JP2006219458A - Agent for inhibiting ischemic enteropathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine and/or food and drink having preventing and/or treating effect on ischemic enteropathy, having low side action and high safety and ingestible over a long period. <P>SOLUTION: The agent for the prevention and/or treatment of ischemic enteropathy contains milk whey protein and/or hydrolyzed milk whey protein as an active component. The invention further provides a food or drink containing milk whey protein and/or hydrolyzed milk whey protein, having preventing and/or treating effect on ischemic enteropathy and labeled to notice that the food, etc., is to be used for the prevention and/or treatment of ischemic enteropathy. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a prophylactic and / or therapeutic agent for ischemic bowel disease and / or a food or drink comprising whey protein and / or whey protein hydrolyzate as an active ingredient.

  Ischemic enteritis occurs due to low blood flow (shock), major arteries, small vascular lesions, venous occlusion, or ileus. In each case, the mucosa and submucosa are damaged depending on the extent and duration of ischemia. This process causes inflammation and mucosal ulcers. The bent portion is a watershed-dominated watershed, and is particularly susceptible to ischemic enteritis (Non-patent Document 1).

  Causes of shock include 1) hemorrhagic shock due to increased loss of circulating blood volume due to trauma, surgery and gastrointestinal bleeding, 2) burn shock due to massive loss of plasma components due to burns, 3) diarrhea, Dehydration shock due to loss of large amounts of water and electrolytes from the kidney and skin due to vomiting and diabetic acidosis 4) Rapid cardiac output associated with acute myocardial infarction, acute myocarditis, pulmonary infarction, and cardiac tamponade Cardiogenic shock caused by a decline, 5) Infectious shock caused by sepsis, 6) Cerebral injury, traumatic mental tension, spinal cord injury, vasomotor reflex hypotension, vasodilation, bradycardia 7) Neurogenic shock caused by the occurrence of histamine is released from mast cells due to allergic reactions caused by food, drugs, blood products, etc., causing vasodilation, laryngeal edema, asthma-like dyspnea The accompanying anaphylactic shock etc. are mentioned (nonpatent literature 2).

  When the physical barriers such as intestinal mucus and tight junctions and the immunological barrier caused by IgA fail due to the lack of enteral nutrition, the reduction of bacterial translocation and systemic mucosal defense mechanism is reduced. It causes inflammatory response in the whole body and infectious diseases.

  Pathogens or toxins that enter the body activate cells of the immune system and release various inflammatory mediators. The function of these mediators and the action of the nerve and endocrine system increase local and systemic inflammation, exhibiting the so-called hyper dynamic state (increased cardiac output, increased heart rate, peripheral vasodilation), immune system The cells are further activated. These reactions are inherently indispensable as biological reactions at the time of infection, and are useful for enhancing the biological defense ability. However, anorexia associated with infection, energy production, peripheral skeletal muscle breakdown, hypermetabolism such as increased gluconeogenesis and lipolysis in the liver, and loss of body tissue due to the state of hyper catabolism, exacerbation of infection, It leads to organ failure and an unfortunate outcome.

  The most common complications observed during enteral nutrition after major invasive surgery are abdominal symptoms such as diarrhea, abdominal distension, and abdominal pain. About 60% of patients complain of abdominal symptoms, including mild abdominal symptoms. Massive invasive surgery results in hypercytokinemia, and gastrointestinal mucosal cells fall off due to the direct action of cytokines, which may be involved in abdominal symptoms during enteral nutrition. It has been reported that the frequency of abdominal symptoms is high in cases in which the post-operative hypercytokinemia period is prolonged (Non-patent Document 3).

  In recent years, inflammatory cytokines such as TNF-α and IL-6 have been reported to be involved in the undernutrition of various diseases. In particular, it is known that blood IL-6 rises with the progress of gastrointestinal diseases and correlates well with serum CEA and serum CRP. Hypercytokinemia associated with an increase in serum CRP is thought to increase resting energy consumption and cause undernutrition (Non-patent Document 4).

  IL-6 is produced from various cells such as T / B cells, fibroblasts, vascular endothelial cells, and macrophages. IL-6 has been reported to be elevated in mRNA and protein, and in serum level, in the active mucosa of active Crohn's disease. Considering that TNF-α has an effect of inducing IL-6 production, there is a possibility that TNF-α and IL-6 are deeply involved in the pathophysiology of gastrointestinal inflammation (non-patent literature). 5). By regulating the production of inflammatory cytokines, various effects including prevention and / or treatment of ischemic bowel disease can be expected.

  Prior art documents related to preparations and foods and drinks that control the production of inflammatory cytokines include human antibodies that bind to human TNF-α (Patent Document 1), disease treatment methods involving cytokines containing nucleosides as active ingredients ( Patent Document 2, Patent Document 3) and the like are disclosed. Although human antibodies that bind to human TNF-α have the effect of neutralizing TNF-α activity in vitro and in vivo, they do not control the cytokine production itself from the immune-competent cells of the intestinal tract associated with ischemia. Further, in such human antibodies that bind to human TNF-α, the appearance of neutralizing antibodies by multiple administrations becomes a problem that the effect is attenuated.

  In an ischemic bowel disease, undernutrition is caused in the pathological condition, so that “nutrition therapy” is always considered when selecting a treatment. Specifically, a nutrient component whose main source is an amino acid having a lower antigenicity is used instead of a protein having an antigenic nitrogen source. However, since the component nutrients have higher osmotic pressure than general nutrients and semi-digested nutrients, abdominal symptoms such as diarrhea, abdominal fullness, and abdominal pain are likely to occur. Therefore, when abdominal symptoms are exhibited, the administration rate of the component nutrients is slowed down and the total dose is reduced, but there is a problem that calorie consumption is insufficient.

Whey protein is a protein fraction present in a soluble fraction (whey) when milk is adjusted to pH 4.6 at 20 ° C., for example, in the case of milk, α-lactalbumin, β-lactoglobulin, serum Albumin, immunoglobulin, lactoferrin, transferrin, proteose peptone, β ₂ -microglobulin and the like are included as whey proteins (Non-patent Document 6).

  α-Lactalbumin is a globular protein with a molecular weight of 14,000 that accounts for about 25% of whey protein. Structural analysis of the protein shows structural similarity with lysozyme having lytic activity, and the gene structure is known to have high homology. (Non-patent document 6) α-lactalbumin is also a calcium-binding protein. Furthermore, since α-lactalbumin has a high cystine content and the protein of the casein fraction contains less cystine, α-lactalbumin has the nutritional appeal of supplementing it. In addition, α-lactalbumin is known to have a gastrointestinal motility regulating action (Patent Document 4), a small intestinal injury prevention or repair promoting action (Patent Document 5), and an anti-ulcer action (Non-Patent Document 7).

  β-Lactoglobulin is a globular protein with a molecular weight of 18,000 that accounts for about 50% of whey protein. Protein structure analysis shows that the structure is very similar to vitamin A (retinol) binding protein in serum (Non-patent Document 6). The analysis of both gene structures shows a remarkable similarity, and they are considered to be proteins with a common origin. It has been shown that β-lactoglobulin is responsible for transporting vitamin A to the small intestine in vivo, and absorption of vitamin A from the small intestine is promoted by β-lactoglobulin. Recently, it has been shown that this absorption enhancement is mediated by a receptor selective for β-lactoglobulin. Also, β-lactoglobulin has an intestinal permeation inhibitory action (Patent Document 6), a lipid metabolism improving action (Patent Document 7), an antioxidant action (Patent Document 8), and a melanin production inhibitory action (Patent Document 9). ing.

  However, there has been no report that α-lactalbumin, β-lactoglobulin, or a hydrolyzate thereof has an effect of preventing and / or treating ischemic bowel disease.

α-Lactalbumin, β-Lactoglobulin, or their degradation products and peptides have been reported to be useful for oral immunonutrition therapy during living body invasion, but this action prevents cytokine production by lipopolysaccharide stimulation in macrophages. It has a suppressing effect (Patent Document 10). In addition, the effect of inhibiting cytokine production by lipopolysaccharide stimulation in macrophages (Patent Document 11), the effect of inhibiting gastric excretion and the ability to transport the small intestine based on the action of regulating gastrointestinal motility (Patent Document 4), and diarrhea caused by lactose Prevention and repair promotion effect of infectious diseases and small intestinal tract injury (Patent Document 5), gastric ulcer inhibitory action by water invasion restraint stress (Non-Patent Document 7), prevention of food allergies and the like through suppression of permeation of allergen substances in the intestine and Therapeutic effects (Patent Document 6), lipid metabolism improving action (Patent Document 7), antioxidant action (Patent Document 8), melanin production inhibitory action (Patent Document 9), etc. have been reported. It does not disclose the effect of preventing and / or treating ischemic bowel disease based on the action of controlling the intestinal immune system in the disease.
Special Table 2000-507810 Special Table 2002-515892 Japanese Patent No. 3479077 JP 2004-231643 A Japanese Patent No. 3529770 Japanese Unexamined Patent Publication No. 08-073375 Japanese Patent Laid-Open No. 08-259461 JP 2000-104064 A Japanese Patent Laid-Open No. 10-218755 JP 2004-155751 A JP 2004-196707 A Edited by Masanori Fukushima, “Merck Manual 17th Edition, Japanese Edition”, Nikkei BP Section 3 Gastrointestinal Diseases (1999) Naoki Aikawa, Katsunori Aoki, "SIRS / Shock / MODS", School of Medicine, pp.86-97, (2001) Yoshifumi Inoue, National Questionnaire Survey Report on Nutrition Therapy Implementation Status (1), Parenteral Enteral Nutrition, 19 (1), pp.41-53, (2004) Akihiko Kato, The relationship between nutritional status and inflammatory response before cancer treatment, Journal of Japanese Society of Pathophysiology, 7 (2), pp.113-117 (2004) Izumi H, Mechanism of production and release of tumor necrosis factor implicated in inflammatory diseases., Folia Pharmacol Jpn, 121 (3), pp.163-173 (2003) Kunio Yamauchi, Kenkichi Yokoyama, `` Milk Comprehensive Dictionary '', Asakura Shoten, pp.33-38 (1997) Ushida Y, Shimokawa Y, Matsumoto H, Toida T, Hayasawa H, Effects of Bovine α-Lactalbumin on Gastric Defense Mechanisms in Naive Rats., Biosci Biotech Biochem, 67 (3), pp. 577-583 (2003)

  Conventionally, in general treatment of ischemic bowel disease, initial treatment is performed by infusion (infusion), and when it is determined that it is necessary, blood transfusion is performed and lifesaving treatment is performed. If bleeding is massive, a minimal amount of blood transfusion may be done to save lives. The standard treatment for ischemic bowel disease in the upper gastrointestinal tract is to stop bleeding (endoscopic hemostasis) with an endoscope after initial treatment and diagnosis. Most ischemic bowel diseases of the lower gastrointestinal tract can be improved by conservative treatment such as refraining from infusion and infusion. Steroid therapy is given to cases with severe inflammation.

  As described above, symptomatic therapies such as infusion and blood transfusion are the mainstream in the pathological conditions in which rest of the living body or digestive tract that has been greatly invaded is essential. In addition, side effects such as obesity, hypertension, diabetes, and infectious diseases appear in steroids used in severely inflammatory cases, fractures easily occur, and mental states may become unstable.

  The present invention has been made in view of the circumstances described above, has few side effects, has high safety, can be ingested over a long period of time, and is ischemic that can be applied to medicines and foods and drinks An object of the present invention is to provide a therapeutic agent for intestinal diseases.

  The present inventor has played an important role in the generation of inflammatory cytokines due to circulatory disturbance in the intestinal tract and the translocation of enteric bacteria due to decreased mucosal barrier function in the pathogenesis of multiple organ failure due to shock. As a result of earnest research for the purpose of preventing the inflammatory reaction and reducing the symptoms by controlling the production of inflammatory cytokines, the whey protein α-lactalbumin and / or β -Lactoglobulin has been found to have an effect of remarkably suppressing the production of inflammatory cytokines resulting from ischemic enteritis, leading to the completion of the present invention.

That is, the present invention
[1] A prophylactic and / or therapeutic agent for ischemic bowel disease comprising whey protein and / or whey protein hydrolyzate as an active ingredient,
[2] Prophylaxis and / or treatment of ischemic bowel disease characterized by containing whey protein and / or whey protein hydrolyzate and having an effect of preventing and / or treating ischemic bowel disease Food and drink with a sign indicating that it is used for
[3] The pharmaceutical or the food or drink according to any one of [1] to [2], wherein the whey protein is α-lactalbumin and / or β-lactoglobulin,
[4] The pharmaceutical product or food or drink according to any one of [1] to [3], which is for a patient after invasive surgery,
[5] The pharmaceutical or the food or drink according to any one of [1] to [3], which is for a patient with ischemic bowel disease presenting undernutrition,
Is to provide.

The effects produced by the intake of the product of the present invention are as follows.
(1) By suppressing the production of inflammatory cytokines caused by ischemic enteritis, it is possible to enjoy the preventive and / or therapeutic effect of gastrointestinal inflammation and the preventive and / or therapeutic effect of systemic inflammatory reaction.
(2) Sufficient nutritional management is possible by daily administration or ingestion.
(3) There are few side effects, it is safe and can be taken for a long time.
(4) It can be mixed with foods and drinks or pharmaceuticals, and is more versatile than conventional therapeutic agents.
(5) It can be obtained from relatively inexpensive raw materials such as milk and can be mass-produced.

Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. Whey protein is a protein fraction present in a soluble fraction (whey) when milk is adjusted to pH 4.6 at 20 ° C., for example, in the case of milk, α-lactalbumin, β-lactoglobulin, serum Albumin, immunoglobulin, lactoferrin, transferrin, proteose peptone, β ₂ -microglobulin and the like are included as whey proteins. As the whey protein, α-lactalbumin and β-lactoglobulin are preferable, which are present in a soluble fraction when milk is adjusted to pH 4.6 at 20 ° C. and have a property of being insolubilized by heating. The amino acid sequences of these α-lactalbumin and β-lactoglobulin vary depending on the species. The amino acid sequence and protein_id are registered in the database of European Molecular Biology Laboratory (EMBL). For example, the protein_ids of α-lactalbumin and β-lactoglobulin derived from milk are AAA30615.1 and CAA32835.1, respectively. The CAS Nos. Of α-lactalbumin and β-lactoglobulin derived from milk are 9051-29-0 and 9045-23-2. The contents of α-lactalbumin and β-lactoglobulin in milk vary somewhat depending on the place of production and feed, but are usually about 1.2 g / kg and 3.2 g / kg for domestic milk. The content of α-lactalbumin in human milk is usually 1.6 g / kg, although there are some differences depending on the race. β-lactoglobulin is not detected in human milk.

  In order to prepare α-lactalbumin and β-lactoglobulin of the present invention, milk, whey protein, etc. derived from animals such as humans, cows, sheep, goats, etc., or purified products thereof can be obtained. Moreover, as long as it contains α-lactalbumin or β-lactoglobulin, it may be derived from plants or microorganisms. Alternatively, it may be obtained by known chemical synthesis, synthesis using enzymes or microorganisms, or the like.

  The α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate of the present invention are prepared by enzymatic or chemical hydrolysis of the α-lactalbumin and β-lactoglobulin preparations described above. can do. Alternatively, it may be obtained by known chemical synthesis, synthesis using enzymes or microorganisms, or the like.

  As a method for enzymatically preparing α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate, for example, it can be produced by the method described later, but the present invention is not limited to this method. As long as the product can be prepared, other methods may be used.

First, α-lactalbumin or β-lactoglobulin is dispersed and dissolved in water or hot water. The concentration of the α-lactalbumin or β-lactoglobulin solution is not particularly limited, but if it is mentioned, it is preferably within a concentration range of about 5 to 15% in terms of protein. The pH of the α-lactalbumin or β-lactoglobulin solution is not particularly limited, but is preferably adjusted to the optimum pH of the enzyme or in the vicinity thereof. If the pH is adjusted, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium hydrogen carbonate, or an alkali or acid such as hydrochloric acid, citric acid, phosphoric acid, acetic acid or carbonic acid can be added.
Next, a hydrolase is added to the α-lactalbumin solution or β-lactoglobulin solution. The hydrolase is not particularly limited as long as it is an enzyme that hydrolyzes proteins. Food grade protein hydrolases (proteases) include endo- and exo-proteases and exo-peptidase / endo-protease complex enzymes. Endo-type proteases include, for example, AlcalaseR (from Bacillus licheniformis), Esperase (from B. lentus), NeutraseR (from B. subtilis), Protamex (bacteria), PTN6.0S (porcine pancreatic trypsin), etc. Examples of the peptidase / endo-type protease complex enzyme include flavorzyme (derived from Aspergillus oryzae) and the like (above Novo). In addition, as endo-type proteases, for example, chymotrypsin (Novo, Boehringer), protease N Amano (from Bacillus subtilis, Amano Enzyme), biolase (from Bacillus subtilis, Nagase Sangyo), papain W-40 (Amano Enzyme), Examples of the exo-type protease include carboxypeptidase derived from porcine or bovine viscera. These enzymes are not meant to be limiting. A hydrolase may combine 1 type, or 2 or more types of enzymes. When combining, each enzyme reaction may be performed simultaneously or separately.
Next, the solution to which the hydrolase is added is maintained at an appropriate temperature according to the type of enzyme, and hydrolysis is started. The enzyme reaction temperature is not particularly limited, but is preferably adjusted to the enzyme optimum temperature or the vicinity thereof. The hydrolysis reaction time is not particularly limited, and the reaction time is preferably set while evaluating the average molecular weight and molecular weight distribution after the enzyme reaction. In the present invention, the average molecular weight is preferably 500 to 3000, more preferably 800 to 1000. The molecular weight distribution is preferably from 300 to 5,000, more preferably from 500 to 3,000.
The enzyme reaction can be stopped by enzyme deactivation during hydrolysis, that is, chemical deactivation treatment, heat deactivation treatment, or the like.

  The solution containing α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate thus obtained can be used as it is, and is also necessary. Depending on the above, this solution can be used as a solution concentrated or diluted by a known method. Furthermore, this concentrated liquid can also be used as a dried product by a known method. Depending on the mode of use, these can be used singly or in combination.

The α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate, and β-lactoglobulin hydrolyzate of the present invention are useful for the control of systemic inflammatory response syndrome for the prevention of organ failure due to invasion. Expectations are also expected for use in the production of enteral nutrients. It can also be used for preoperative nutritional management. Specifically, it is an oral and enteral nutrient for surgery, trauma, burns, acute pancreatitis, infections, peritonitis, malignant tumors and the like.
In addition, α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate, and β-lactoglobulin hydrolyzate of the present invention are used for treatment, prevention, or improvement of diseases involving inflammatory cytokines such as IL-6. It is expected to be effective. Diseases involving inflammatory cytokines such as IL-6 are caused by overproduction of inflammatory cytokines such as IL-6. Examples of such diseases include inflammatory diseases (retinopathy, nephropathy, nerves). Diabetic complications such as disorders and macrovascular disorders; rheumatoid arthritis, osteoarthritis, rheumatoid myelitis, arthritis such as gouty arthritis, periosteitis; low back pain; inflammation after surgery / traumatic injury; Neuralgia; sore throat; cystitis; pneumonia; atopic dermatitis; inflammatory bowel disease such as Crohn's disease and ulcerative colitis; meningitis; inflammatory eye disease; pneumonia; Pulmonary diseases, etc.), cardiovascular diseases (eg, angina pectoris, myocardial infarction, congestive heart failure, generalized intravascular coagulation syndrome), asthma allergic disease, chronic obstructive pulmonary disease, systemic lupus erythematosus, Crohn's disease, self Immune hemolytic anemia, , Neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, AIDS encephalopathy, etc.), central nervous system disorders (eg, cerebrovascular disorders such as cerebral hemorrhage and cerebral infarction, head trauma, spinal cord injury, Brain edema, multiple sclerosis, etc.), venomemia (eg, sepsis, septic shock, endotoxin shock, Gram-negative bacterial sepsis, toxin shock syndrome), Addison's disease, Creutzfeldt-Jakob disease, viral infection (cytoro) Virus infections such as megavirus, influenza virus, herpes virus), transplant rejection, dialysis hypotension, osteoporosis and the like.

  In the present embodiment, the intake for enjoying the effect of preventing and / or treating ischemic bowel disease varies depending on the dosage form, symptoms, body weight, etc., and thus is not particularly limited. 30 to 2000 mg can be ingested in terms of conversion, preferably 100 to 1000 mg in terms of protein per kg of body weight per day, more preferably 200 to 500 mg in terms of protein per kg of body weight per day.

The α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate, and β-lactoglobulin hydrolyzate of the present invention can be used in any form of pharmaceuticals or foods and drinks. For example, it is expected to treat and / or prevent various ischemic enteritis by directly administering it as a pharmaceutical, or by directly ingesting it as a special-purpose food such as a food for specified health use or a nutritional functional food. Add to various foods (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, formula milk, liquid foods, food for the sick, nutritional foods, etc.) Also good.
The food containing α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate, β-lactoglobulin hydrolyzate of the present invention includes water, protein, carbohydrate, lipid, vitamins, minerals, Organic acids, organic bases, fruit juices, flavors and the like can be used as main components. Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, their degradation products; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose, etc. And various milk-derived components. Examples include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used in combination of two or more, and synthetic products and / or foods containing a large amount thereof may be used. The form of food may be solid or liquid. It may be in the form of a gel.

  When the α-lactalbumin, β-lactoglobulin, α-lactalbumin hydrolyzate, or β-lactoglobulin hydrolyzate of the present invention is used as a pharmaceutical, it can be administered in various forms. Examples of the form include oral administration using tablets, capsules, granules, powders, syrups and the like. These various preparations can be generally used in the pharmaceutical formulation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. as main ingredients according to conventional methods. It can be formulated using known adjuvants.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this.

[Example 1] (Production method of hydrolyzate of α-lactalbumin and hydrolyzate of β-lactoglobulin)
5 g of commercially available α-lactalbumin (hereinafter also referred to as αLA: Davisco Foods International, Inc.) and β-lactoglobulin (hereinafter also referred to as βLG: Davisco Foods International, Inc.) were each dissolved in 50 ml of water. After adjusting the pH of the solution to about 7, 25 mg of trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred, and immersed in a 37 ° C. warm bath for hydrolysis for 5 hours. After hydrolysis, the permeated solution with an ultrafiltration membrane having a cutoff value of 10,000 was collected and concentrated with an evaporator to obtain a concentrated solution. The α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate thus obtained had an average molecular weight of about 800 and a molecular weight distribution of about 500 to 3000.

[Example 2] (Effect of whey protein on inflammatory cytokine production)
This test was conducted to evaluate the effects of α-lactalbumin and β-lactoglobulin, which are active ingredients of the preventive and / or therapeutic agent for ischemic bowel disease of the present invention, on inflammatory cytokine production. The model of ischemic bowel disease is the method of Chen et al. (Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF, Karin M, The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion., Nature Medicine, 9 (5), pp.575-581 (2003)), and evaluated as an effect based on the inflammatory cytokine production inhibitory effect.

(1) Test animal Seven-week-old male SD rats (purchased from CLEA Japan, Inc.) were randomly divided into 5 groups (5 animals per group) and used after preliminary breeding for 1 week.

(2) Sample Preparation Commercial saline α-lactalbumin (hereinafter also referred to as αLA: Davisco Foods International, Inc.) and β-lactoglobulin (hereinafter also referred to as βLG: Davisco Foods International, Inc.) The test samples were prepared by adding 150 mg / ml each. For the control test, bovine serum albumin (hereinafter also referred to as BSA: Sigma-Aldorich Co.) and casein (sodium casein: Wako Pure Chemical Industries, Ltd.) were prepared to 150 mg / ml each as described above, A control sample was used. The negative sample was physiological saline.

(3) Test method An intestinal ischemia reperfusion model was prepared by the superior mesenteric artery clamp method using rats (SD, male, 7 weeks old, each group n = 5). For blood sampling over time, a catheter was attached to the jugular vein under surgical anesthesia by subcutaneous administration of ethyl carbamate (1 g / kg). The jugular vein catheter was placed in a state filled with heparinized physiological saline. In addition, 6 groups were established: a physiological saline administration group (control group), a βLG administration group, an αLA administration group, a casein administration group, a BSA administration group, and a group that performed only surgical procedures (Sham group). 300 mg / kg (2 ml / kg) was administered into the duodenum. One hour after administration of the test sample, in the groups other than the Sham group, ischemia was performed for 45 minutes by clamping the superior mesenteric artery, and ischemia reperfusion was performed by removing the clamp and performing reperfusion for 3 hours. At 0, 1, 2, and 3 hours after the start of reperfusion, blood was collected from each jugular vein catheter by about 300 μl, and serum was obtained by centrifugation (20,000 × g, 20 minutes). After appropriate dilution, the IL-6 concentration was measured by enzyme immunoassay (rat cytokine ELISA system, IL-6 / Amersham Biosciences). The average value ± standard deviation of the values of 5 animals for each group was determined and used as the test result for each group.

(4) Test results The results of this test are shown in FIG. When βLG and αLA were administered into the duodenum 1 hour before ischemia, production of IL-6 was suppressed in both cases compared to the control group. For IL-6 production at 3 hours after reperfusion, a significant difference test was conducted with Mann-Whitney's U-test using a negative sample as a control. As a result, βLG was significantly significant at 5% and αLA was significantly significant at 1%. -6 production was found to be suppressed. On the other hand, when casein and BSA, which are control samples, were administered into the duodenum 1 hour before ischemia, no significant difference was observed in the Mann-Whitney's U-test, as in the control group.

[Example 3] (Effects of α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate on inflammatory cytokine production)
This test was conducted to evaluate the effects of α-lactalbumin hydrolyzate and β-lactoglobulin hydrolyzate, which are active ingredients of the agent for preventing and / or treating ischemic bowel disease of the present invention, on inflammatory cytokine production. went. In addition, preparation of an ischemic bowel disease model and evaluation of the inflammatory cytokine production inhibitory effect were performed in the same manner as in Example 2.

(1) Test animal Seven-week-old male SD rats (purchased from CLEA Japan, Inc.) were randomly divided into 5 groups (5 animals per group) and used after preliminary breeding for 1 week.

(2) Preparation of Samples Physiologically to α-lactalbumin hydrolyzate (hereinafter also referred to as αLA-HP) and β-lactoglobulin hydrolyzate (hereinafter also referred to as βLG-HP) produced in the same manner as in Example 1. Saline was added to prepare 150 mg / ml each, and used as test samples. In addition, commercially available αLA (Davisco Foods International, Inc.) and βLG (Davisco Foods International, Inc.) were prepared to 150 mg / ml each using physiological saline, and used as comparative samples. The negative sample was physiological saline.

(3) Test method An intestinal ischemia reperfusion model was prepared by the superior mesenteric artery clamp method using rats (SD, male, 7 weeks old, each group n = 5). For blood sampling over time, a catheter was attached to the jugular vein under surgical anesthesia by subcutaneous administration of ethyl carbamate (1 g / kg). The jugular vein catheter was placed in a state filled with heparinized physiological saline. In addition, 5 groups were established: physiological saline administration group (control group), βLG administration group, αLA administration group, βLG-HP administration group, αLA-HP administration group, and each test sample was 300 mg / kg (2 ml / kg). Each was administered into the duodenum. One hour after administration of the test sample, the superior mesenteric artery was clamped for 45 minutes, and the clamp was removed and reperfusion was performed for 3 hours to perform ischemia reperfusion. At 0, 1, 2, and 3 hours after the start of reperfusion, blood was collected from each jugular vein catheter by about 300 μl, and serum was obtained by centrifugation (20,000 × g, 20 minutes). After appropriate dilution, the IL-6 concentration was measured by enzyme immunoassay (rat cytokine ELISA system, IL-6 / Amersham Biosciences). The average value ± standard deviation of the values of 5 animals for each group was determined and used as the test result for each group.

(4) Test results The results are shown in FIG. αLA-HP and βLG-HP showed an IL-6 production inhibitory effect equivalent to αLA and βLG, which are respective undegraded protein materials. IL-6 production at 3 hours after reperfusion was tested for significant difference with Mann-Whitney's U-test using negative samples as a control. As a result, βLG-HP was 5% significant and αLA-HP was 1% significant. Was found to significantly suppress IL-6 production.

[Example 4] (Effective amount of α-lactalbumin for inflammatory cytokine production)
This test was conducted in order to evaluate the effective amount of α-lactalbumin, which is an active ingredient of the agent for preventing and / or treating ischemic bowel disease of the present invention, against inflammatory cytokine production. In addition, preparation of an ischemic bowel disease model and evaluation of the inflammatory cytokine production inhibitory effect were performed in the same manner as in Example 2.

(1) Test animal Seven-week-old male SD rats (purchased from CLEA Japan, Inc.) were randomly divided into 5 groups (5 animals per group) and used after preliminary breeding for 1 week.

(2) Preparation of sample Saline was added to commercially available α-lactalbumin (αLA: Davisco Foods International, Inc.) to prepare 15, 50, 150 mg / mL and used as test samples. The negative sample was physiological saline.

(3) Test method An intestinal ischemia reperfusion model was prepared by the superior mesenteric artery clamp method using rats (SD, male, 7 weeks old, each group n = 5). For blood sampling over time, a catheter was attached to the jugular vein under surgical anesthesia by subcutaneous administration of ethyl carbamate (1 g / kg). The jugular vein catheter was placed in a state filled with heparinized physiological saline. Furthermore, 4 groups were established: a physiological saline administration group (control group), an αLA 30 mg / kg administration group, an αLA 100 mg / kg administration group, an αLA 300 mg / kg administration group, and αLA of 30 mg / kg, 100 mg / kg, 300 mg / kg ( All were administered into the duodenum at a dose volume of 2 ml / kg). One hour after administration of the test sample, the superior mesenteric artery was clamped for 45 minutes, and the clamp was removed and reperfusion was performed for 3 hours to perform ischemia reperfusion. At 0, 1, 2, and 3 hours after the start of reperfusion, blood was collected from each jugular vein catheter by about 300 μl, and serum was obtained by centrifugation (20,000 × g, 20 minutes). After appropriate dilution, the IL-6 concentration was measured by enzyme immunoassay (rat cytokine ELISA system, IL-6 / Amersham Biosciences). The average value ± standard deviation of the values of 5 animals for each group was determined and used as the test result for each group.

(4) Test results The results are shown in FIG. Analysis using Mann-Whitney's U-test with a negative sample as a control revealed that a significant IL-6 production inhibitory effect was obtained when the dose was 100 mg / kg or more.

[Example 5] (Mechanism of action of α-lactalbumin on inflammatory cytokine production)
This test was conducted to evaluate the action mechanism of α-lactalbumin, which is an active ingredient of the preventive and / or therapeutic agent for ischemic bowel disease of the present invention, on inflammatory cytokine production. Specifically, it was verified whether stabilization of the NF-κB / IκB complex by NO (nitrogen monoxide) is involved in the anti-inflammatory action mechanism by α-lactalbumin.

(1) Test animal Seven-week-old male SD rats (purchased from CLEA Japan, Inc.) were randomly divided into 5 groups (5 animals per group) and used after preliminary breeding for 1 week.

(2) Preparation of sample Commercially available α-lactalbumin (αLA: Davisco Foods International, Inc) as a test sample, and N ^G -nitro-L-arginine-methyl ester (hereinafter also referred to as L-NAME) as a NO synthesis inhibitor: Wako Pure Chemical Industries, Ltd.) was used by dissolving in physiological saline. The negative sample was physiological saline.

(3) Test method An intestinal ischemia reperfusion model was prepared by the superior mesenteric artery clamp method using rats (SD, male, 7 weeks old, each group n = 5). For blood sampling over time, a catheter was attached to the jugular vein under surgical anesthesia by subcutaneous administration of ethyl carbamate (1 g / kg). The jugular vein catheter was placed in a state filled with heparinized physiological saline. In addition, 4 groups were set: physiological saline administration group (control group), αLA 300 mg / kg administration group, αLA 300 mg / kg + L-NAME 1 mg / kg administration group, L-NAME 1 mg / kg administration group, αLA 300 mg / kg, αLA 300 mg / kg + L-NAME 1 mg / kg and L-NAME 1 mg / kg were administered into the duodenum at a dose volume of 2 ml / kg. One hour after administration of the test sample, the superior mesenteric artery was clamped for 45 minutes, and the clamp was removed and reperfusion was performed for 3 hours to perform ischemia reperfusion. At 0, 1, 2, and 3 hours after the start of reperfusion, blood was collected from each jugular vein catheter by about 300 μl, and serum was obtained by centrifugation (20,000 × g, 20 minutes). After appropriate dilution, the IL-6 concentration was measured by enzyme immunoassay (rat cytokine ELISA system, IL-6 / Amersham Biosciences). The average value ± standard deviation of the values of 5 animals for each group was determined and used as the test result for each group.

(4) Test results The results of this test are as shown in FIG. The anti-inflammatory effect by αLA was partially lost by L-NAME. L-NAME 1 mg / kg used here is a single ineffective dose. NO has been reported to promote the stabilization of the NF-κB / IκB complex and to inhibit the binding of NF-κB to the transcriptional region of inflammatory cytokines such as IL-6. (Stefano GB, Goumon Y, Bilfinger TV, Welters ID, Cadet P., Basal nitric oxide limits immune, nervous and cardiovascular excitation: human endothelia express a mu opiate receptor., Prog Neurobiol. Apr; 60 (6): 513-30 ., (2000))
From this, it was confirmed that a mechanism mediated by NO is partly involved in the anti-inflammatory effect of αLA.

  The α-lactalbumin and β-lactoglobulin or their hydrolysates, which are active ingredients in the present invention, can be mass-produced from relatively inexpensive raw materials such as milk. In addition, since it has few side effects, is highly safe, and can be ingested over a long period of time, it can be mixed with pharmaceuticals or ingested as food to produce inflammatory cytokines caused by ischemic enteritis It is possible to enjoy the effect of preventing and / or treating gastrointestinal inflammation and the effect of preventing and / or treating systemic inflammatory reaction. In addition, since sufficient nutritional management is possible by daily administration or ingestion, it can be applied to applications such as the production of functional foods for post-operatively invasive patients and patients with undernutrition. is there.

It is a figure which shows the inhibitory effect with respect to inflammatory cytokine production of (beta) -lactoglobulin and (alpha) -lactalbumin. The mean ± standard deviation (n = 5) of serum IL-6 concentration (ng / ml) over time when β-lactoglobulin or α-lactalbumin is pre-administered to an ischemic bowel disease model rat is shown. *: P <0.05 (vs. control, Mann-Whitney's U-test), **: p <0.01 (vs. control, Mann-Whitney's U-test), #: p <0.05 (vs. casein, Mann-Whitney's U-test), ##: p <0.01 (vs. casein, Mann-Whitney's U-test). It is a figure which shows the inhibitory effect with respect to inflammatory cytokine production of (alpha) -lactalbumin enzyme hydrolyzate and (beta) -lactoglobulin enzyme hydrolyzate. Mean value ± standard deviation of serum IL-6 concentration (ng / ml) over time when pre-administration of α-lactalbumin enzyme hydrolyzate or β-lactoglobulin enzyme hydrolyzate to ischemic bowel disease model rats ( n = 5). *: P <0.05 (vs. control, Mann-Whitney's U-test), **: p <0.01 (vs. control, Mann-Whitney's U-test). It is a figure which shows the dose dependence of the inhibitory effect with respect to inflammatory cytokine production of (alpha) -lactalbumin. The mean ± standard deviation (n = 5) of serum IL-6 concentration (ng / ml) over time when each dose of α-lactalbumin was pre-administered to ischemic bowel disease model rats is shown. *: P <0.05 (vs. control, Mann-Whitney's U-test), **: p <0.01 (vs. control, Mann-Whitney's U-test). It is a figure which shows the action mechanism of the inhibitory effect with respect to inflammatory cytokine production of (alpha) -lactalbumin. Serum IL-6 concentration over time (ng / ml) when pre-administered with α-lactalbumin alone or in combination with α-lactalbumin alone and NO synthesis inhibitor (L-NAME) to rat model of ischemic bowel disease ) Mean value ± standard deviation (n = 5). *: P <0.05 (vs. control, Mann-Whitney's U-test), **: p <0.01 (vs. control, Mann-Whitney's U-test), #: p <0.05 (vs. casein, Mann-Whitney's U-test), ##: p <0.01 (vs. casein, Mann-Whitney's U-test).

Claims (5)

  A prophylactic and / or therapeutic agent for ischemic bowel disease comprising whey protein and / or whey protein hydrolyzate as an active ingredient.   It contains whey protein and / or whey protein hydrolyzate, and has a preventive and / or therapeutic effect on ischemic bowel disease, for the prevention and / or treatment of ischemic bowel disease A food or drink with an indication that it is used.   Whey protein is alpha-lactalbumin and / or beta-lactoglobulin, The pharmaceutical or food-drinks of any one of Claims 1-2.   The pharmaceutical product or food or beverage according to any one of claims 1 to 3, which is for a patient after invasive surgery.   The pharmaceutical or food or drink according to any one of claims 1 to 3, which is for patients with ischemic bowel disease presenting undernutrition.

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