testis). The fish is not particularly limited, but is preferably a fish selected from the group consisting of salmon, trout, herring, and cod. Among them, salmon milt is preferable from the viewpoint of obtaining raw materials.

  The water-soluble nucleoprotein used in the present invention can be prepared, for example, according to the methods described in JP-A-2003-325149 and JP-A-2004-16143. In addition, a commercially available water-soluble nucleoprotein can also be used.

  The water-soluble nucleoprotein is, for example, after removing skins, muscles, blood vessels and the like from the milt of the fish as required, and further purifying as necessary to remove the oil, and then performing a treatment with a protease and a nuclease. Can be prepared. There are no particular restrictions on the nature of the protease used, but serine proteases such as trypsin are preferred. Serine proteases are suitable for hydrolyzing arginine-rich protamine because they selectively hydrolyze peptide bonds on the carboxyl side of arginine and lysine. The nature of the nuclease to be used is not particularly limited. For example, a nuclease that cleaves while leaving a phosphate group at 5 'is preferable, and preferably has a certain degree of thermostability. As these proteases and nucleases, commercially available products may be appropriately selected and used. These enzyme-treated products can be used as the water-soluble nucleoprotein in the present invention as it is, or in the form of a powder by spray drying or the like, if necessary. If necessary, it can be used after further purification.

  The molecular weight and the like of the water-soluble nucleoprotein are not particularly limited, but those having a low molecular weight containing a fraction having a molecular weight of 5,000 or less, preferably 3000 or less, up to about 50 to 100% are preferable. The molecular weight distribution can be measured by gel permeation chromatography (GPC), by quantifying the sample with a UV detector after fractionating the sample based on the molecular weight.

  The functional food used for suppressing LDL oxidation in the present invention is a functional food to which the use of LDL oxidation suppression is applied. Examples of the form of the functional food include powders, capsules, tablets, and granules. In addition to powders, ordinary food forms such as beverages and foods can be employed. Further, as a preparation in the form of a functional food, a form of a preparation such as a capsule, a tablet, a granule, a drink, and the like, whose intake amount is easily adjusted, can be suitably exemplified.

  Further, the dosage form of the LDL oxidation inhibitor can be produced by a conventionally known ordinary preparation method. For example, it can be molded by mixing with carriers, excipients, flavors, emulsifiers, preservatives, dispersants and the like that are permitted in the production of the preparation. Further, the LDL oxidation inhibitor of the present invention includes, in addition to crude drugs, vitamins and minerals, lactose, starch, cellulose, maltitol, dextrin and other excipients, glycerin fatty acid esters, as long as the effects of the present invention are not impaired. , Surfactants such as sucrose fatty acid esters, coating agents such as gelatin, pullulan, shellac, zein, oils and fats such as wheat germ oil, rice germ oil and safflower oil, waxes such as beeswax, rice bran wax, carnauba wax, Sweeteners such as sucrose, glucose, fructose, stevia, saccharin, and sucralose, and acidulants such as citric acid, malic acid, gluconic acid, and the like can be appropriately added. Examples of crude drugs include ginseng, American ginseng, seven ginseng, reishi, propolis, agaricus, blueberries, ginkgo biloba and extracts thereof. Examples of vitamins include oil-soluble vitamins such as vitamins D and K, and water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid, and biotin.

  As described above, various dosage forms can be prepared using the water-soluble nucleoprotein. That is, according to the present invention, use of a water-soluble nucleoprotein in the production of a functional food used for suppressing LDL oxidation and use of a water-soluble nucleoprotein in the production of an LDL oxidation inhibitor are also provided.

  An LDL oxidation inhibitor containing the water-soluble nucleoprotein of the present invention as an active ingredient, a prophylactic / ameliorating agent for atherosclerosis using the LDL oxidation inhibitor, or a prophylactic / ameliorating agent for inhibiting LDL oxidation or for atherosclerosis The amount of functional food to be taken depends on age, therapeutic effect, disease state, etc., and is not particularly limited, but is usually 20 mg or more, preferably 50 mg or more, more preferably 100 mg or more per day in terms of dry weight of water-soluble nucleoprotein. In addition, it can be adjusted in the range of usually 2000 mg or less, preferably 1500 mg or less, more preferably 1000 mg or less.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.

(1) Preparation of Serum Two volunteers (one male and one female in their 20s) collected blood on an empty stomach using plain blood collection tubes. After blood collection, it was left at room temperature for 1 hour. Centrifugation was performed at 3,500 rpm for 10 minutes to obtain serum.

(2) Separation of lipoprotein by ultracentrifugation The LDL fraction was separated from serum by ultracentrifugation by the following method. The ultracentrifuge used was Himac CP60E ultracentrifuge (Hitachi), and the rotor used was RPV-50T rotor (Hitachi).
d = 1.006 kg / L solution (0.20 mol / L NaCl, 0.27 mmol / L EDTA-2Na (pH 7.4), 1 mmol / L NaOH) and potassium bromide (KBr, manufactured by Kanto Chemical Co., Ltd.) , D = 1.023 kg / L and 1.160 kg / L, respectively.
The serum was mixed with a solution of d = 1.023 kg / L to adjust d = 1.019 kg / L, filled in an ultracentrifuge tube (manufactured by Hitachi, Ltd.), and then subjected to 40,000 rpm for 20 hours at 4 ° C. And centrifuged. After completion of the centrifugation, the upper layer of CM-IDL (chylomicron-intermediate density lipoprotein) fraction (d <1.019 kg / L) was removed.

  The lower layer was adjusted to d = 1.63 kg / L with a d = 1.160 kg / L solution, filled in a tube, and centrifuged at 50,000 rpm for 18 hours at 4 ° C. After the completion of the centrifugation, the upper layer LDL fraction (d = 1.019-1.063 kg / L) was collected. The collected LDL was dispensed into an Eppendorf tube, degassed at 4 ° C. for 5 minutes, sealed with Ar gas, and stored at 4 ° C. protected from light.

(3) Preparation of native-LDL EDTA was removed from the collected EDTA-containing LDL using an ultrafiltration filter having a molecular weight of 100,000 filled with 1 × Dulbecco's Phosphate-Buffered Salines (DPBS) and allowed to stand for 5 minutes. -LDL (n-LDL: unoxidized LDL) was prepared. After each measurement, EDTA-containing LDL was washed 6 times with DPBS in the same manner to remove EDTA before use.

(4) Protein quantification of native-LDL The protein concentration of n-LDL was measured by the modified Lowry method described in detail below.
Solution A (2% sodium carbonate, 0.4% sodium hydroxide, 0.16% tartrate, 1% aqueous sodium dodecyl sulfate (SDS)) and solution B (4% aqueous copper sulfate solution) at a ratio of 100: 1. 3 ml of the mixed solution was added to 1 ml of n-LDL or a standard solution (bovine serum albumin, BSA) and incubated at room temperature for 30 minutes.
Next, a solution obtained by mixing equal amounts of a phenol reagent (Folin &Ciocalteu's reagent, manufactured by Wako Pure Chemical Industries, Ltd.) and deionized water was added to each sample and standard solution while vigorously stirring, and the mixture was incubated at room temperature for 45 minutes.
The absorbance (660 nm) was measured with a spectrophotometer, and the protein concentration of n-LDL was calculated from the standard curve drawn by plotting the concentration of the standard solution (BSA) and the absorbance.

(5) Oxidation of native-LDL Copper sulfate was added to n-LDL having an apoprotein concentration of 0.36 mg / mL to a concentration of 0.8 μM (final concentration), followed by incubation at 37 ° C. for 1 hour. -LDL was oxidized to produce oxidized LDL (ox-LDL).

(6) Suppression of native-LDL oxidation by water-soluble nucleoprotein Suppression of oxidation of n-LDL by water-soluble nucleoprotein was confirmed by the following method.
A water-soluble nucleoprotein (manufactured by Nissei Bio Co., Ltd.) dissolved in PBS so as to have a concentration of 0.5 mg / mL and n-LDL having an apoprotein concentration of 0.36 mg / mL are mixed in advance, and then mixed at 37 ° C. Incubated for hours. After keeping the temperature, n-LDL was oxidized by adding copper sulfate. The produced ox-LDL and the water-soluble nucleoprotein were separated using an ultrafiltration filter, and the amount of ox-LDL was measured by the following method.
By comparing with the amount of ox-LDL in the case where no water-soluble nucleoprotein was added, the effect of suppressing the oxidation of n-LDL by the water-soluble nucleoprotein was confirmed.

(7) Preparation of Carbon Nanotube (CNT) Electrode and Measurement of ox-LDL Amount by CNT Electrode Method The preparation of the electrode substrate and the CNT solution to be added thereon is described in Sensors and Actuators B: Chemical 166-167, 833-836, Prepared according to the method described in 2012. A CNT electrode prepared on a glass substrate was used as a working electrode, and a silver-silver chloride electrode was used as a reference electrode. The reference electrode was positioned right above the CNT electrode, 30 μL of DPBS was injected so as to be sandwiched between the two, and equilibration was performed for 10 minutes to stabilize the electrode. 7 μL of DPBS on the CNT electrode was removed, 7 μL of DPBS was newly injected, the measurement was performed for 200 seconds, and the baseline was measured. Similarly, 7 μL was removed, 7 μL of a water-soluble nucleoprotein was injected, and the mixture was stirred by pipetting. Water soluble nucleoprotein was measured for 500 seconds after injection.

  In the analysis, the measurement time (s) was plotted on the horizontal axis and the potential (mV) was plotted on the vertical axis, and an approximate curve of the baseline was created. The difference between the measured potential 500 seconds after the start of substitution with ox-LDL and the potential on the approximate curve at that time was determined, and the value was defined as the potential difference due to ox-LDL. It is known that the potential change increases as the oxidation proceeds.

  FIG. 1 shows an example of a potential change curve due to oxidation of n-LDL in the presence and absence of n-LDL and a water-soluble nucleoprotein. The potential change of LDL before oxidation is represented by the potential change (A), the potential change of LDL in the presence of the water-soluble nucleoprotein is represented by the potential change (B), and the potential change of LDL in the absence of the water-soluble nucleoprotein is represented by the potential It was shown as the change (C).

  A large potential change was observed in the potential change (C) in the absence of a water-soluble nucleoprotein (in the absence of an antioxidant). On the other hand, the potential change (B) in the presence of the water-soluble nucleoprotein (in the presence of an antioxidant) was smaller than (C), confirming that the oxidation of n-LDL was suppressed. The potential difference BA divided by the potential difference CA and multiplied by 100 was defined as the degree of LDL oxidation progress (%).

Measurement of ox-LDL by TBARS method The amount of lipid peroxide in ox-LDL was measured as MDA (malondialdehyde) concentration using a commercially available TBARS measurement kit (manufactured by Cayman Chemical Co.) as follows.
A reaction reagent was prepared by dissolving thiobarbituric acid in an equal volume mixture of acetic acid and an aqueous solution of sodium hydroxide such that the concentration of thiobarbituric acid became 36.8 mM. 25 μL of ox-LDL or MDA standard was mixed with 25 μL of SDS and 500 μL of reaction reagent. The mixture was incubated at 90 ° C. for 1 hour, and then cooled on ice for 10 minutes. The fluorescence intensity of the generated red substance was measured with a spectrofluorometer (FP-6500; manufactured by Jasco Corp.) (excitation wavelength: 530 nm, measurement wavelength: 550 nm). TBARS of ox-LDL obtained in the absence of a water-soluble nucleoprotein was divided by the value of TBARS of ox-LDL obtained in the presence of a water-soluble nucleoprotein, multiplied by 100, and the degree of oxidation of LDL by the TBARS method was calculated. Was calculated. Water-soluble nucleoprotein was added to LDL in the range of 0 to 0.5 mg / mL.

  The degree of oxidation progression of LDL was also evaluated by the method for evaluating the ability of human LDL to inhibit oxidation using the CNT electrode described in Example 1. FIG. 2 shows the results. In both the TBARS method and the CNT electrode method, the degree of oxidation progression was reduced in a concentration-dependent manner of the water-soluble nucleoprotein, suggesting that LDL oxidation was suppressed. At a sample concentration of 0.08 mg / mL, it was confirmed that the degree of oxidation progress was about 50%.

  The low-density lipoprotein (LDL) oxidation inhibitor containing the water-soluble nucleoprotein of the present invention as an active ingredient is useful in the field of pharmaceuticals and functional foods (supplements) as a preventive or ameliorating agent for atherosclerosis. It is.

Claims (2)

A water-soluble nucleoprotein containing a water-soluble nucleoprotein as an active ingredient , wherein the water-soluble nucleoprotein is a hydrolyzate of a milt of fish, and contains 50 to 100% of a fraction having a molecular weight of 5,000 or less. low density lipoprotein (LDL) oxidation inhibitor, characterized in that it. The LDL oxidation inhibitor according to claim 1, which is in the form of a functional food.