JP7333968B2 - lipid peroxidation inhibitor - Google Patents

Description

The present invention relates to a lipid peroxide production inhibitor and a preventive or therapeutic agent for diseases associated with lipid peroxide, which contain orotic acid as an active ingredient, and furthermore, reduce heat stress by suppressing the production of lipid peroxide due to heat stress. Regarding agents.

Orotic acid (also called orotic acid, uracil 6-carboxylic acid, orotic acid, or vitamin B13) is a major intermediate in the pyrimidine nucleotide biosynthesis system and is derived from dihydroorotate by dihydroorotate dehydrogenase to form phosphoribosyl orotate. It becomes orotidyl acid by transferase (PRPP). Orotidylic acid is further rapidly converted to uridine monophosphate (UMP), after which pyrimidine nucleotides such as uridine triphosphate and cytidine triphosphate are synthesized.

In recent years, studies on the physiological effects of orotic acid have been progressing. For example, orotic acid has a blood uric acid level lowering effect (see Patent Document 1), orotic acid has an endurance improving effect (see Patent Documents 2 and 3), orotic acid has oxygen consumption and energy consumption. (see Patent Document 3), orotic acid activates the sympathetic nerve, improves drowsiness, raises body temperature, promotes the decomposition of fat, or has the effect of maintaining concentration ( See Patent Document 4) and the like are known.

On the other hand, the following are known to reduce heat stress in livestock or poultry.
1) Livestock and poultry, especially chickens that lack sweat glands, which are significantly vulnerable to heat, are damaged by peroxides, especially lipid peroxides, which increase through heat stress due to heat, resulting in a decrease in meat quality at the time of dismantling. feed containing a feed additive composition containing zeolite, ferrite and charcoal to livestock or poultry in order to prevent this (see Patent Document 5).
2) A heat stress reducing agent characterized by containing at least sake lees and rice koji (see Patent Document 6).
3) Administering a vegetable feed composition for egg-laying hens, which consists essentially of a vegetable feed and is enriched with a glycine content in the range of 0.8 to 1.2% by weight, to egg-laying hens in the summer. Therefore, heat stress can be alleviated (see Patent Document 7).

4) Genetically improved prophylactic and ameliorating agents for production diseases caused by heat and cold stress in livestock and poultry, comprising quinones, particularly coenzyme Q6 to coenzyme Q10 (see Patent Document 8).
5) Heat stress or heat death in chickens can be suppressed by administering at least one selected from adrenal medullary hormones and their analogues or agonists to chickens (see Patent Document 9).
6) By orally administering or feeding trehalose as an active ingredient to livestock (e.g., dairy cows, beef cattle, etc.), it is possible to reduce the feed intake of livestock in hot weather when the temperature and humidity index (THI) is 72 or more. Suppress and reduce the heat stress (see Patent Document 10).

On the other hand, orotic acid is known to have an antioxidant effect (see Non-Patent Document 1).

JP 2011-98896 A JP 2011-136907 A JP 2012-246280 A JP 2012-126683 A JP 2013-128493 A JP 2007-001937 A JP-A-2002-027920 JP-A-2000-053565 JP 2008-013521 A JP 2015-140347 A

Hassani, A., et al AAPS Pharm Sci, Tech. 2019, 20:53.

However, in Non-Patent Document 1, only a radical scavenging test using DPPH (2,2-diphenyl-1-picrylhydrazyl) at a relatively high concentration in vitro was performed, and peroxidation was performed in vivo. It is not disclosed to suppress lipid production. Furthermore, it is not known that orotic acid has the effect of alleviating heat stress.
An object of the present invention is to provide a novel drug that suppresses the production of lipid peroxide, which adversely affects animals such as humans, pets, and livestock.

As a result of diligent efforts to solve the above problems, the present inventors have found that by feeding broilers a feed containing orotic acid under hot conditions, the production of lipid peroxide in the blood can be suppressed, and heat stress The inventors have found that the problem can be solved, and have completed the present invention.

That is, one aspect of the present invention is as follows specified by the following matters.
(1) A lipid peroxide production inhibitor characterized by containing orotic acid or a salt thereof as an active ingredient.
(2) A prophylactic or therapeutic agent for diseases associated with lipid peroxide, characterized by containing orotic acid or a salt thereof as an active ingredient.
(3) The preventive or therapeutic agent according to (2), wherein the therapeutic agent for diseases associated with lipid peroxide is a heat stress reducing agent.
(4) The preventive or therapeutic agent according to (3), wherein the reduction in heat stress is reduction in calorie intake due to heat stress.
(5) The preventive or therapeutic agent according to (3), wherein the reduction of heat stress is the reduction of decreased fertility.
(6) A method for reducing heat stress, comprising orally administering or feeding orotic acid or a salt thereof to humans or animals continuously in summer.
(7) A method of reducing heat stress by orally administering or feeding orotic acid or a salt thereof to humans or animals when the air temperature reaches or exceeds the upper critical temperature of humans or animals.

Another aspect of the present invention includes those specified by the following items.
(8) A method for inhibiting the production of lipid peroxide, which comprises administering or feeding orotic acid or a salt thereof to humans or animals.
(9) A method for preventing or treating a disease associated with lipid peroxide, which comprises administering or feeding orotic acid or a salt thereof to humans or animals.
(10) Use of an agent containing orotic acid or a salt thereof as an active ingredient as a lipid peroxide production inhibitor.
(11) Use of an agent containing orotic acid or a salt thereof as an active ingredient as a prophylactic or therapeutic agent for diseases associated with lipid peroxide.
(12) Use of orotic acid or a salt thereof for producing a lipid peroxidation inhibitor.
(13) Use of orotic acid or a salt thereof for producing a prophylactic or therapeutic agent for a disease associated with lipid peroxide.
(14) Orotic acid or a salt thereof for use in suppressing lipid peroxide production.
(15) orotic acid or a salt thereof for use in preventing or treating diseases associated with lipid peroxides;

By using the lipid peroxide production inhibitor of the present invention, it is possible to prevent or treat diseases said to be associated with lipid peroxide. For example, by orally administering or feeding the lipid peroxide production inhibitor of the present invention to humans or animals continuously in summer or when the temperature reaches or exceeds the upper critical temperature of humans or animals, humans and animals can reduce oxidative stress caused by heat stress. More specifically, it reduces the decrease in calorie intake (diet amount) (decreased appetite) in hot weather, so it reduces or suppresses the daily weight gain of humans or animals or the decrease in body weight before hot weather, and suppresses summer fatigue. It can be prevented or treated.
In addition, heat stress becomes oxidative stress in vivo, which adversely affects the development of embryos, disrupts hormonal balance, or causes weakening or decreased expression of estrus, resulting in a decrease in conception rate, Although this causes problems such as a reduction in the milk yield of dairy cows and a decrease in the number of calves and piglets produced, the lipid peroxide production inhibitor of the present invention is expected to solve these problems.
Furthermore, an increase in lipid peroxides due to oxidative stress such as heat stress causes problems such as offensive odors, offensive odors, meat juice loss, discoloration, shortened shelf life, decreased nutritional value, and generation of harmful substances in meat products. The lipid peroxide inhibitor of the present invention is expected to solve these problems.

It shows the body temperature of the broiler at the time of heat exposure in each test section 1 to 4 of the example.

The lipid peroxide production inhibitor and the preventive or therapeutic agent for diseases associated with lipid peroxide (hereinafter referred to as "lipid peroxide inhibitor, etc.") of the present invention contain orotic acid or a salt thereof as an active ingredient. It is not particularly limited as long as it is an agent that The orotic acid is also called uracil-6-carboxylic acid, but according to the IUPAC nomenclature, a heteroaromatic ring represented by "1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid" It is a kind of compound.

The above-mentioned orotic acid is produced, for example, by culturing a bacterium of the genus Corynebacterium having orotic acid-producing ability, producing and accumulating orotic acid in the culture, and collecting this (see Japanese Patent Publication No. 7-10235). It can be produced and accumulated in a culture solution by a fermentation method using microorganisms, and the above-mentioned culture is purified by conventional known purification means such as precipitation, chromatography using ion exchange resins, activated carbon, etc. It can be purified and collected by using a separation purification method.

In addition, Bacillus natto with orotic acid-producing ability is added to soybeans, defatted soybean flour, full-fat soybean flour, soybean protein, bean curd refuse, soybean meal, soybean broth, rice husk, corn cob, soybean husk, rice straw, bagasse, buckwheat husk, Barley husk, rice husk, rice, rice bran, wheat, wheat bran, malt cake, gluten feed, wheat flour, corn, corn bran, cornmeal, beet, beet meal, groundnut, groundnut husk, oil cake, fishmeal, crab shell, shrimp shell , krill fine powder, sawdust, pulp waste, waste paper, starch residue, soluble starch, sugars, yeast extract, skimmed milk powder, bone meal, peat moss, etc. is sporulated by drying or the like, orotic acid-containing probiotics containing spores of Bacillus natto having orotic acid-producing ability can be produced. Orotic acid used in the lipid peroxide production inhibitor and the like of the present invention can also be ingested in the form of probiotics as described above.
Moreover, it can also be prepared using a known chemical synthesis method or the like. Furthermore, a commercial item can also be used.

Examples of orotic acids include free orotic acid (free form) and salts of orotic acid, and free orotic acid (free form) is preferable. The orotic acid free form may be a hydrate.
Examples of salts of orotic acid include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, acetate, maleate, fumarate, citrate, malate, lactate and α-ketoglutarate. , organic acid salts such as gluconate and caprylate. Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts and zinc salts. Examples of ammonium salts include salts such as ammonium and tetramethylammonium. Examples of organic amine addition salts include salts of morpholine, piperidine and the like. Examples of amino acid addition salts include salts of glycine, phenylalanine, lysine, aspartic acid, glutamic acid and the like.

In addition, the preventive or therapeutic agent for diseases associated with lipid peroxides of the present invention is not particularly limited as long as it contains orotic acid or a salt thereof as an active ingredient.
Lipid peroxide is a general term for oxidized lipids such as cholesterol and triglycerides, and is produced by active oxygen. In the body, active oxygen is generated as a by-product in the process of oxygen consumption by aerobic organisms, but most of it is eliminated by enzymes and antioxidants that remove it. However, when the antioxidant capacity (the ability to remove active oxygen) cannot catch up with the generated active oxygen, the oxidation reaction causes harmful effects on the body, resulting in a so-called oxidative stress state, and lipid peroxide is generated. be done. There are various factors that cause oxidative stress, but for example, diseases such as ischemia and psychological/physical stress, ultraviolet light or radiation, air pollution, tobacco, drugs, metals, and intake of oxidized foods. , excessive exercise, etc.

Diseases associated with lipid peroxide include hypertension, diabetes, stroke, arteriosclerosis, liver dysfunction, retinopathy, cataract, angina pectoris, myocardial infarction, cerebral infarction, Alzheimer's disease, Parkinson's disease, liver cirrhosis, Examples include hepatitis, allergic diseases, cancer, metabolic disorders, rough skin, and the like.

Oxidative stress is also known to be caused by heat stress, and the preventive or therapeutic agent for diseases associated with lipid peroxides of the present invention can also be used as a heat stress reducing agent. Here, heat stress reduction means not causing or suppressing in vivo reactions caused by summer heat, and among others, 1) alleviation of decrease in calorie intake due to heat stress, 2) alleviation of reduction in fertility rate. It is preferably used for meaning.

A specific example of heat stress is summer fatigue. Summer fatigue is also known as shokiatari, heat loss, or summer loss. Symptoms such as general malaise, dullness, difficulty falling asleep, and loss of energy appear. The cause is the deterioration of gastrointestinal function due to the imbalance of the autonomic nervous system, malnutrition due to unbalanced eating habits, insufficient recovery of the body due to the experience of heat stroke I in midsummer, and dehydration tendency in the late summer heat (hidden dehydration). ), etc., resulting in an oxidative stress state.
Since the heat stress reducing agent of the present invention has the effect of reducing the decrease in calorie intake due to heat stress, it is thought that it can improve anorexia and malnutrition that can cause summer fatigue, and can prevent and eliminate summer fatigue.

Taking animals such as livestock and pets, especially cattle, as an example of heat stress reduction, suppression of reduction in lying time, suppression of increase in respiration rate, suppression of decrease in feed intake, suppression of decrease in milk production, body temperature and/or It means at least one or more selected from suppression of rectal temperature rise and suppression of locomotion decrease.

The agent for reducing heat stress of the present invention can reduce the decrease in the fertility rate of livestock such as dairy cows, beef cattle, laying hens, meat chickens, and domestic pigs.
Decreased signs of estrus, inhibition of embryo development, and the like are thought to be factors for the decrease in the conception rate. Suppression of lipid production means that the oxidative stress is eliminated, and as a result, heat stress is reduced.

As the lipid peroxide production inhibitor or the like of the present invention, orotic acid or a salt thereof can be directly administered. It is preferable to provide various compositions such as cosmetics, food and drink, or feed.

Hereinafter, the case of providing the lipid peroxide production inhibitor and the like of the present invention as a pharmaceutical composition will be described.
The salt of orotic acid provided as a pharmaceutical product is not particularly limited as long as it is a pharmaceutically acceptable salt. Choline salts, lysine salts, arginine salts, and ornithine salts, which are less likely to precipitate or separate out, can be mentioned, and these water-soluble salts are preferred in the case of beverages. In the case of ingestion as a capsule or tablet, it is not necessary to be water-soluble, and therefore metal salts such as sparingly soluble sodium salts, potassium salts, magnesium salts, calcium salts and ammonium salts can be used. In addition, orotic acid can also be solubilized by forming a salt with carnitine, and carnitine salt (L-carnitine orotic acid) has good solubility, but the aqueous solution has a low pH, so if necessary, a purine base such as guanosine It is preferable to raise the pH to weak acidity by adding a basic amino acid.

When providing the lipid peroxide production inhibitor or the like of the present invention as a pharmaceutical product, appropriate additives that are pharmaceutically acceptable and appropriately selected according to the dosage form, such as carriers, excipients, diluents, binders, Lubricants, disintegrants or disintegration aids, solubilizers, stabilizers, preservatives, preservatives, bulking agents, thickeners, emulsifiers, dispersants, suspending agents, buffers, etc. It may be prepared into various pharmaceutical forms that can be administered systemically or locally, orally or parenterally, by various methods.

The dosage form of the formulation may be oral administration or parenteral administration such as intravenous, intraperitoneal or subcutaneous administration, with oral administration being more preferred. Dosage forms for administration include, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions/decoctions, capsules, syrups, liquids, elixirs, extracts, tinctures, and liquid extracts. Oral agents, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, suppositories (e.g., rectal suppositories, vaginal suppositories), inhalants, transdermal - Parenteral agents such as transmucosal absorbers, ointments, and patches may be used, but oral agents are more preferred.

When preparing the lipid peroxide production inhibitor and the like of the present invention, in addition to orotic acid or a salt thereof, an antioxidant, a bacteria inhibitor, and the like can be used as necessary.

When the lipid peroxide production inhibitor or the like of the present invention is a liquid preparation such as syrup suitable for oral administration, water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, Oils such as sesame oil, olive oil, and soybean oil, preservatives such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and peppermint, and the like can be added to prepare formulations.

In addition, in the case of tablets, powders, granules, etc., which are suitable for oral administration, the lipid peroxide production inhibitor, etc. of the present invention may be prepared from saccharides such as lactose, sucrose, glucose, sucrose, mannitol, sorbitol, etc., potatoes, wheat, Excipients such as starch such as corn, inorganic substances such as calcium carbonate, calcium sulfate, sodium bicarbonate and sodium chloride, plant powder such as licorice powder and gentian powder; starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, Disintegrants such as carmellose calcium, calcium carbonate, sodium hydrogen carbonate, sodium alginate; Lubricants such as magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicon oil; polyvinyl alcohol, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose , carmellose, gelatin, starch paste, etc.; surfactants such as fatty acid esters; plasticizers such as glycerin;

Formulations suitable for oral administration may also contain additives generally used in food and drink, such as sweeteners, coloring agents, preservatives, thickening stabilizers, antioxidants, coloring agents, bleaching agents, antifungal agents, and gum bases. , bittering agents, enzymes, brighteners, acidulants, seasonings, emulsifiers, enhancers, manufacturing agents, flavors, spice extracts and the like may be added.

When the lipid peroxide production inhibitor or the like of the present invention is provided as a pharmaceutical, the content of orotic acid or a salt thereof, which is an active ingredient, is appropriately selected according to the type of pharmaceutical and the expected effects of administration of the pharmaceutical. However, it is usually 0.01 to 100% by mass, preferably 0.05 to 100% by mass, more preferably 0.1 to 100% by mass.

Humans to whom the lipid peroxide production inhibitor of the present invention is administered can be applied regardless of gender, race, age, and the like.
When the lipid peroxide production inhibitor or the like of the present invention is used as a pharmaceutical, the dosage can be appropriately determined according to the age, sex, body weight, degree of symptoms, administration method, etc. of the subject to be administered. , Orotic acid or its salt is administered once or several times a day in an amount of 10 mg to 10 g, preferably 50 mg to 5 g, more preferably 100 mg to 1 g per day for an adult.

Next, the case where the lipid peroxide production inhibitor of the present invention is provided as an additive for quasi-drugs, cosmetics, food and drink (hereinafter referred to as food and drink, etc.) will be described.
The lipid peroxide production inhibitor of the present invention may be added to food and drink as a food and drink additive. In this case, the amount to be added may be appropriately set in consideration of the general intake of the target food and drink, the form of the food and drink, efficacy, taste, palatability, cost, etc., but usually it is an active ingredient. The content of orotic acid or a salt thereof in food or drink is usually 0.01 to 99% by mass, preferably 0.05 to 99% by mass, more preferably 0.1 to 99% by mass. can be done.

When the lipid peroxide production inhibitor of the present invention is used as an additive for food and drink, other ingredients such as various proteins, sugars, fats, trace elements, vitamins, citric acid and the like are used as long as the effects of the present invention are not impaired. Organic acid salts such as acetic acid may be contained. In addition, depending on the type of food or drink to be added, additives that are acceptable and commonly used in food or drink, for example, sweeteners such as aspartame and stevia, acidulants such as citric acid, malic acid and tartaric acid, dextrin , In addition to excipients such as starch, coloring agents, flavoring agents, bittering agents, buffering agents, thickening agents, gelling agents, stabilizers, gum bases, binders, diluents, emulsifiers, dispersing agents, suspending agents , antioxidants, preservatives, preservatives, antifungal agents, color formers, bleaching agents, brightening agents, enzymes, seasonings, spice extracts and the like.

Beverages to which the lipid peroxide production inhibitor of the present invention can be added include beverages such as tea beverages, beer-based beverages, coffee, mineral water, milk beverages (including concentrated undiluted solutions and preparation powders of these beverages). ); Carbohydrate-containing food and drink such as rice, noodles, bread, pasta; Western confectionery such as cookies and cakes, Japanese confectionery such as manju and yokan, candy, gum, pudding, jelly and other frozen desserts and ice desserts Various confectioneries; Processed marine and livestock foods such as fish cakes, chikuwa, hamburgers, hams, sausages; Dairy products such as processed milk, fermented milk, yogurt, butter, and cheese; Oils and fats such as margarine, mayonnaise, shortening, whipped cream, and dressings and oil-and-fat processed foods; and seasonings such as sauces and sauces.

The form of the food and drink to which the lipid peroxide production inhibitor of the present invention is added is not particularly limited as long as it can be ingested by humans and is suitable for eating. Semi-liquid, granule, granular, powder, capsule, cream, paste, jelly and the like can be mentioned.

Foods and drinks to which the lipid peroxide production inhibitor of the present invention is added include health foods, functional foods, foods for specified health uses, dietary supplements, foods for the sick, and supplements.
The intake amount of the food or drink to which the lipid peroxide production inhibitor of the present invention is added is not particularly limited as long as it is an amount that can exhibit the lipid peroxide production inhibitory action or the heat stress reducing action, but is usually , the daily intake of orotic acid or its salt for adults is 10 mg to 10 g, preferably 50 mg to 5 g, more preferably 100 mg to 1 g.

Next, the case where the lipid peroxide inhibitor of the present invention is provided as a feed, a feed additive, or a veterinary drug will be described.
The lipid peroxide production inhibitor of the present invention can be used as it is or as a feed additive mixed with other ingredients, and can be used as a veterinary drug as it is or mixed with other veterinary drugs. It can also be used as a composition. A formulation obtained by adding an excipient such as starch or dextrin and granulating or tableting may also be used. Furthermore, for example, those coated (bypassed) with oil or the like so as not to be decomposed in the rumen can also be used.

When the lipid peroxide production inhibitor of the present invention is provided as a feed composition, commonly used feed ingredients can be added and mixed. Specific examples of commonly used feed ingredients include those listed in Attached Table 3 in the official specifications for feed notified by the Ministry of Agriculture, Forestry and Fisheries, proteins, lipids, minerals (potassium, sodium, magnesium, etc.), vitamins (vitamin A, nutrient ingredients such as vitamin E, etc.).

Furthermore, the lipid peroxide production inhibitor of the present invention can be provided as a veterinary drug or veterinary drug composition. A veterinary pharmaceutical composition may be formulated according to a conventional method, and may be used in combination with other active ingredients having physiological functions. Examples of the dosage form include oral administration such as tablets, capsules, granules, powders and syrups, parenteral administration such as injections, etc. Oral administration is preferred. These various formulations are prepared according to the usual methods, and the formulation technical field of veterinary pharmaceutical compositions such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, coating agents, etc. can be formulated using known adjuvants that are commonly used in

When the lipid peroxide production inhibitor or the like of the present invention is provided as a feed, a feed additive, or a veterinary drug, the animals to which it is administered or fed include dairy cows (especially Holstein, Jersey, etc.), beef cattle, and sheep. , livestock (including poultry) such as goats, buffaloes, camels, yaks, pigs, horses and chickens, and pets such as dogs and cats.

The administration or feeding amount (especially orally) of the lipid peroxide production inhibitor of the present invention to animals is preferably about 0.01 to 1.0 g/day, preferably 0.05 to 0.4 g, per 1 kg of body weight of the animal. /day is more preferable. For example, for dairy cows (weight of about 500 to 1000 kg), a suitable (oral) feeding amount is 10 to 500 g/head, preferably 50 to 200 g/head per day. For other domestic animals, the (oral) feeding amount may be set in consideration of their weight, etc., but the (oral) administration or feeding amount other than the above, including dairy cows, is not completely excluded.

Next, the case of using the lipid peroxide production inhibitor and the like of the present invention as a heat stress reducing agent for animals such as humans and livestock, or the case of using it in a method for reducing heat stress for humans and livestock will be described.
When the lipid peroxide production inhibitor or the like of the present invention is used as a heat stress reducing agent or in a method for reducing heat stress, (oral) administration or feeding should be continued during hot weather. For example, it is more preferable to administer or feed continuously (continuously every day) for 7 to 120 days or more (preferably 30 to 90 days) in summer. In particular, in the case of humans, the temperature and humidity index (THI) is 91 or higher (for example, when THI is 91 to 106), and in the case of livestock, the temperature and humidity index (THI) is 72 or higher. Continuous (oral) administration or feeding (on all days when the maximum THI is 91 or more, or 72 or more) to animals (for example, when THI is 72 to 84) is calorie intake It is very effective in terms of suppressing the amount decrease/decrease.
Moreover, it is preferable to administer or feed the animal when the atmospheric temperature reaches or exceeds the animal's upper critical temperature. The upper critical temperature of an animal is the temperature at which the body temperature maintenance function fails and causes an increase in body temperature, and varies depending on the animal. 30-32°C, 28°C for broilers.

EXAMPLES The present invention will be described in more detail with reference to examples below, but these examples do not limit the technical scope of the present invention.

The presence or absence of lipid peroxidation suppression effect and heat stress reduction effect by feeding orotic acid was investigated using broilers.
One-day-old male broiler chicks (Chunky, Ross 308) were obtained from a commercial hatchery (Kumiai Hina Center, Kagoshima, Japan). Chicks were placed in an electrically heated battery room and fed with water and a commercial diet (23% crude protein, 12.8 MJ/kg, Nichiwa Sangyo Co., Ltd.) until 14 days of age. On day 14, 32 broilers were randomly selected from a group of 100 birds. The broilers were housed individually in wire-bottomed aluminum cages (50 x 40 x 60 cm) and fed the basal diet shown in Table 2 for 3 days until the start of the experimental period.
After that, the selected broilers were divided into 4 groups of 8 heads each, each designated as test plots 1 to 4, and the rearing temperature and test feed to be provided for each test plot were set as shown in Table 1.

The tests were conducted in a temperature-controlled room with a relative humidity of 50-70% with a photoperiod of 23 hours light and 1 hour dark. Feed and water were allowed ad libitum.
When the rearing temperature was 35°C, the animals were reared at 35°C for 8 hours from 11:00 to 19:00, and at 25°C for the rest of the time.
Table 2 shows the composition of the basal feed.

＊１；ビタミンミネラル混合１ｋｇあたりの各成分の組成：ビタミンＡ ３００，０００ＩＵ、ビタミンＤ３ ４０，０００ＩＵ、酢酸ＤＬ－α－トコフェロール ２，０００ｍｇ、メナジオン亜硫酸水素ナトリウム ３６８ｍｇ、硝酸チアミン ４４４ｍｇ、リボフラビン ７２０ｍｇ、Ｄ－パントテン酸カルシウム ２，１７４ｍｇ、ニコチン酸 ７，０００ｍｇ、塩酸ピリドキシン ８５１ｍｇ、ビオチン ３０ｍｇ、葉酸 １１０ｍｇ、シアノコバラミン ２ｍｇ、ヨウ素酸カルシウム １０８ｍｇ、酸化マグネシウム １９８，９９１ｍｇ、硫酸マンガン（ＩＩ） ３２，９８５ｍｇ、硫酸亜鉛 １９，７５３ｍｇ、硫酸鉄（ＩＩ） ４３，５２３ｍｇ、硫酸銅（ＩＩ） ４，０１９ｍｇ、及び塩化コリン ２９９，６０８ｍｇ

*1; Composition of each component per 1 kg of vitamin-mineral mixture: vitamin A 300,000 IU, vitamin D3 40,000 IU, DL-α-tocopherol acetate 2,000 mg, menadione sodium hydrogen sulfite 368 mg, thiamine nitrate 444 mg, riboflavin 720 mg, D - calcium pantothenate 2,174 mg, nicotinic acid 7,000 mg, pyridoxine hydrochloride 851 mg, biotin 30 mg, folic acid 110 mg, cyanocobalamin 2 mg, calcium iodate 108 mg, magnesium oxide 198,991 mg, manganese(II) sulfate 32,985 mg, zinc sulfate 19 , 753 mg, iron(II) sulfate 43,523 mg, copper(II) sulfate 4,019 mg, and choline chloride 299,608 mg.

The basal diet was designed so that crude protein and metabolizable energy were 20% and 3.1 Mcal/kg, respectively. 0.7% by mass of orotic acid-free monohydrate added basic feed is obtained by adding 0.7% by mass of orotic acid-free monohydrate to the basic feed.

The test was conducted from 17 days old to 32 days old. During the test period, food intake was measured every day and body weight was measured once every 3 days. As feeding results, body weight at the end of the test (32 days old), body weight gain and body temperature during the test period (17 days old to 32 days old) were measured.

The average body weight at the end of the two groups of test groups 1 and 2 reared at an appropriate temperature was 1178.0 ± 64.68 g in test group 1, and 1175.0 ± 70.06 g in test group 2 administered with orotic acid. and no difference was found.
The average body weight at the end of the two groups of test groups 3 and 4 exposed to heat was 1085.4 ± 33.39 g in test group 3, and 1140.8 ± 40.13 g in test group 4 where orotic acid was administered. and the average weight gain from 17 days old to 32 days old exposed to heat was 746.3 ± 35 .77 g, and the effect of adding orotic acid was confirmed. Each of the above values represents the mean±standard error of the mean in each test section.

Also, from FIG. 1, the body temperature of the broilers was increased in the test group 3 where heat sensitization was given, but it was confirmed that the body temperature rise was suppressed in the test group 4 where the test feed containing orotic acid was fed. .
These results demonstrated the effectiveness of the lipid peroxide production inhibitor of the present invention, which contains orotic acid or a salt thereof as an active ingredient, as a heat stress reducing agent.

[Metabolomics of each tissue, measurement of lipid peroxide]
At the end of the test period, each broiler tested was _CO2 anesthetized, sacrificed by cervical dislocation, pectoralis major, pectoralis minor, leg muscles, liver, heart, and abdominal adipose tissue. dissected by Blood samples were also collected into heparinized tubes and centrifuged at 5,900 xg for 10 minutes at 4°C to separate plasma and stored at -30°C until analysis.

[Measurement of lipid peroxide in plasma]
The collected plasma was processed by the following procedure to obtain a measurement sample, and fluorescence was measured. Fluorescence measurement was performed using a spectral fluorescence detector (RF-20Axs, manufactured by Shimadzu Corporation).
1. Using a pipette, take 0.05 ml of blood.
2. The collected blood is placed in 1.0 ml saline in a centrifuge tube and gently shaken.
3. After centrifugation at 4000 rpm for 10 minutes, transfer 0.5 ml of supernatant to another centrifuge tube.
4. Add 4.0 ml of 12N sulfuric acid to the remaining solution and mix.
5. Add 0.5 ml of 10% phosphotungstic acid, mix, leave at room temperature for 5 minutes, then centrifuge the mixture at 4000 rpm for 10 minutes.
6. The supernatant is discarded, the precipitate is mixed with 2.0 ml of 12N sulfuric acid and 0.3 ml of 10% phosphotungstic acid, and the mixture is centrifuged at 4000 rpm for 10 minutes.
7. Suspend the precipitate in 4.0 ml of distilled water and add 1.0 ml of TBA (thiobarbituric acid) reagent. The reaction mixture is heated in an oil bath at 95° C. for 60 minutes.
8. After cooling with tap water, 5.0 ml of n-butanol are added and the mixture is vigorously shaken.
After centrifugation at 9.4000 rpm for 15 minutes, the n-butanol layer was collected for fluorescence measurement, excited by irradiation with light of 515 nm, and fluorescence intensity was measured at a fluorescence wavelength of 553 nm.
10. Fluorescence intensity of the standard solution obtained by reacting 0.5 nmol of tetraethoxypropane with TBA in step 7-9 is F, fluorescence intensity of the sample is f, lipid peroxide concentration in blood (Lp, unit: nmol) /ml) was determined by the following formula as the concentration of malonaldehyde (MDA).
Lp=0.5×f/F×1.00/0.05×1.05/0.5=f/F×21
The results are shown in Table 3 below. The values in Table 3 represent the mean ± standard error of the mean for the broilers tested in each test plot.

Under normal breeding conditions, the blood lipid peroxide concentration in test group 2 fed with orotic acid-added feed was different from the blood lipid peroxide concentration in test group 1 fed with orotic acid-free feed. However, under hot conditions, the concentration of lipid peroxide increased significantly in the test group 3, which was fed the diet without orotic acid, whereas the test group, which was fed with the orotic acid-added feed, showed a significant increase in the lipid peroxide concentration. At 4, lipid peroxide concentrations did not increase.

[Measurement of lipid peroxide in pectoralis major muscle]
Sodium dodecyl sulfate and acetate buffer (pH 3.5) were added to a 10 w/v% homogenate solution in which the collected pectoralis major muscle was pulverized and suspended in physiological saline. After heating for 60 minutes, the produced red pigment was extracted with a mixed solution of n-butanol-pyridine, the resulting extract was irradiated with light of 515 nm for excitation, and fluorescence intensity was measured at a fluorescence wavelength of 553 nm. Fluorescence measurement was performed using a spectrofluorometric detector RF-20Axs manufactured by Shimadzu Corporation (for details of the procedure, see Ohkawa, H. et al., Anal Biochem 1979, 95, 351-358).
The method for determining the lipid peroxide concentration in the pectoralis major muscle is the same as the method for determining the lipid peroxide concentration in plasma. Table 4 shows the results. The values in Table 4 represent the mean ± standard error of the mean for the broilers tested in each test plot.

Under both normal and hot conditions, the lipid peroxide concentration in test plots 2 and 4 fed with orotic acid-added feed was and significantly lower than the lipid peroxide concentration in Test Group 3. This indicates that the production of lipid peroxide in the pectoralis major muscle was suppressed by feeding orotic acid.

(Measurement of metabolomics)
A measurement sample was prepared as follows.
50 μL of plasma or 10 w/v% homogenized solution of pectoralis major muscle was suspended in 250 μL of methanol:chloroform:water (5:2:2) and 5 μL of 1 mg/mL 2-isopropylmalic acid was added internally. Added as a standard. The resulting solution was then mixed on a shaker at 1,200 rpm at 37°C for 30 minutes and then centrifuged at 16,000 xg at 4°C for 5 minutes. Next, 200 μL of distilled water was added to the resulting 225 μL of supernatant, mixed by vortexing, and then centrifuged at 16,000×g at 4° C. for 5 minutes. Subsequently, after cooling at -80°C for 10 minutes, 250 µL of the supernatant was dried under reduced pressure using a centrifugal evaporator (RD-400, manufactured by Yamato Scientific Co., Ltd.). Methoxyamine hydrochloride (20 mg/mL, 40 μL) in pyridine was then added to the tube, mixed by vortexing, and then shaken at 1,200 x g at 30°C for 90 minutes in the dark for oximation. -Methyl-N-trimethylsilyl trifluoroacetamide (20 μL) was added to the tube, the contents were mixed by vortexing, and the tube was shaken at 1,200 xg for 45 minutes at 37°C in the dark to prepare the trimethylsilyl derivative; This was used as a measurement sample.
The measurement was performed using the following analytical instruments.
Analysis equipment: GC/MS/MS (GCMS-TQ8050, manufactured by Shimadzu Corporation)
Column: BPX-5 column (length: 30 m, inner diameter: 0.25 mm, df: 0.25 μm)

Table 5 shows the measurement results of ascorbic acid, β-alanine, uridine, 2'-deoxyuridine, uracil, and orotic acid in plasma. The values in Table 5 are the mean ± standard error of the mean of the test animals in each test group, and the values are relative values using the peak area ratio to the internal standard, with test group 1 set to 100. .

Free amino acids in plasma and carnosine in pectoralis major muscle were measured using a high-performance liquid chromatography device (Nexera X2, manufactured by Shimadzu Corporation) having a pre-column equipped with an automatic derivatization function (for details, see Azuma , K et al., Metabolites 2016, 6, 3).
Table 6 shows the measurement results of aspartic acid, glutamic acid and tyrosine in plasma, and the measurement results of carnosine in pectoralis major muscle.

There was a significant difference in the ascorbic acid concentration under rearing temperature conditions, but there was no significant difference in the feeding of orotic acid compared to the control.
Analysis of amino acids in plasma showed that aspartic acid, glutamic acid, and tyrosine were significantly affected by feeding orotic acid-added feed, and aspartic acid and glutamic acid tended to increase even under hot conditions. Ta.
Aspartic acid is converted to glutamic acid through the citric acid cycle, and glutamic acid is a constituent amino acid of glutathione, an antioxidant. It was suggested that there is a possibility that

Analysis of carcinone in pectoralis major muscle showed that the addition of orotic acid to the diet had a significant effect on the concentration of carnosine in pectoralis major muscle of broilers.
Metabolomics analysis of plasma revealed that the addition of orotic acid to feed had a significant effect on pyrimidine-related metabolites such as orotic acid, uracil, uridine, 2'-deoxyuridine and β-alanine. As a result of metabolic enrichment analysis, it was confirmed that orotic acid feeding significantly changed not only pyrimidine metabolism but also β-alanine metabolism and aspartate metabolism.
From the above, it was suggested that orotic acid supplementation increased muscle carnosine content via pyrimidine metabolism and β-alanine metabolism. Since carnosine has an antioxidant effect, it was suggested that this may suppress the production of lipid peroxide due to heat stress.

Oxidative stress can be reduced by using the lipid peroxide production inhibitor of the present invention. As a result, for example, oxidative stress due to heat stress in humans or animals during hot summer months can be reduced, so that summer heat fatigue can be prevented or eliminated, a decrease in the fertility rate of livestock can be reduced, and a reduction in meat quality can be reduced. can be done. Therefore, the lipid peroxide inhibitor of the present invention is useful in the medical field, health food field, and livestock production field.

Claims (7)

An in vivo lipid peroxidation inhibitor comprising orotic acid or a salt thereof as an active ingredient. A prophylactic or therapeutic agent for diseases associated with in vivo lipid peroxides, comprising orotic acid or a salt thereof as an active ingredient. 3. The preventive or therapeutic agent according to claim 2, wherein the therapeutic agent for diseases associated with in vivo lipid peroxides is a heat stress reducing agent. 4. The preventive or therapeutic agent according to claim 3, wherein the reduction in heat stress is reduction in calorie intake due to heat stress. 4. The preventive or therapeutic agent according to claim 3, wherein the reduction of heat stress is the reduction of decreased fertility. A method for reducing heat stress by continuously feeding orotic acid or a salt thereof to animals (excluding humans) in summer . A method for reducing heat stress, comprising feeding orotic acid or a salt thereof to an animal (excluding humans) when the air temperature reaches or exceeds the upper critical temperature of the animal (excluding humans) .

Images