JP5155609B2 - Antioxidants and blood pressure improvers - Google Patents

Description

  The present invention relates to an antioxidant, an oxidative stress improving agent, and a blood pressure improving agent that suppress active oxygen.

In modern people, lifestyle habits (eating habits, exercise habits, rest, smoking, drinking, etc.) are often disturbed. (Excluding familial), hyperuricemia, cardiovascular disease (excluding congenital), colon cancer (excluding familial), hypertension, squamous cell lung cancer, chronic bronchitis, emphysema, alcoholic liver disorder or It is thought that periodontal disease and the like develop and progress, and these disease groups are collectively called lifestyle-related diseases.
Of these disease groups, cancer, heart disease, and cerebrovascular disease are the three leading causes of death among Japanese people, accounting for 60% of all deaths. And it is said that the active oxygen produced in the body is deeply involved as a cause of various pathological conditions including life-style related diseases such as hypertension, arteriosclerosis, diabetes and cancer (Non-patent Document 1).

  Where appropriate, the active oxygen is used to maintain homeostasis such as biological regulation, antibacterial activity, and antiviral activity, but when production is excessive due to stress, ultraviolet rays, etc., nucleic acid degradation, protein denaturation, lipid Causes peroxidation, etc., damages cells and contributes to the induction of various pathological conditions. Suppressing excessive active oxygen production in the body is thought to lead to the prevention, improvement and treatment of symptoms and diseases induced by excessive production of active oxygen, such as lifestyle-related diseases. Research and development on substances to be suppressed is underway.

By the way, liquor is a beverage mainly composed of water and alcohol, but since it contains components useful for health, it is considered to be a hundred medicines and moderate drinking is considered to be good for health.
Japanese sake is produced by squeezing moromi brewed and fermented using koji molds, but in the process of squeezing, a by-product called sake lees (also called sake squeezed lees) is produced. Sake lees are sometimes used as raw materials for producing shochu shochu, Nara-zuke, koji vinegar, etc., but most of them are discarded without being effectively used. Therefore, the effective use is demanded.

Under such circumstances, the anti-obesity action of Amazake, the blood pressure lowering action and the amnesia inhibiting action (Patent Document 1), the superoxide anion scavenging action of the extract with alcoholic solvents such as ethanol of sake lees (Patent Document 2), Reactive oxygen removal action of water or ethanol extract of sake lees or rice shochu residue (Patent Document 3), NK cell activity promotion of water extract of sake lees, insulin-like action, physiological activity action of amylase inhibition and toxohormone-L inhibition (Patent document 4), antihyperlipidemic action of protein extracted from sake lees with water (non-patent document 2), angiotensin converting enzyme inhibitory action of peptides isolated from liquor digested with protein enzyme (patent document) 5 and Non-Patent Document 3), and antihypertensive action by this (Non-Patent Documents 4 and 5) are reported.
However, it is not known that the hydrophobic organic solvent extract of sake lees has an antioxidant effect and a blood pressure lowering effect.
JP 2004-261119 A JP 2002-284632 A JP-A-5-310590 JP-A-10-146166 JP-A-5-294844 Itakura Hiroshige, Eiyo-Hyouka To Tiryo. Vol. 19, No. 3, 293-298, 2002 Tsutsui N, Yamamoto Y, Iwami K., J Nutr Sci Vitaminol. 1998 Feb; 44 (1): 177-86. Vol. 66, No. 7, 1081-1087, 1992 Saito Yoshiyuki, Nakamura Kyoko, Kawato Shogo, Imano Nada, Nippon Nogeikagaku Kaishi. Saito Y, Wanezaki K, Kawato A, Imayasu S. Biosci Biotechnol Biochem. 1994 58 (10): 1767-71 Saito Y, Wanezaki K, Kawato A, Imayasu S. Biosci Biotechnol Biochem. 1994 58 (5): 812-816

  The present invention relates to providing an antioxidant and an oxidative stress ameliorating agent that suppresses active oxygen produced excessively in the body or a hypertension improving agent that improves hypertension.

  When the present inventors searched for a substance having an active oxygen inhibitory action or a blood pressure lowering action, the hydrophobic organic solvent extract of sake lees surprisingly showed an excellent active oxygen inhibitory action and an antihypertensive action, It has been found that it is effective as a pharmaceutical, food, etc. that exhibits an antioxidant effect or an antihypertensive effect.

That is, the present invention provides the following inventions.
(1) An antioxidant comprising a hydrophobic organic solvent extract of sake lees as an active ingredient.
(2) An oxidative stress ameliorating agent comprising a hydrophobic organic solvent extract of sake lees as an active ingredient.
(3) An antihypertensive agent comprising a hydrophobic organic solvent extract of sake lees as an active ingredient.
(4) A food for improving oxidative stress containing a hydrophobic organic solvent extract of sake lees.

  The antioxidant, oxidative stress ameliorating agent or hypertension ameliorating agent of the present invention can suppress excessive active oxygen in the body, or can lower blood pressure, hypertension, arteriosclerosis, diabetes, hyperlipidemia. It is useful as a pharmaceutical or food that exhibits an effect of preventing, treating and / or improving lifestyle-related diseases such as cancer.

  Sake (sake) is generally made from steamed rice and rice bran by adding water and yeast to fermented sake moromi, and after aging, squeezed to obtain a clarified sake, which is further filtered and fired, Manufactured. The sake (sake) mentioned here is generally classified into ordinary sake and specially named sake (honjozo, ginjo sake, junmai sake, etc.) depending on the raw materials and production method, but includes all of these. The sake lees used in the present invention are by-products that remain when squeezed to obtain a clarified liquor, and are also called squeezed lees and residues.

  The sake lees extract of the present invention can be obtained by extracting the sake lees with a hydrophobic organic solvent at room temperature or under heating.

The extraction method may be any known method such as liquid-liquid separation, solid-liquid separation, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, and the like.
Further, during extraction, the sake lees may be appropriately prepared in the form of a suspension (water and / or water-soluble organic solvent), a paste, a concentrated dry solid, a dry powder or the like.

  Examples of the hydrophobic organic solvent include chloroform, dichloromethane, diethyl ether, petroleum ether, benzene, cyclohexane, n-hexane, and the like. Two or more types may be combined, and n-hexane is preferable.

  1/2 to 10 parts by weight of hexane is used with respect to 1 part by weight of sake lees, at a temperature of 0 to 40 ° C., preferably 15 to 25 ° C., for 1/10 hours to 12 hours, particularly 1/2 to 5 hours. It is preferable to extract.

  The obtained hydrophobic organic solvent extract of sake lees may be appropriately adjusted to the state of an extract, a diluted solution, a concentrated solution, a dried product, or the like. Further, inert impurities may be appropriately removed and further purified by a known separation / purification technique such as liquid-liquid separation, solid-liquid separation, filtration membrane, activated carbon, adsorption resin, ion exchange resin, or the like. .

As shown in the examples below, the hexane extract of sake lees showed excellent active oxygen suppression effect on leukocytes and blood pressure lowering effect in spontaneously hypertensive model rats. It can be used as an oxidizing agent, an oxidative stress improving agent, or a hypertension improving agent (hereinafter referred to as an antioxidant or the like), and can be used for the production of an antioxidant or the like. That is, the antioxidant of the present invention is a quasi-drug or pharmaceutical product that exhibits the effect of preventing, ameliorating or treating lifestyle-related diseases such as hypertension, arteriosclerosis, diabetes and cancer caused by active oxygen in vivo. Can be used as food, etc.
“Oxidative stress” refers to a state unfavorable for a living body in which an active oxygen producing system and an erasing system in the living body are out of balance and excessive active oxygen is produced. Thus, “improving oxidative stress” means returning this balance to normal. Active oxygen that has been produced excessively in the body causes protein oxidation, lipid oxidation, nucleic acid degradation, and the like, damages cells, causes dysfunction, and affects the progression of pathological conditions. Currently, diseases induced or promoted by excessive production of active oxygen include, for example, cardiovascular disease, cranial nervous system disease, digestive system disease, kidney disease, respiratory system disease, metabolic / endocrine disease, allergic disease, ocular disease And aging / senile diseases.
Here, foods include foods and drinks, functional foods, foods for the sick, and foods for specified health use that are based on the concept of antioxidants, prevention and improvement of lifestyle-related diseases, in addition to general foods and drinks. These foods are useful for bed rest persons, life style disease reserves such as hypertension, arteriosclerosis, diabetes and cancer (groups that have not yet reached life style disease but are close to that state (border zone)).

Examples of the form when the antioxidant of the present invention is used as a pharmaceutical include oral administration such as tablets, capsules, granules, powders, and syrups, or injections, suppositories, inhalants, and transdermal absorption agents. And parenteral administration by external preparations. Moreover, in order to prepare each formulation of such various dosage forms, the hydrophobic organic solvent extract of the sake lees of the present invention alone or other pharmaceutically acceptable excipients, binders, extenders , Disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, flavoring agents, fragrances, coating agents, carriers, diluents, colorants and the like can be used in appropriate combinations.
The total amount of antioxidants and the like in the above-mentioned preparation of the present invention is usually 0.0001 to 20% by mass, particularly preferably 0.0002 to 5% by mass of the total composition as a dried product.

  Examples of the form of using the antioxidant of the present invention as food include breads, cakes, noodles, cooked rice, soups, confectionery (various snacks, baked confectionery, fried confectionery, chocolate, gum , Rice cakes, etc.), jellys, frozen foods, dairy products, beverages, etc., oils and fats such as triglycerides and diglycerides, edible oils such as soybeans and rapeseed oil, fats such as butter and margarine Various types of foods, processed oils and fats such as shortenings and peanut butter, water-in-oil foods such as margarines and spreads, oil-in-water types such as ice creams, dressings, toppings and mayonnaises In addition to foods, the same forms (tablets, capsule preparations, syrups, etc.) as the above-mentioned oral administration preparations can be mentioned. Moreover, you may use the antioxidant etc. of this invention as pet food.

  To prepare various forms of food, the hydrophobic organic solvent extract of the sake lees of the present invention alone or other food materials, solvents, softeners, fats and oils, emulsifiers, preservatives, fragrances, stabilizers , Colorants, antioxidants, humectants, thickeners, and the like can be used in appropriate combinations. When using the food or pet food, the content of the extract of the present invention is usually preferably 0.0001 to 20% by mass, particularly preferably 0.0002 to 5% by mass of the total composition.

  The dose (effective intake amount) of the hydrophobic organic solvent extract of the sake lees according to the present invention is preferably 60 to 60000 mg / 60 kg body weight, more preferably 600 to 6000 mg / 60 kg body weight per day.

Example 1 (Sample preparation from sake lees)
A sake hexane extract (14.3 mg) was obtained by concentrating the hexane phase obtained by adding 25 mL of hexane to 25 g of wet weight of sake lees (Kobayashi Shuzo), stirring and filtering.
Similarly, sake water extract (889.0 mg), sake 50% ethanol extract (646.3 mg), sake 100% ethanol extract (283.1 mg) were obtained with water, 50% ethanol aqueous solution and 100% ethanol. .

Example 2 (Active oxygen suppression effect of sake lees extract)
The animals used were SD rats (10-16 weeks old, male). Carotid artery blood was collected under the anesthesia of Foren (Abbott Japan). This blood sample was overlaid with a blood cell separation reagent (SIGMA) and centrifuged, and then the leukocyte fraction was collected. Each extract (10 μg / mL) PBS solution was added to the collected leukocytes and reacted at room temperature for 1 hour, and then fluorescent reagent 10 μM 5,6-CM-H ₂ DCFDA (Invitrogen) was added at room temperature for 10 minutes (10%). v / v) Fixing reagent (BECKMAN COULTER) was added at room temperature for 20 minutes, and intracellular active oxygen was measured by Flow cytometry (Becton Dickinson).

As shown in FIG. 1, when the control was taken as 100%, the water extract, 50% ethanol extract, and 100% ethanol extract of sake lees did not suppress active oxygen, but the hexane extract showed 73. 2% significantly suppressed.
Therefore, as shown in FIG. 2, the amount of active oxygen at the concentration of sake hexane extract 0.1 μg / mL, 1 μg / mL, and 10 μg / mL was further measured.
The active oxygen was 21.5% at 0.1 μg / mL, 45.6% at 1 μg / mL, and 75.8 at 10 μg / mL depending on the concentration, depending on the concentration of sake hexane extract compared to the control. % Was significantly suppressed.

Example 3 (In vivo active oxygen suppression effect by single administration of sake hexane extract)
To SHR rats (10 weeks old, male: n = 3), a sake hexane extract solution was administered once at a dose of 100 mg / Kg. Blood samples were collected before administration and 60 minutes and 120 minutes after administration, and the amount of active oxygen produced from leukocytes was measured according to Example 2.
SHR rats are known to produce more active oxygen than normal rats.
The sake lees hexane extract solution is obtained by suspending sake lees hexane extract at 10 mg / mL in methylcellulose.
As shown in FIG. 3, the amount of active oxygen in leukocytes was significantly reduced in the sake hexane extract administration group as compared to the control group. Active oxygen was suppressed by 23.6% 60 minutes after administration and 33.0% by 120 minutes.

Example 4 (Reactive oxygen suppression effect by long-term continuous administration of sake hexane extract)
SHR rats (10 weeks old, male: n = 3) were administered with sake hexane extract solution at 10 mg / Kg and 100 mg / Kg, and this was administered once a day for 7 consecutive days. Blood samples were collected before administration and 4 days and 7 days after administration to fractionate white blood cells, and the amount of active oxygen was measured in the same manner as in Example 3. Blood was collected 12 hours after the previous day's administration. SHR rats are known to have more active oxygen in vivo than normal rats.
The sake lees hexane extract solution is obtained by suspending sake lees hexane extract in methylcellulose at 1 mg / mL and 10 mg / mL.

  As shown in FIG. 4, as a result of measuring the amount of active oxygen when the sake hexane extract was administered and when not administered, the sake hexane extract showed an active oxygen concentration of 10 mg on the fourth day compared to the control. 22.4% in the / Kg administration group, 18.1% in the 100 mg / Kg administration group, and 26.2% in the 100 mg / Kg administration group on the seventh day.

Example 5 (Blood pressure lowering effect by single administration of sake hexane extract)
SHR rats (9 weeks old, male: n = 6) were given a single dose of sake hexane extract solution at a dose of 100 mg / Kg. Blood pressure was measured with a non-invasive blood pressure measuring device for rats (manufactured by Softron) at 1 hour, 3 hours, 6 hours, and 24 hours after administration. SHR rats are spontaneously hypertensive model rats.
As shown in FIG. 5, the blood pressure of rats administered with sake hexane extract significantly decreased 1, 3, and 6 hours after administration compared to controls. The changes in blood pressure at each time are shown in Table 1.
The sake lees hexane extract solution is obtained by suspending sake lees hexane extract at 10 mg / mL in methylcellulose.

The active oxygen inhibitory effect in each extract (water, 50% ethanol water, ethanol, n-hexane) of sake lees is shown (control: no extract added). The active oxygen inhibitory effect of the hexane extract of sake lees at each concentration (0.1, 1, 10 μg / mL) is shown (control: no extract added) The amount of active oxygen which white blood cells produce after one administration of hexane extract in a SHR rat is shown. (Control: no extract added). The amount of reactive oxygen produced by leukocytes after continuous administration of hexane extract once a day for 7 days in SHR rats is shown. (Control: no extract added). The blood pressure value after one administration of the hexane extract in a SHR rat is shown. (Control: no extract added).

Claims (2)

An active oxygen inhibitor containing a hexane extract of sake lees as an active ingredient. Antihypertensive agent containing hexane extract of sake lees as an active ingredient.