JP2007022989A - Anti-fatigue agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new safe and inexpensive anti-fatigue agent having production promoting actions on ATP (adenosine 5'-triphosphate) in muscles, effective for recovery from fatigue and having great effects. <P>SOLUTION: The anti-fatigue agent comprises lactoferrin or an iron-bound type lactoferrin in which iron is bound to the lactoferrin as an active ingredient. The anti-fatigue agent has production promoting actions on the ATP and is effective for the recovery from the fatigue. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an anti-fatigue agent that improves fatigue effectively and safely by increasing ATP production and mitochondrial enzyme activity. The anti-fatigue agent of the present invention is characterized by using as an active ingredient iron-binding lactoferrin obtained by binding lactoferrin or lactoferrin and iron.

  Fatigue recovery and anti-fatigue agents that can recover from accumulated fatigue caused by work or exercise, or prevent fatigue when taking work or exercise after taking in advance, are some vitamins that are active ingredients In particular, the demand for drinks is increasing year by year. These contain various ingredients in addition to vitamins as active ingredients, and each product has its characteristics. The product features are the type and amount of the compound, for example, sugars such as glucose and glucuronolactone, amino acids such as taurine, arginine salt and aspartate, caffeine, inorganic substances such as iron and manganese, There are a wide variety of garlic, ginseng, and herb extracts. However, these conventional products are not always satisfactory in terms of effect and price.

  Lactoferrin, which is a milk protein extracted and separated from milk, is immunostimulatory (for example, see Patent Document 1), cell proliferation (for example, see Patent Document 2), anti-rheumatic (for example, see Patent Document 3), antibacterial (for example, Patent Document 3). For example, it is known to have various actions such as non-patent document 1) and anti-tumor (for example, refer to non-patent document 2). Moreover, lactoferrin is mentioned as one of the immunostimulants contained in the anti-stress agent of aquatic animals such as fish, shrimp, and invertebrates that live in water (see, for example, Patent Document 4). Furthermore, an antistress agent for terrestrial animals containing lactoferrin as an active ingredient has been disclosed (for example, see Patent Document 5). These are all intended to suppress various reactions due to hormone secretion in an animal body that has been subjected to emotional stress due to factors such as attack, handling, and transportation, and are different from the recovery or prevention of physical fatigue of the present invention. The anti-fatigue action related to energy production has not been clarified so far.

Lactoferrin is also known to promote iron absorption from the intestinal tract. Iron is abundant in the body, but cannot be synthesized in the body, so it relies only on food intake. Incidentally, it is well known that most of the anemia found in women is iron deficiency anemia, which is caused by a lack of iron. Fat is a high-calorie, high-energy nutrient, but fatty acids, which are constituents of fat, are taken up by mitochondria in the cell and undergo β-oxidation, tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation. After that, a large amount of oxygen is used to produce adenosine triphosphate (ATP), making it easy to use as energy. Moreover, it is well known that hemoglobin in blood contains iron, and this iron plays an important role in transporting oxygen into the body. For this reason, when the intake of iron is insufficient, the supply of oxygen to the body decreases, so that metabolic activity that requires a large amount of oxygen decreases, and conversion to ATP, which is an energy source, is limited. In other words, an increase in the amount of oxygen supplied to the body is expected to increase the effects of physical strength enhancement, fatigue recovery, and stamina improvement.
Cytochromes are also involved in the oxidative phosphorylation reaction, but this protein also contains iron. Thus, it can be said that iron is greatly involved in the production of energy by the decomposition of fat, and that the increase in the amount of absorbed iron has the effect of enhancing physical strength, recovering from fatigue, and improving stamina.
From these aspects, search for a substance having an action of promoting ATP production and oxidative phosphorylation is required.

It is known that carnitine is required for the incorporation of fatty acids into mitochondria that produce energy. In the process of carnitine synthesis in vivo, two hydroxylases (hydroxylase) are required, and it is known that these two hydroxylases require iron for the expression of activity (for example, non-patent literature). 3 and 4). In addition, when an iron-deficient diet was given to a mother rat during pregnancy and lactation, blood neutral fat in the pup rat increased 8 times, and the hemoglobin concentration and the amount of liver carnitine decreased significantly (for example, see Non-Patent Document 5). .) In other words, in the iron deficient state, the transport of fatty acids into the mitochondria is suppressed due to the decrease in the content of carnitine in the tissue, and it is not used as an energy source, but the fatty acids are re-synthesized into fat and accumulated in the body. Become.
Therefore, by increasing the enzyme activity of hydroxylase in mitochondria, fatty acid is taken into mitochondria, energy production is promoted, and fatigue recovery effect can be expected.
Conventionally, vitamin groups such as vitamin B1 (including derivatives), B2, nicotinic acid, and pantothenic acid have been used as anti-fatigue agents. These vitamin groups are all coenzymes or combinations associated with the TCA cycle reaction. As a result, these vitamin groups are said to efficiently produce ATP and indirectly promote lactic acid metabolism. The production of ATP is based on the glycolysis process and oxidative phosphorylation, but in the energy production at the mitochondrial level, the use of oxygen depends on the presence of ubiquinone. Ubiquinone is used for the purpose of improving metabolism and ATP production. However, ubiquinone is limited to the anti-fatigue action of the myocardium. If ubiquinone is not mixed with a large amount of raw materials such as dry yeast extract, the effect is not recognized, and the effect cannot be said to be sufficient. Met.
In addition, regarding suppression of energy production caused by iron deficiency, anemia can be solved by simply supplementing with iron, but it has been difficult to eliminate anti-fatigue action.
JP-A-7-179355 Japanese Patent Laid-Open No. 6-48955 JP-A-5-186368 Japanese National Patent Publication No. 11-514773 Japanese Patent Laid-Open No. 2001-354583 Journal of Pediatrics, 94, 1, 1979 Cancer Research, 54, 2310, 1994 J. D. Halse (Jul), Journal of Biological Chemistry (J. Biol. Chem.), 253, 1654-1659, 1978 Lindstedt, G., Biochemistry, 5, 1271-1281, 1967 S. J. Bartholomey (SJ), Journal of Nutrition, 115, 138-145, 1985

As described above, vitamins have been conventionally used as anti-fatigue agents. However, in energy production at the mitochondrial level, the use of oxygen depends on the presence of ubiquinone, and ubiquinone is limited to the anti-fatigue action of the myocardium and cannot be said to be sufficient. It was. In addition, regarding suppression of energy production caused by iron deficiency, anemia can be solved by simply supplementing with iron, but it has been difficult to eliminate anti-fatigue action.
Accordingly, an object of the present invention is to provide a novel anti-fatigue agent that is safe, inexpensive and highly effective.

As a result of intensive studies to solve the above problems, the present inventors have found that lactoferrin contained in milk has an effective anti-fatigue action, and have achieved the present invention. Furthermore, the present inventors have found that iron-bound lactoferrin obtained by binding iron to lactoferrin has a remarkable effect on anti-fatigue, and has led to the present invention.
The present inventors have found that fatigue caused by various factors can be improved by orally ingesting lactoferrin or iron-bound lactoferrin in which iron is bound to this, and further based on this finding, Completed anti-fatigue agent. That is, the present invention is an anti-fatigue agent characterized by containing lactoferrin separated from milk or iron-bound lactoferrin obtained by binding iron to this as an active ingredient.

The effect as an anti-fatigue agent can be confirmed by the following test.
The first is a test for confirming the ATP production promoting action of the administered test substance by measuring the change in the total amount of ATP in the gastrocnemius muscle before and after forced exercise of rats. That is, using a rat administered with the test feed and general feed, a treadmill forced exercise experiment led to a running overwork state, and from the start of the experiment, the 31P NMR spectrum of the gastrocnemius muscle was taken, at rest, during exercise, immediately after exercise The amount of muscle ATP after 5 minutes of exercise is calculated. ATP is widely present in living bodies including muscles, and is involved in many energy metabolisms as an energy transmitter, and plays an important role in the acquisition and use of energy. Especially in aerobic metabolism, it is more efficiently produced by oxidative phosphorylation. That is, when one molecule of glucose is metabolized, 38 molecules of ATP are produced when it is completely oxidized by oxygen respiration via the glycolysis. If the amount of ATP consumed by exercise is regenerated, it can be said that there is a fatigue recovery effect by increasing the energy used in various tissues including muscles.

  The second is a test to confirm changes in mitochondrial cytolate synthetase activity due to continuous exercise. Cytolate synthase is an early stage enzyme for introducing acetyl-CoA produced by glycolysis or fatty acid degradation into a citrate cycle that plays an important role in energy production. It is known to be important for producing CoA and citric acid from acetyl CoA and oxaloacetic acid and smoothly turning the citric acid cycle. Using rats fed the test diet and general diet, forced exercise with a treadmill is performed for 10 days, and mitochondria are separated from the gastrocnemius muscle 20 days after the start of the experiment. Measure the enzymatic activity of cytolate synthetase in mitochondria with a spectrophotometer and compare it before and after the start of exercise. When the cytosolic synthase enzyme activity in mitochondria is increased by administration of the test substance, acetyl CoA generated by glycolysis and fatty acid degradation is led to the citrate cycle, which plays an important role in energy production, and CoA and citrate It can be said that the citric acid circuit is smoothly rotated to generate a fatigue recovery action.

  The third is measurement of immobility time (seconds) in forced swimming. That is, after oral administration of the test substance, the rat is placed in a water bath, and the immobility time (seconds) is measured until 15 minutes at 5-minute intervals. When the immobility time when the muscle activity becomes impossible is shortened by administration of the test substance, it is confirmed that the test substance is resistant to fatigue.

  Furthermore, after forced swimming, by measuring the lactic acid concentration in the blood, it is possible to confirm the presence or absence of the action of suppressing the increase in the lactic acid concentration in the blood by administering the test substance. Lactic acid is known as one of the fatigue substances produced when energy is consumed, and when this accumulates in the blood and tissues, it interferes with various motor functions and causes subjective symptoms such as muscle fatigue. It is said that it becomes.

As described above, the ability to improve physical fatigue is such that the amount of muscle ATP changes during forced exercise experiments, the increase of enzyme activity in mitochondria by continuous forced exercise, the measurement of immobility time (seconds) during forced swimming, and lactic acid in blood It can be confirmed by measuring four points of concentration measurement.
The present invention was able to confirm that lactoferrin isolated from milk and iron-bound lactoferrin bound with iron had unexpected pharmacological and therapeutic effects by these tests.
That is, the present invention relates to an anti-fatigue agent containing lactoferrin as an active ingredient. The present invention also relates to an anti-fatigue agent comprising iron-bound lactoferrin obtained by binding iron to lactoferrin as an active ingredient.

The anti-fatigue agent of the present invention is less fatigued by taking it, and is also effective for recovery from fatigue. In other words, when you feel physical fatigue during sports or other muscle exercises, you can take it to recover from fatigue, but if you take it in advance and then do labor, sports, etc., you will prevent fatigue You can also. In addition, endurance can be expected by taking it before or during sports.
Lactoferrin used in the anti-fatigue agent of the present invention is a milk-derived protein made from milk that has been eaten since a long history, so it is highly safe and has no side-effect problems. Expected to be used for health foods and functional foods. Further, it can be used for a long time, and is a highly versatile protein that itself is almost tasteless and odorless. Further, it has an advantage that it can be obtained at a relatively low cost, and is extremely useful in practical use. Moreover, since iron-binding lactoferrin is obtained by binding iron to the above-mentioned lactoferrin, there is no problem in safety.

Lactoferrin isolated from milk, which is an active ingredient of the anti-fatigue agent of the present invention, is ionized from, for example, milk from cows, goats, sheep, horses, humans, etc., or skim milk, whey, etc., processed products of these milks. It can be prepared separately by exchange chromatography or the like. For example, when skim milk is passed through a column and the adsorbed protein is eluted, the resin is thoroughly washed with 0.3 M saline, then 1.5 M saline is passed through, and lactoferrin adsorbed on the ion exchange resin. Can be obtained. This eluate is ultrafiltered using an ultrafiltration membrane module (trade name: SLP0053, manufactured by Asahi Kasei Co., Ltd.), then water is added, and desalting is performed by diafiltration using the same apparatus. Then, powdery lactoferrin can be obtained by freeze-drying. The purity of lactoferrin obtained by the above method has a purity of 95% or more as a result of measurement by electrophoresis.
In addition, lactoferrin can usually chelate up to two atoms of iron per molecule, but in the present invention, iron non-binding lactoferrin that does not bind iron at all, iron per molecule prepared from milk is an average. Either lactoferrin bonded to about 0.2 atoms or lactoferrin bonded with 2 atoms of iron per molecule can be used, and those obtained by degrading them with proteolytic enzymes can also be used.

An iron-bound lactoferrin obtained by binding iron to lactoferrin used in the present invention is conventionally known.
Lactoferrin usually chelates only up to 2 atoms of iron per molecule, but the iron-binding lactoferrin used in the present invention provides at least 3 atoms of iron per molecule of lactoferrin by performing a specific treatment on lactoferrin. It can be held stably. Such iron-binding lactoferrin can be obtained, for example, by adding an iron salt to a lactoferrin solution and adding an alkali to increase the pH of the solution. Lactoferrin powder that stably holds at least 3 atoms of iron per molecule of lactoferrin includes a heat-resistant lactoferrin-iron conjugate in which iron is bound to the amino group of lactoferrin via bicarbonate ions (“heat-resistant lactoferrin-iron conjugate” And its production method ", Japanese Patent No. 2835902), or an iron-lactoferrin complex obtained by adding a solution containing carbonic acid and / or bicarbonate and a solution containing iron at a certain ratio to lactoferrin (""Iron-lactoferrin complex and method for producing the same", Japanese Patent No. 2884045) and the like are known.
The iron-binding lactoferrin that can be used in the present invention may be any of these iron-lactoferrins. Iron-bound lactoferrin is in a state where iron and lactoferrin are bound, and iron and lactoferrin are bound, or iron and lactoferrin are bound via another substance. Any so-called iron may be used as long as it does not exist in an ionic state. In particular, the above-mentioned “iron-lactoferrin conjugate” and “iron-lactoferrin complex” obtained by adding a solution containing carbonic acid and / or bicarbonate to a lactoferrin and a solution containing iron can be exemplified. It is desirable to use iron-binding lactoferrin. In addition, these iron-bound lactoferrin has a characteristic that there is no iron astringency, metal taste, etc., so there is no problem in flavor.
Moreover, what decomposed | disassembled the iron binding type lactoferrin with the proteolytic enzyme retains the iron binding ability, and can be used for this invention. Furthermore, the thing which combined iron with what decomposed | disassembled the lactoferrin with the proteolytic enzyme can also be used for this invention.

  Examples of iron that can be used in producing the iron-binding lactoferrin as described above include ferrous sulfate, ferric sulfate, iron lactate, iron citrate, sodium ferrous citrate, and ammonium iron citrate. And ferrous pyrophosphate, ferric pyrophosphate, ferric chloride, ferrous nitrate, ferric nitrate and the like.

  Lactoferrin, which is an active ingredient of the anti-fatigue agent of the present invention, is a natural product derived from milk, is highly safe when ingested, is contained in foods such as milk, and is ingested on a daily basis. It shows no toxicity and has almost no side effects even when ingested continuously for a long period of time. Therefore, it can be appropriately used depending on the administration method such as oral.

  The anti-fatigue agent using lactoferrin and iron-binding type lactoferrin which are the active ingredients of the present invention is used as it is or if necessary, other known additives such as excipients, disintegrants, binders, lubricants, Mixing antioxidants, coating agents, coloring agents, flavoring agents, surfactants, plasticizers, etc., and preparing oral preparations such as granules, powders, capsules, tablets, dry syrups, and liquids by conventional methods Can do. Excipients include, for example, mannitol, xylitol, sorbitol, glucose, sucrose, lactose, crystalline cellulose, crystalline cellulose / sodium carboxymethylcellulose, calcium hydrogen phosphate, wheat starch, rice starch, corn starch, potato starch, carboxymethyl starch sodium , Dextrin, α-cyclodextrin, β-cyclodextrin, carboxyvinyl polymer, light anhydrous silicic acid, titanium oxide, magnesium aluminate metasilicate, polyethylene glycol, medium chain fatty acid triglyceride and the like. In the case of a drink, palatability can also be imparted by mixing other physiologically active ingredients, minerals, vitamins, nutritional ingredients, fragrances and the like as necessary.

  Moreover, the anti-fatigue agent which used the lactoferrin of this invention or the iron binding type lactoferrin as an active ingredient can be mix | blended with food-drinks, and can be made to contain in a foodstuff, The function which has an anti-fatigue effect as one aspect | mode of an anti-fatigue agent It is also possible to process into a natural food or feed. When blended in foods and drinks, lactoferrin or iron-binding lactoferrin can be blended as it is in the liquid state immediately after it is prepared, and further powdered by drying by freeze drying, spray drying, etc. Can be blended.

  Examples of the food and drink containing the lactoferrin of the present invention or iron-binding lactoferrin include cheese, butter, milk drinks, juice, yogurt, jelly, bread, ice cream, noodles, sausage, infant formula and baby food Can do.

  The effective dose of lactoferrin separated from milk in the present invention and iron-bound lactoferrin in which iron is bound thereto may be appropriately determined in consideration of the degree of fatigue, purpose, weight, age, sex, etc. In general, administration of 10 to 2,000 mg per day for adults can provide a recovery or prevention effect of fatigue. Thus, the present invention is effective at low doses. The component having an action of recovering fatigue according to the present invention exhibits an action of recovering fatigue in a living body as an anti-fatigue agent or by orally ingesting a food or drink containing them.

  EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited by these.

[Test Example 1]
(Preparation of iron-bound lactoferrin (70FeLF))
90 g of lactoferrin (manufactured by DMV), 20 g of ferric chloride hexahydrate and 5 g of sodium bicarbonate were dissolved in 10 liters of water to prepare a solution containing iron-bound lactoferrin. This solution was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 5,000 cut, and water was added to obtain an iron-binding lactoferrin solution with a capacity of 10 liters. After freeze-drying this solution, the iron content of the iron-bound lactoferrin freeze-dried product was measured with an inductively coupled plasma emission spectrometer (ICP) (ST-3000, manufactured by Leeman Labs). Since it contained 70 atoms, this lyophilized product was used as iron-bound lactoferrin (70FeLF) powder.

(Changes in total amount of ATP in rat gastrocnemius muscle)
The following experiment was conducted using Wistar male rats (body weight 250 to 300 g). The diet was freely ingested with general feed (CE-2: manufactured by Oriental Yeast Co., Ltd.) and water until one week before the start of the forced exercise experiment. Thereafter, the control group was given 20 mg / kg body weight / day soy protein supplemented with ferric chloride, the test group was 20 mg / kg body weight / day, 2 mg / kg body weight / day, 0.2 mg / kg body weight / day, 0.02 mg / kg body weight / day of lactoferrin (manufactured by DMV) and iron-bound lactoferrin (70FeLF) were ingested. The group consisted of 5 animals. Following a rest of 1 minute, the vehicle was overworked for 3 minutes at 100 m / min with a treadmill. The recovery period was 5 minutes after the end of exercise. From the start of the experiment, a 31P NMR spectrum of the gastrocnemius muscle of the right hind leg was recorded every minute using MRI for animals, and the amount of ATP in the muscle was calculated. The amount of ATP in the muscles at rest, during exercise, immediately after exercise, and after 5 minutes of exercise is shown in Table 1 as relative values when the value at rest is 1.0.

  As seen in Table 1, when the amount of ATP produced was compared with that of the control group (soy protein + iron), significant effects were observed in lactoferrin and iron-bound lactoferrin. Moreover, the effect was remarkable with iron binding type lactoferrin. From these results, it was shown that the ingestion of lactoferrin and iron-binding lactoferrin has an ATP production promoting action that is not predicted by existing data, and it is clarified that it is effective for recovery from fatigue.

[Test Example 2]
(Change in mitochondrial cytolate synthetase activity due to continuous exercise)
The following experiment was conducted using 20 Wistar male rats (body weight 250 to 300 g). The diet was freely ingested with general feed (CE-2: manufactured by Oriental Yeast Co., Ltd.) and water until one week before the start of the forced exercise experiment using the treadmill. Thereafter, the rats were divided into 5 groups, the control group was fed the general diet as it was, and the tests were 20 mg / kg body weight / day, 2 mg / kg body weight / day, 0.2 mg / kg body weight / day, 0.02 mg / kg body weight / day. Iron-bound lactoferrin (70FeLF) was ingested. Forced exercise with a treadmill was performed for 120 days a day for 10 days at 40 m / min. On the 20th day after the experiment was started, the gastrocnemius muscle was separated, homogenized, and subjected to differential centrifugation to separate mitochondria. According to the method of Oscai et al. [Am. J. Physiol., Vol. 220, p. 1238-1241, 1971], citrate synthetase, which is a mitochondrial enzyme activity, was measured with a spectrophotometer and compared before and after the start of exercise. . The results are shown in Table 2.

  As shown in Table 2, significant increase in enzyme activity in the (20 mg / kg body weight / day) group, the (2 mg / kg body weight / day) group, and the (0.2 mg / kg body weight / day) group after 10 days of exercise. Was seen. On the other hand, there was only a slight increase in the control group and the (0.02 mg / kg body weight / day) group.

[Test Example 3]
(Preparation of non-iron-binding lactoferrin (0FeLF))
A commercially available 1% aqueous solution of lactoferrin (manufactured by Sigma) was sealed in a dialysis tube and dialyzed against a 0.1M aqueous citric acid solution containing 20 times the amount of 0.05% EDTA at 4 ° C. for 30 hours. Subsequently, the dialysis tube is taken out and dialyzed against distilled water for 24 hours to prepare an iron non-binding lactoferrin (0FeLF) solution. The solution is freeze-dried to obtain iron non-binding lactoferrin (0FeLF) powder. Obtained.

(Confirmation of iron content of commercial lactoferrin)
When the iron content of commercially available lactoferrin (manufactured by Sigma) was measured with an inductively coupled plasma emission spectrometer (ICP), it contained 2 atoms of iron per molecule of lactoferrin, so this product was called lactoferrin (2FeLF) powder. did.

(Preparation of iron-binding lactoferrin (200FeLF))
To 2 liters of water, 400 g of sodium bicarbonate was added and stirred with a stirrer. In a supersaturated solution of sodium bicarbonate, 8 liters of water were added 90 g of commercially available lactoferrin (DMV) and ferric chloride hexahydrate. A solution in which 60 g of the product was dissolved was added with stirring to prepare a solution containing iron-bound lactoferrin. This solution was desalted and concentrated with an ultrafiltration membrane having a molecular weight of 5,000 cut, and water was added to obtain an iron-binding lactoferrin solution with a capacity of 10 liters. After freeze-drying this solution, the iron content of the iron-bound lactoferrin freeze-dried product was measured with an inductively coupled plasma emission spectrometer (ICP). As a result, it contained 200 atoms of iron per molecule of lactoferrin. The dried product was iron-bonded lactoferrin (200FeLF) powder.

(Forced swimming experiment in rats)
After purchasing Wistar male rats (7 weeks old), the body weight was measured during a quarantine habituation period of 5 days or more and the general condition was observed, and animals judged healthy were used for the test. All animals were housed under conditions of temperature 24 ± 2 ° C., humidity 55 ± 10%, ventilation rate 13 times / hour, lighting 12 hours (7 am-7pm) (mouse, rearing room in the rat area). A lactoferrin and iron-binding lactoferrin (number of iron-binding molecules per lactoferrin molecule: 0,2,70,200) administration group and a group to which 5% gum arabic physiological saline was administered were set as a control group. In the measurement of blood lactate level, an untreated group was set in which only 30 minutes preload was performed. The test substance was administered orally and was forcibly administered using a plastic gastric sonde. The dose was 5 ml / kg body weight / day. Eight 8-week-old rats with a body weight range of 299-323 g were used per group. Place animal in a 100 L (bottom diameter 40 cm, top diameter 50 cm x height 60 cm) polybucket containing water at 25 ± 1 ° C to a depth of 19.5 cm for 30 minutes (preload), and immediately after that, the test substance is orally administered. did. Next, according to the method of Porsolt et al. [Nature, 266, 730-732, 1977], 1 hour after oral administration of the test substance, the animal was placed in cylindrical vinyl chloride containing 25 ± 1 ° C water to a depth of 24 cm. One animal was placed in each container, and the immobility time (seconds) was measured until 15 minutes after 5 minutes. A cylindrical vinyl chloride container having a height of 40 cm and a diameter of 20 cm was used. In addition, about 5 ml of heparinized blood was collected from the abdominal vena cava under ethyl ether anesthesia after completion of measurement of immobility time (after completion of preload in the untreated group). For the measurement of the amount of lactic acid, serum obtained by deproteinization with perchloric acid and then centrifugation at 3,500 rpm for 10 minutes was used.
Table 3 shows the results of after-load forced swimming for 15 minutes.
Table 4 shows the measurement results of the amount of lactic acid.

As shown in Table 3, it was revealed that administration of lactoferrin (0FeLF, 2FeLF) and iron-bound lactoferrin (70FeLF, 200FeLF) significantly shortened immobility time and increased resistance to fatigue. Moreover, the tendency for immobility time to become short was seen, so that there was much iron binding amount.
Furthermore, as seen in Table 4, the blood lactate concentration in the untreated group was 24.0 mg / ml after preload swimming, whereas in the control group 43.3 mg after afterload forced swimming. / ml, and the blood lactate concentration increased significantly. Blood lactate concentration was significantly reduced by oral administration of lactoferrin and iron-bound lactoferrin, indicating that lactoferrin and iron-bound lactoferrin have the effect of suppressing the increase in blood lactate concentration.

(Preparation of lactoferrin)
A column (5 cm in diameter x 30 cm in height) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki) was thoroughly washed with deionized water, and then 40 L (pH 6.7) of non-sterilized skim milk was added to this column. At a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, then washed with 0.02 M carbonate buffer (pH 7.0) containing 0.7 M sodium chloride, and then 0.02 M carbonate buffer (0.98 M sodium chloride (pH 7.0)). The fraction adsorbed on the resin at pH 7.0) was eluted. The eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and lyophilized to obtain 11 g of lactoferrin powder.
The lactoferrin powder thus obtained contains 93% by weight of lactoferrin and can be used as it is as the anti-fatigue agent of the present invention.

(Preparation of iron-binding lactoferrin)
A solution 1 L containing 10 μmol of lactoferrin obtained in Example 1 and 1.2 mol of sodium bicarbonate was prepared as solution A. 1 L of a solution containing 1.5 mmol of ferric sulfate as iron ions was prepared as solution B. After adding solution B to solution A, desalting and concentrating with an ultrafiltration membrane with a molecular weight of 5,000 cut, and diluting with simulated buffer (pH 8.9) so that the iron concentration becomes 94 mg / 100 ml. A conjugated lactoferrin was obtained.
The iron-bound lactoferrin thus obtained contains 0.9 g of lactoferrin and 94 mg of iron per 100 ml of the solution, and can be used as it is as an anti-fatigue agent of the present invention.

(Preparation of tablets containing lactoferrin)
After sufficiently mixing 36 parts by weight of lactoferrin obtained by the method of Example 1, 36 parts by weight of lactose (manufactured by DMV), 25 parts by weight of crystalline cellulose (manufactured by Wako Pure Chemical Industries, Ltd.) and 3 parts by weight of water, tableting is performed. Tableting was carried out using a machine (Fuji Yakuhin Co., Ltd.) to obtain a tablet-type anti-fatigue agent.

(Preparation of tablets containing iron-binding lactoferrin)
After sufficiently mixing 36 parts by weight of iron-bound lactoferrin obtained by the method of Example 2, 36 parts by weight of lactose (DMV), 25 parts by weight of crystalline cellulose (manufactured by Wako Pure Chemical Industries), and 3 parts by weight of water Tableting was performed with a tableting machine (Fuji Pharmaceutical Co., Ltd.) to obtain a tablet-type anti-fatigue agent.

(Preparation of soft drink containing anti-fatigue agent)
Commercially available lactoferrin (manufactured by DMV) 0.1 parts by weight, 50% lactic acid solution 0.12 parts by weight, maltitol 7.5 parts by weight, perfume 0.2 parts by weight, water 92.08 parts by weight were mixed, using a plate type heat sterilizer at 90 ° C. Sterilized for 15 seconds to produce a soft drink with anti-fatigue effect.

(Preparation of soft drink containing anti-fatigue agent)
Mixing 0.1 parts by weight of iron-bound lactoferrin obtained by the method of Example 2, 0.12 parts by weight of 50% lactic acid solution, 7.5 parts by weight of maltitol, 0.2 parts by weight of fragrance, and 92.08 parts by weight of water, It was sterilized at 90 ° C. for 15 seconds to produce a soft drink having an anti-fatigue effect.

(Preparation of milk beverages containing anti-fatigue agents)
The lactoferrin obtained by the method of Example 1 is added to raw milk so as to be 100 mg / 100 g, homogenized at 150 kgf / cm ² , sterilized at 130 ° C. for 2 seconds using a plate type heat sterilizer, A milk beverage having an anti-fatigue effect was produced.

(Preparation of milk beverage with iron-binding lactoferrin added)
The iron-binding lactoferrin obtained by the method of Example 2 is added to raw milk so as to be 100 mg / 100 g, homogenized at 150 kgf / cm ² , and sterilized at 130 ° C. for 2 seconds using a plate sterilizer. A milk beverage having an anti-fatigue effect was produced.

Claims (2)

  An anti-fatigue agent comprising lactoferrin as an active ingredient. The anti-fatigue agent according to claim 1, wherein the lactoferrin is iron-bound lactoferrin in which lactoferrin and iron are bound.