JP4971597B2 - Antioxidant composition - Google Patents

Description

The present invention relates to a composition comprising a combination of specific components having antioxidant ability derived from animal natural extracts and plant natural extracts, and more specifically, it effectively resists various reactive oxygen species. it relates antioxidative set Narubutsu can exhibit oxidizing ability. The present invention eliminates oxidative stress caused by active oxygen generated in the human body and prevents lifestyle-related diseases in a broad sense, preferably, for example, for prevention of diabetes-related diseases in diabetic patients whose active oxygen in the body is significantly high. An antioxidant composition is created and provided.

  In recent years, the increase in various adult diseases accompanying the increase in the elderly population and the increase in human and economic burdens related to medical care have been taken up as extremely serious problems. Since the aging of living organisms and the occurrence of diseases are inseparable, it is inevitable that the number of sick people will increase as the number of elderly people increases. Therefore, it is important to slow down the aging rate of humans as much as possible and suppress the occurrence of diseases.

  The primary cause of human aging is the limited number of times the cells that make up the body divide and proliferate for tissue regeneration. The ability to regenerate the tissue is reduced. And aging is accelerated | stimulated by the effect | action by the factor which damages a cell, for example, irradiation of an ultraviolet-ray or a radiation, a medicine etc., or the active oxygen generated in the living body. In particular, active oxygen is inevitably generated in organisms that perform energy metabolism using oxygen, and in this sense, it can be said that our aging inevitably occurs. Therefore, as a main measure for delaying aging and suppressing the occurrence of diseases associated with aging, it is important to suppress the harmful effects of active oxygen generated in the living body.

Until now, various antioxidants have been supplied to the market, but most of them were prepared from a single component or a single raw material. However, there are various types of active oxygen generated in a living body. Specifically, for example, superoxide (O ₂ ⁻ ) and O ₂ ⁻ generated from oxygen gas taken into the body are superoxide display. Hydrogen peroxide (H ₂ O ₂ ) is produced by an enzyme called TACE (SOD), which further generates hydroxyl radicals (OH.) By the catalytic action of transition metals. In addition, leukocytes produce active oxygen in order to sterilize bacteria that have entered the living body. Among them, there are hypochlorite radicals, nitrogen oxide radicals and the like in addition to H ₂ O ₂ .

  On the other hand, various antioxidants are used to eliminate these active oxygens, but are these antioxidants effective against all these active oxygens, or are the active oxygen species that act on them limited? There hasn't been enough consideration. The present inventors examined the action of antioxidants on the protein oxidative degradation action of various active oxygens, and there are types of active oxygens that they act on, and are effective against all active oxygens in vivo. It has been clarified that there are very few antioxidants.

  That is, peptides such as anserine, carnosine, reduced glutathione (GSH), sulfur-containing amino acids, or amino acid analogs present in various animal living bodies are vitamin C (L-) against hypochlorous acid radicals (ClO.). Ascorbic acid, VC) is strong against peroxynitrite radicals (ONOO.), And vitamin E (α-tocopherol, VE) is strong against hydroxyl radicals (OH.). In order to completely remove injuries of active oxygen that occur in the living body, the combination and composition of antioxidants that use them together is effective, rather than using them alone. It was confirmed (Patent Document 1).

  Furthermore, when the antioxidant activity of the natural antioxidant component is examined in detail, the antioxidant component consisting of amino acids and peptides is similar to Anserine and Carnosine against ClO. As with E, hydrophobic carotenoids, polyphenols, caffeic acid analogues, etc., which are derived mainly from plants and have phenol groups, have been found to have strong antioxidant activity against OH.

  Until now, various antioxidants have been tested to prevent various diseases, particularly lifestyle-related diseases. For example V. C, V.D. And polyphenols such as E, β-carotene and catechin. However, looking at the test results of examining the disease prevention effect of using these antioxidants alone or in combination, there are not many findings that they were necessarily effective in preventing diseases such as cancer. Conversely, there are many findings that there is no clinical effect (Non-patent Document 1), and antioxidants have not been effective in preventing diseases and aging as expected.

  The following points can be considered as the cause. First, there are many types of active oxygen generated in the body, and it is difficult to eliminate their harmful effects with a single antioxidant, and the antioxidant activity is similar even in the combination of antioxidants Combinations of the components to be treated, such as V. A combination of E and carotenoids or polyphenols; C and V. E or V. For example, the combination of C and polyphenols was not a combination of suppressing three types of active oxygen having different properties.

  Furthermore, plant-derived antioxidants, especially polyphenols, are known not only to have antioxidant effects but also to damage human genetic DNA, and sometimes have been reported to promote carcinogenesis and mutation ( Non-patent documents 2, 3).

JP 2004-231902 A J. et al. Kaikkonen et al., Free Radicals Research, 33, 329-340, 2000 C. A. Austin, et al., Biochemistry Journal, 282, 883-889, 1992. R. Sanyal et al., Mutageness, 12, 297-303, 1997

Under such circumstances, the present inventors have found that in view of the above prior art, the goal of developing effective antioxidative set Narubutsu capable of exhibiting antioxidant properties for a variety of reactive oxygen species As a result of extensive research, vitamin C (L-ascorbic acid or L) was used as a peroxynitrite-based active oxygen scavenger per 100 m of a histidine-containing dipeptide mixture obtained from animal extracts as a hypochlorous acid-based active oxygen scavenger. - ascorbate) was 20mg or greater, and have succeeded in developing an anti-oxidative pairs Narubutsu the ubiquinones were formulated into the proportion of more than 4mg such coenzyme Q10 as hydroxyl radicals based active oxygen eliminator, the present invention It came to complete.

  An object of this invention is to provide the antioxidant composition which can exhibit antioxidant ability effectively with respect to various reactive oxygen species.

The present invention for solving the above-described problems comprises the following technical means.
(1) As a hypochlorite-based active oxygen scavenger for reactive oxygen species produced in vivo, as a peroxynitrite-based active oxygen scavenger per 100 mg of a histidine-containing dipeptide mixture obtained from animal extracts Vitamin C (L-ascorbic acid or L-ascorbate) is 20 mg or more and, as a hydroxyl radical active oxygen scavenger, ubiquinone containing coenzyme Q10 is blended in a ratio of 4 mg to 10 mg. to a radical hypochlorite, antioxidative set Narubutsu having an action to suppress the three hydroxyl radicals and peroxynitrite radicals.
(2) The above-mentioned hypochlorous acid-based active oxygen scavenger is an anserine and carnosine histidine-containing dipeptide contained in an animal extract obtained by hot water extraction of poultry, cow, pig, or migratory fish muscle. antioxidative sets composition as claimed in a mixture of the (1).

Next, the present invention will be described in more detail.
In the present invention, in order to make the above-mentioned three types of active oxygen suppressive, it is a mixture of antioxidants capable of dealing with a wide range of active oxygen species, and sufficiently copes with OH · generated in the process of energy metabolism. It is important to select materials that are safer than possible. As described above, the antioxidant peptides anserine, carnosine and sulfur-containing amino acids obtained from animal raw materials have a strong antioxidant action against ClO. C has a strong antioxidant action against ONOO. And the present inventors have already clarified as a component having a strong antioxidant action against OH. In addition to E, carotenoids, polyphenols, and caffeic acid analogs, it was discovered that there are ubiquinones present in living cells.

Coenzyme Q10 (hereinafter abbreviated as CoQ10), which is a typical component of ubiquinone, is mainly present in mitochondria that perform energy metabolism and is an important electron transport system component, but it is known to have a strong antioxidant effect. ing. For humans, CoQ10 is not derived from plants, but is a component that exists in the human body like anserine and carnosine, and does not have a DNA-damaging effect like plant-derived antioxidants. Moreover, in the mitochondria, heavy active oxygen is produced in the process of energy metabolism, and they generate OH · from O ₂ ⁻ via H ₂ O ₂ , but when examining the antioxidant effect of CoQ10 present in mitochondria This is V. It was stronger than E and had strong OH / antioxidant action equivalent to catechin and caffeic acid analogues. However, CoQ10 did not exhibit an antioxidant action against ClO. Or ONOO. At concentrations that can be physiologically present as well as plant-derived antioxidants.

As a result of searching for a material for an antioxidant with excellent safety and strong OH / erasing ability, the present inventors have found CoQ10, which is one of ubiquinones, and have a ClO / erasing ability. Strong anserine-carnosine mixture and V. By blending C and CoQ10 at a predetermined mixing ratio, and we found that it is possible to synergistic ideal antioxidant activity Preparation of Kosan of sets Narubutsu with the three active oxygen.

In the present invention, vitamin C (L-ascorbic acid or L-ascorbine is used as a peroxynitrite-based active oxygen scavenger per 100 mg of a histidine-containing dipeptide mixture obtained from animal extracts as a hypochlorous acid-based active oxygen scavenger. Acid salt) is added in an amount of 20 mg or more, and ubiquinones such as coenzyme Q10 are blended in a proportion of 4 mg or more as a hydroxyl radical active oxygen scavenger. Thus, in the present invention, it is important to blend the above components at a predetermined blending ratio. Outside these ranges, the specific antioxidant action on the three active oxygens of the present invention cannot be obtained.

In this case, as the above hypochlorous acid-based active oxygen scavenger, preferably, for example, histidine contained in an animal extract obtained by hot water extraction of muscle of poultry, cattle, pigs, or migratory fish mixture of dipeptides containing anserine and carnosine are illustrated, but can be similarly used as long as the these same effect.

So far, anti-oxidative pairs formed of 2-3 components, effective compositions against three active oxygen, and the example is not reported. This also antioxidative sets composition as the present invention, it is apparent that those achieving the no good remarkable effect in conventional products.

  Until now, foods containing a single antioxidant have been widely used for the purpose of preventing lifestyle-related diseases and aging, but have not always been sufficiently effective in clinical trials. This is because the active oxygen in the living body that promotes illness and aging is not a single thing, but there are various reactive oxygen species, while antioxidants suppress all of the various active oxygen species. Is considered to be due to the presence of reactive oxygen species that are extremely rare and unable to exert strong antioxidant effects. The inventors of the present invention have examined the action of various antioxidants by classifying the active oxygen species already generated in the living body into chlorine, hydroxyl radicals, and nitrogen oxides, and found that histidine-containing peptides present in animal extracts. And sulfur-containing amino acids or peptides against chlorinated active oxygen, polyphenols, carotenoids, caffeic acid analogues, etc. that are derived from plants and that are fat-soluble against hydroxylated radicals, and further against nitrogen oxides It has been revealed that vitamin C exhibits a strong antioxidant effect.

And it became possible to prepare the antioxidant composition which has an ideal antioxidant effect by mix | blending these appropriately. The present invention has a strong antioxidant action against hydroxyl radicals, but CoQ10 of ubiquinones, which is an antioxidant component having no DNA damaging action or mutagenicity like polyphenols, as a hydroxyl radical scavenger. by blending with other components, it is to allow for the preparation of Kosan of sets Narubutsu with the safety is high, and complete antioxidant. This onset Ming, more safety even when ingested long time is high, thus provides the antioxidative sets Narubutsu effect of suppressing aging and diseases caused by various active oxygen generated in vivo that can be expected It is possible to do that.

Next, the present invention will be described in more detail based on test examples.
(1) Inhibiting action of various antioxidants against ovalbumin (Ovalbumin, hereinafter abbreviated as Ova) degradation action by three kinds of active oxygen 3 of ClO., OH. And ONOO. As typical active oxygen produced in vivo Seeds were prepared. In the method ClO · is to dilute the sodium hypochlorite of distilled water pH 6.5, OH · the Fenton method (B.Halliwell et al, Analytical Biochemistry 165, pp. 215-219, 1987) was partially modified ONOO. Was prepared by modifying the quenching flow reactor method (R. Radi et al., Journal of Biological Chemistry 266, 4244-4250, 1991).

  Next, Ova was dissolved in buffered saline, pH 7.4 to 2.5 mg / mL, and an antioxidant of each concentration was added at a volume ratio of 10: 1 to the protein solution, and room temperature was added. The active oxygen of each concentration was added at a volume ratio of 10: 1 to the protein solution, and the ClO / oxidation was 37 ° C. for 30 minutes, the OH / oxidation was 37 ° C., 60 The ONO / oxidation was allowed to react at 37 ° C. for 120 minutes. After the reaction, each sample solution was electrophoresed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel stained with Coomassie Brilliant Blue R250 staining solution was subjected to densitometer to calculate the Ova decomposition inhibition rate. . In addition, the accurate decomposition inhibition rate was calculated by applying the reaction sample solution directly to GPC-HPLC of a TSKG-3,000SW column and calculating the proteolysis inhibition activity by the following formula.

  Decomposition inhibition rate (%) = peak area of active oxygen degrading protein when antioxidant is added / peak area of undegraded control protein × 100

  The proteolysis-inhibiting action of various antioxidants and the three compounding agents against Ova oxidative degradation by three active oxygens is as shown in FIG. For the ClO radical (ClO.), The purified anserine-carnosine mixture and sulfur-containing amino acids or peptides derived from chicken extract were the strongest, followed by vitamin C (VC), but CoQ10 was proteolytic It did not show any blocking action. For OH radicals (OH.), CoQ10 at a concentration of 50 μM exhibits the strongest proteolytic inhibitory action, and anserine-carnosine and V. C showed little antioxidant effect.

  On the other hand, for the ONOO radical (ONOO.), V.I. Although C completely prevented the proteolytic action, the proteolytic inhibitory action of chicken extract-derived anserine-carnosine, sulfur-containing amino acid-peptide and CoQ10 were all weak. Chicken extract-derived anserine-carnosine mixture 5 mM, CoQ10 50 μM, and V.I. In the case of the three mixed antioxidant compositions containing C at a concentration of 5 mM, as shown in FIG. 1, the result was that the proteolytic action by the three active oxygens was almost completely prevented. These proteolytic inhibitory activities are summarized in Table 1 as “inhibitory action (%) of each antioxidant against proteolysis of various active oxygens”.

(2) Impaired microchannel passage ability of human erythrocytes due to active oxygen and anti-acid preventive effect Microchannel (abbreviated as MC) is a device that measures blood rheology, that is, microvascular circulation ability of blood. is there. Factors affecting blood rheology include diet and oxidative stress. In the present invention, the effects of three types of active oxygen produced in vivo on the ability of human erythrocytes to pass through MC are tested, and prevention of human erythrocyte fluidity disorder effects by the active oxygen of various antioxidants and three kinds of combination agents. The effect was tested.

  In the test method, red blood cells were collected from blood of healthy volunteers using Ficoll-Paque centrifugation (Pharmacia) and treated with various active oxygens using MCFAN KH-3A (channel width: 7 μm, MC Laboratory). The MC transit time (final hematocrit concentration 10%) was measured. As a result, all three types of active oxygen, ClO., OH. And ONOO., Caused human erythrocytes to pass through. In ClO., Chicken extract-derived anserine-carnosine mixture, in OH., CoQ10, and , ONOO. C was the result which prevented the fall of MC passage ability most. In the case of the combination of the three antioxidants, no decrease in MC passage ability due to active oxygen was observed in any case (FIG. 2).

(3) Blood rheology improvement effect in healthy persons Targeting healthy males (58 years old, 35 years old, 29 years old), one bottle contains chicken extract-derived anserine-carnosine mixture 400 mg, vitamin C 300 mg and CoQ10 20 mg 50 mL of fruit juice was drunk daily for 10 days, and the blood fluidity improvement effect before and after drinking was tested by measuring the MC passage time using the above MCFAN KH-3A. As a result, the average MC passage time before drinking the three kinds of antioxidant-containing beverages was 55.1 seconds, but the average MC passage time after drinking was improved to 46.6 seconds (FIG. 3).

The following effects are exhibited by the present invention.
(1) The present invention can provide an anti-oxidative pairs Narubutsu can effectively exhibit antioxidant properties for a variety of reactive oxygen species.
(2) An anti-antigen comprising a combination of specific components having a synergistic antioxidant action on three active oxygen species, hypochlorous acid radical, hydroxyl radical and peroxynitrite radical produced in vivo. it is possible to provide an acid of sets Narubutsu.
(3) The present invention can provide an effective new anti-oxidative pairs Narubutsu the prevention and anti-aging lifestyle diseases.

  EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.

  Concentrate 1,200 L of chicken extract obtained by hot water extraction from 400 kg of whole chicken, adjust the salt concentration so that the electric conductivity is 25 mS / cm, and equilibrate it to 0.05 M citrate buffer beforehand. After passing through a column packed with the Dowex 50-X8 cation exchanger, the anserine and carnosine in the chicken extract were adsorbed to the ion exchanger, and the ion exchanger was thoroughly washed with the same buffer, and then diluted with alkaline solution. The anserine and carnosine were eluted from the ion exchange column and recovered. Concentrate the mixture of anserine and carnosine eluted from the cation exchanger under reduced pressure, adjust the pH to neutral with hydrochloric acid, add 2% (W / W) of activated carbon, decolorize, and filter with filter paper. Activated carbon was removed. The purity of the purified anserine-carnosine obtained here was about 78% per solid.

  400 mg of the chicken extract-derived anserine-carnosine mixture in 50 mL of mango juice (60%); 300 mg of C (manufactured by Wako Pure Chemical Industries) and 0.2 g of CoQ10 water-solubilized emulsion (manufactured by Yokohama Oil and Fats) (20 mg as CoQ10) were added to prepare a juice drink containing three antioxidant components. As shown in Test Example (3), the effect of improving the blood fluidity of healthy people was observed in this beverage.

  After concentrating the chicken extract obtained by extracting the whole chicken with hot water, the anserine-carnosine content per solid was adjusted to 10% and spray-dried to prepare a chicken extract powder. V. per 2.0 g of the chicken extract powder. 150 mg of C (Wako Pure Chemical Industries), 200 mg of CoQ10 powder (manufactured by Nisshin Pharma) (10 mg as CoQ10) were added, and 8 g of corn powder, 1 g of onion powder, and 1 g of seasoning were further added to prepare instant corn soup powder. By dissolving 12.6 g of the corn soup powder in 100 mL of hot water at 80 ° C., it was possible to easily provide the table as an antioxidant enhanced corn potage soup. Further, when the corn soup solution was tested for its in vitro proteolytic action by the above three active oxygens, the proteolytic action by any active oxygen was completely suppressed.

As described in detail above, the present invention according to the antioxidative group formed product, the present invention can provide an antioxidant composition acting effectively against a wide range of reactive oxygen species. Until now, foods containing a single antioxidant have been widely used for the purpose of preventing lifestyle-related diseases and aging. For example, arteriosclerosis, hypertension, cancer, various diseases associated with diabetes and diabetes. However, in the clinical trials for preventing lifestyle-related diseases and aging prevention of antioxidants that have been conducted so far, there is a reality that has not necessarily been sufficiently effective. There are many possible causes for this, but first, if the clinical effects obtained by inhalation in vitro or in animals are actually taken orally by humans, problems with intestinal absorption and biodistribution may occur. There was a problem. In particular, plant-derived polyphenols are applicable. Second, reactive oxygen in the living body that causes aging and lifestyle-related diseases is not a single one, but various types of oxygen are generated depending on human lifestyle and physiological conditions. . Conventionally, the strength of the activity of an antioxidant has been evaluated for active oxygen that is poorly related to the active oxygen generated in the living body, and the order of strength has been defined by that standard. However, as confirmed in the test of the present invention, the action of various antioxidants at physiologically adjustable concentrations has specificity for reactive oxygen species, and it has been conventionally evaluated that the antioxidant activity is weak. It has been clarified that even those that have been demonstrated to exhibit a strong antioxidant action against specific active oxygen, and conversely, even those that have been evaluated as strong antioxidants have active oxygen that has a weak action. From these facts, it is considered that foods mainly composed of a single antioxidant could not sufficiently prevent various diseases caused by various active oxygens generated in the living body.

In the present invention, the active oxygen species generated in the living body are classified into three categories, and the antioxidants for OH · system generated from oxygen gas, chlorine-based and nitrogen oxide-based active oxygen generated by white blood cells were specified. . As a result, the histidine-containing dipeptide mixture is strong against chlorinated active oxygen, ubiquinones such as coenzyme Q10 are strong against OH, and vitamin C is strong against nitrogenous active oxygen. It was clarified to show activity. From these findings, it is important to formulate antioxidants that act effectively on a wide range of reactive oxygen species, and the final glycation product in vivo caused by oxidative damage caused by the actual incorporation of antioxidants is important. The effect which suppresses generation | occurrence | production remarkably was recognized, and from this, it became possible to provide the antioxidant composition more effective than before in the suppression of the occurrence of aging and lifestyle-related diseases. Also, a more effective antioxidant composition can be produced by blending natural materials containing these antioxidant components.

The inhibitory effect of ovalbumin degradation action by active oxygen of various antioxidants is shown. A is 10 mM ClO radical, B is 10 mM OH radical, and C is oxidative decomposition of ONOO radical. In each panel, R is untreated ovalbumin, C is oxidatively decomposed ovalbumin control, 1 is a purified anserine-carnosine mixture derived from chicken extract 5 mM, 2 is vitamin C 5 mM, 3 is coenzyme Q10 50 μM, It is a blend of oxidizing agents. The prevention effect by the various antioxidant component and its compounding agent with respect to the microchannel (MC) passage ability decreasing effect of the human erythrocyte by various active oxygen is shown. Panel A is 10 mM ClO radical, Panel B is 10 mM OH radical, and Panel C is oxidative decomposition by 5 mM ONOO radical. Each panel is, in order from the left, an antioxidant-free control, a chicken extract-derived purified anserine-carnosine mixture, vitamin C, coenzyme Q10, and three types. It shows the blood fluidity improving effect of healthy people.

Claims (2)

Vitamin C as a peroxynitrite-based active oxygen scavenger per 100 mg of a histidine-containing dipeptide mixture obtained from animal extracts as a hypochlorite-based active oxygen scavenger for reactive oxygen species produced in vivo (L-ascorbic acid or L-ascorbate) is 20 mg or more, and, as a hydroxyl radical active oxygen scavenger, ubiquinones containing coenzyme Q10 are blended in a ratio of 4 mg to 10 mg, hypochlorite radical, anti-oxidation set Narubutsu having an action to suppress the three hydroxyl radicals and peroxynitrite radicals. The hypochlorous acid-based active oxygen scavenger is a mixture of an anserine and carnosine, a histidine-containing dipeptide contained in an animal extract obtained by hot-water extraction of poultry, cow, pig, or migratory fish muscle. antioxidative sets composition as claimed in certain claim 1.