JP4278028B2 - Peptide having inflammatory cytokine production inhibitory activity - Google Patents

Description

【００４７】
［試験例４］ IL-6産生抑制作用
6週齢のICRマウス（雄）を購入し、1週間AIN-93M飼料および飲水を自由摂取させ馴化させた。1群7匹として、体重の平均値が等しくなるように、カゼイン分解物投与群（5mg/マウス）、β-Lgトリプシン分解物投与群（5mg/マウス）、α-Laトリプシン分解物投与群（5mg/マウス）、FKIDALNE（配列番号６）投与群（1mg/マウス）、FKIDALNE（配列番号６）投与群（5mg/マウス）、LDQWLCEK（配列番号３）投与群（1mg/マウス）、LDQWLCEK（配列番号３）投与群（5mg/マウス）の７群に分けた。試料は3日間連続（13:00）してゾンデを用いて経口投与した。最終投与から24時間後、LPSを50μg/マウスで腹腔内投与した。90分後に眼窩より採血し遠心操作により血清を得た。血清中のIL-6濃度をELISAにより測定した（図13）。Fisher PSLDによる統計学的処理により、カゼイン投与群に対する有意差を検定した。
β-Lgトリプシン分解物投与群、FKIDALNE（配列番号６）投与群およびLDQWLCEK（配列番号３）投与群のIL-6濃度は、カゼインに対して濃度依存的に有意に低下（**p<0.05）し、後者２群の低下は濃度依存的であった。
【００４８】
【発明の効果】
本発明により、TNFやIL-6などの炎症性サイトカイン産生に刺激を加えることなく、それを抑制しつつ、免疫能を賦活化する作用を有する、経口摂取可能なペプチドを提供することができた。また、生体侵襲時の代謝異常や臓器障害を防止しかつ免疫能を亢進させる経口・経腸栄養剤の製造に使用する該ペプチドを提供することができた。さらにまた、抗炎症作用を有する該ペプチドを提供することができた。
【００４９】
【配列表】

【図面の簡単な説明】
【図１】THP-1細胞の単球・マクロファージへの分化誘導後における、未分解のWPI、β-ラクトグロブリン、α-ラクトアルブミンあるいはカゼインの、LPS誘導TNF-α産生抑制効果を示す。
【図２】WPI、β-ラクトグロブリン、α-ラクトアルブミンおよびカゼインのトリプシン加水分解物の、同上TNF-α産生抑制効果を示す。
【図３】WPIのトリプシン（ブタ膵臓由来、Wako社）加水分解物、WPIのAアマノG加水分解物、WPIのトリプシンとAアマノGの組み合わせによる加水分解物、およびAアマノGとトリプシンの加水分解物の、同上TNF-α産生抑制作用を示す。
【図４】β-ラクトグロブリンのトリプシン加水分解物のUFパーミエイトの逆相HPLCクロマトグラムを示す。
【図５】β-ラクトグロブリン由来のペプチドALPMH、ALPMHIRおよびFKIDALNEの同上TNF-α産生抑制作用を示す。
【図６】α-ラクトアルブミンのトリプシン加水分解物のUFパーミエイトの逆相HPLCクロマトグラムを示す。
【図７】α-ラクトアルブミン由来のペプチドWLAHK、LAHKAL、CEVFRおよびLDQWLCEKの同上TNF-α産生抑制作用を示す。
【図８】β-ラクトグロブリンのトリプシン加水分解物の各濃度における同上TNF-α産生抑制作用を示す。
【図９】α-ラクトアルブミンのトリプシン加水分解物の各濃度におけるTNF-α産生抑制作用を示す。
【図１０】β-ラクトグロブリン由来のペプチドIDALNENK、IPAVFK、IPAVFKIDALNEおよびIPAVFKIDALNENKの、同上TNF-α産生抑制作用を示す。
【図１１】ウシラクトフェリンのトリプシン加水分解物の逆相HPLCのクロマトグラムを示す。
【図１２】ウシラクトフェリン由来のペプチドWQWR、EDLIWK、ETAEEVKおよびLGAPSILCVRの各濃度におけるTNF-α産生抑制作用を示す。
【図１３】カゼイントリプシン加水分解物、β-Lgトリプシン分解物、α-Laトリプシン加水分解物、β-Lg由来のペプチドFKIDALNE、あるいはα-La由来のLDQWLCEKマウスにおける経口摂取後の血清中のLPS誘導IL-6の産生抑制作用を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel peptide having an action of suppressing the production of inflammatory cytokines such as TNF-α and IL-6. The present invention also relates to a peptide useful for oral and enteral nutrition therapy during living body invasion. The present invention also relates to a peptide effective for treating, preventing or ameliorating inflammatory diseases caused by abnormal production of TNF-α and IL-6.
[0002]
[Prior art]
Systemic inflammatory response syndrome (SIRS) is a concept that has been proposed to define sepsis. Inflammatory cytokines (IL-) are caused by invasion (trauma, burns, acute inflammation, or cancer surgery, etc.). 1, TNF, IL-6, GM-CSF, etc.) are induced excessively and a systemic inflammatory reaction is induced. On the other hand, it is known that anti-inflammatory cytokine (IL-10) is induced as a homeostasis reaction to SIRS at the same time as inflammatory cytokine is induced. This condition in which anti-inflammatory cytokines are excessively induced is called compensatory anti-inflammatory response syndrome (CARS). Cytokines as background factors related to these two pathological conditions are related to each other to varying degrees, and can be said to be a pathological condition that always exists at the same time. From the concept of SIRS / CARS balance, Attempts have been made to understand the reaction. It is considered that an excessive inflammatory reaction progresses in the SIRS-dominated state, and progresses from prolonged or severe infection to organ damage in the CARS-dominated state.
[0003]
Nutritional management at the time of invasion is recommended not only to suppress the inflammatory response but also to enhance the patient's immunity. When an invaded patient is associated with subsequent infection, there are many cases that often have unfortunate outcomes, and infection prevention is a top priority item, especially in pathological conditions that are dominated by CARS. Therefore, control of SIRS and CARS by enteral nutrition is important. In order to prevent metabolic abnormalities and organ damage at the time of invasion by cytokines, local cytokines are produced normally, but it is considered reasonable to prevent this spread to the whole body.
[0004]
After administering rats with parenteral or enteral nutrition with the same composition of infusion, the peritoneal exudate cells were cultured in vitro under LPS stimulation, and the enteral nutrition group produced more TNF-α than the parenteral nutrition group. The number of bacteria in the peritoneal cavity after administration of intraperitoneal E. coli was small (for example, see Non-Patent Document 1). The blood TNF-α concentration after administration of bacteria was significantly lower in the enteral nutrition group than in the parenteral nutrition group. In other words, enteral nutritional administration maintains the ability of the peritoneal exudate cells to produce the inflammatory cytokine TNF-α, which is necessary for the local defense of the infected area, while maintaining local immunity, while suppressing excessive inflammatory responses throughout the body. . Such enteral nutrition is thought to be useful for controlling the occurrence of SIRS and CARS during bioinvasion (surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor).
[0005]
Although local cytokines are normally produced, enteral nutrition or administration of n-3 polyunsaturated fatty acids (N3PUFA) can be considered as a method for preventing this spread to the whole body. The effects of invasive administration of N3PUFA on the invasive response were investigated using a rat burn model and clinically using an esophageal cancer surgical case, one of the highly invasive surgery. As a result, administration of N3PUFA was found to reduce the inflammatory reaction under invasion and prevent the cellular immune function from decreasing, and the effectiveness of N3PUFA administration was shown (for example, see Non-Patent Document 2).
[0006]
Now, milk protein is a source of biologically active peptides in addition to its nutritional value.
The biologically active peptides derived from milk proteins are comprehensively compiled by Yoshikawa (for example, see Non-Patent Document 3) and Otani (for example, see Non-Patent Document 4), and Otani (for example, (See Non-Patent Documents 5 and 6).
M. Zimecki et al. Monitored bovine lactoferrin (BLFT) -containing capsules for 10 days by healthy individuals and monitored several immune parameters. As a result, the level of serum TNF-α showed a tendency to decrease during the monitoring period, but IL-6 did not change (for example, see Non-Patent Document 7). In addition, it has been reported that bovine casein-derived peptides, LLY, PGPIPN, and TTMPLW enhanced TNF-α and IL-6 production by LPS-stimulated murine macrophages (see, for example, Non-Patent Document 8).
Kuriiwa discloses that a peptide consisting of 25 amino acid residues in the N-terminal region derived from lactoferrin suppresses endotoxin-induced inflammation (see, for example, Patent Document 1).
[0007]
[Non-Patent Document 1]
Lin, MT et.al. “Route of nutritional supply influences local, systemic, and remote organ responses to intraperitoneal bacterial challenge.” Ann. Surg, Vol 223, 1996, p.84-93
[0008]
[Non-Patent Document 2]
Akira Inaba “Possibility of Growth Factor Therapy Under Intravenous Nutrition” History of Medicine, Vol 198, No. 13, 2001, p.1041
[0009]
[Non-Patent Document 3]
Masaaki Yoshikawa et al. “Advanced Functions of Milk”, Kogaku Publishing, Masaaki Yoshikawa et al., 1998, p.188
[0010]
[Non-Patent Document 4]
Hajime Otani et al. “Advanced Functions of Milk” Kogaku Publishing, Masaaki Yoshikawa et al., 1998, p.97
[0011]
[Non-Patent Document 5]
Gen Otani “Nutrition and physiology of milk casein-derived peptides” Milk Science, Vol 47, No. 3, 1998, p.183-193
[0012]
[Non-Patent Document 6]
Gen Otani “Immunoregulatory Function of Peptides Generated by Digestion of Milk Casein” Milk Science, Vol 50, No. 3, 2001, p.139-144
[0013]
[Non-Patent Document 7]
Zimecki, M et. Al. `` Immunoregulatory effects of a nutritional preparation containing bovine lactoferrin taken orally by healthy individuals '' Arch. Immunol. Ther. Exp., Vol 46, 1998, p.231-240
[0014]
[Non-Patent Document 8]
Ziao, C. et. Al. `` Bovine casein peptides co-stimulate naive macrophages with lipopolysaccharide for proinflammatory cytokine production and nitric oxide release '' J. Sci. Food. Agric .., Vol 81, 2000, p.300-304
[0015]
[Patent Document 1]
JP-A-8-165248
[0016]
[Problems to be solved by the invention]
It is an object of the present invention to provide an orally ingestible peptide having an action of stimulating immune capacity while suppressing the production of inflammatory cytokines such as TNF and IL-6 without stimulating it. To do. Another object of the present invention is to provide a peptide and an acid addition salt thereof for producing an oral and enteral nutritional agent for alleviation of inflammatory reaction and nutritional treatment in systemic inflammatory response syndrome (SIRS) accompanying biological invasion. And Furthermore, an object of the present invention is to provide a peptide that is effective in treating, preventing, or improving inflammatory diseases caused by abnormal production of TNF-α and IL-6.
[0017]
[Means for Solving the Problems]
Since the isolation of opioid peptide (β-casomorphin) from milk casein by Brantl et al. In 1979, numerous bioactive peptides have been obtained from enzymatic digests of casein and whey proteins. Therefore, at first, it was expected that all of these physiologically active peptides exist with appropriate purpose. However, it is now known that similar bioactive peptides are derived not only from animals but also from plants, and bioactive peptides exist with a certain probability. It cannot be determined that it exists with appropriate purpose. In any case, it is true that bioactive peptides derived from milk proteins are much more likely to have biological fitness than other sources [Masaaki Yoshikawa: Advanced Functions of Milk [Edited by Masaaki Yoshikawa et al., P. 188, Kogaku Publishing (1998)].
[0018]
On the other hand, it has been clarified that enteral nutrition modifies the production of cytokines in the lung, which is a local organ, whole body, or distant organs, and how the modification varies depending on the type of cytokine (Fong, Y et al .: Ann. Surg., 210: 449-457, 1989; Lin, ML et al .: Ann. Surg., 223: 84-93, 1996).
Therefore, the present inventors have suppressed the excessive production of inflammatory cytokines during invasion and obtained milk protein hydrolyzate using TNF-α production inhibitory activity as an index in order to obtain a food material that can be incorporated into oral and enteral nutrients. The degradation product was screened for active peptides.
The present inventors have identified protease hydrolysates of whey protein isolate (WPI), β-lactoglobulin (β-Lg), α-lactalbumin (α-La) and bovine lactoferrin (Lf). It was found that the permeate of ultrafiltration (UF) has an action of suppressing LPS-induced TNF-α production or IL-6 production. Furthermore, these permeates were fractionated by reversed-phase HPLC, and there were several peaks having TNF-α production or IL-6 production inhibitory activity in fractions having a molecular weight of about 2,000 daltons or less. The peptide was isolated and purified from the amino acid sequence. As a result of synthesizing corresponding peptides based on these sequences and examining TNF-α production inhibitory activity, it was confirmed that they had similar activities. Furthermore, new peptides were synthesized from regions adjacent to each other on the β-Lg molecule or overlapping several amino acids, and several peptides having TNF-α production inhibitory activity could be obtained.
[0019]
That is, the present invention
(1) a peptide selected from the group consisting of LDQWLCEK, FKIDALNE, IDALNENK, IPAVFK, IPAVFKIDALNENK, IPAVFKIDALNE, ETAEEVK and LGAPSITCVR, and an acid addition salt thereof,
(2) A medicament comprising the peptide of (1) and an acid addition salt thereof as an active ingredient,
(3) an anti-inflammatory agent comprising the peptide of (1) and an acid addition salt thereof as active ingredients,
(4) An oral and enteral nutritional agent for alleviating inflammatory response and nutritional treatment in systemic inflammatory response syndrome (SIRS) associated with biological invasion, comprising an effective amount of the peptide of (1) and its acid addition salt,
(5) The oral or enteral nutrient of (4), wherein the biological invasion is surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor,
(6) X ₁ -LAHK-X ₂ -X _Three (Where X ₁ Represents 0 or W and X ₂ Represents 0 or A and X _Three Represents 0 or L), Y ₁ -LPMH-Y ₂ -Y _Three (Where Y ₁ Represents 0 or A, Y ₂ Represents 0 or I and Y _Three Represents 0 or R), Y _Four -IDALNE-Y _Five (Where Y _Four Represents 0 or K, Y _Five Represents 0 or N), IPAVFK-Y ₆ -Y ₇ -Y ₈ -Y ₉ -Y _Ten (Where Y ₆ Represents 0 or I, Y ₇ Represents 0 or D, Y ₈ Represents 0 or A, Y ₉ Represents 0 or L, Y _Ten Represents 0 or N), an anti-inflammatory agent comprising a peptide selected from the group consisting of WQWR and EDLIWK and an acid addition salt thereof as an active ingredient,
(7) an anti-inflammatory agent comprising the peptide of (6) and an acid addition salt thereof as active ingredients,
(8) An oral and enteral nutritional agent for alleviating inflammatory response and nutritional treatment in systemic inflammatory response syndrome (SIRS) associated with biological invasion, containing an effective amount of the peptide of (6) and its acid addition salt,
(9) The oral and enteral nutrition of (8), wherein the biological invasion is surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor,
(10) Use of the peptide of (1) and an acid addition salt thereof for producing a medicament,
(11) Use of the peptide of (1) and an acid addition salt thereof for producing an anti-inflammatory agent,
(12) Use of the peptide of (1) and its acid addition salt to produce an oral and enteral nutrient for alleviation of inflammatory response and nutritional treatment in systemic inflammatory response syndrome (SIRS) associated with biological invasion,
(13) The use of (12), wherein the biological invasion is surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor,
(14) Use of the peptide and acid addition salt of (6) for producing an anti-inflammatory agent,
(15) Use of the peptide of (6) and its acid addition salt to produce an oral and enteral nutrient for mitigating inflammatory response and nutritional treatment in systemic inflammatory response syndrome (SIRS) associated with biological invasion,
(16) The use of (15), wherein the biological invasion is surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor,
Consists of.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
As a starting material, milk protein is optimal for reasons described below. Corn and soy protein are also candidates, but not as advantageous as milk protein. Milk proteins are bovine-derived casein and whey proteins. In particular, fractions such as whey protein isolate (WPI), β-lactoglobulin, α-lactalbumin and lactoferrin having a protein content of 90% or more are advantageous from the viewpoint of the purity of the active substance. In addition, whey protein concentrate (WPC) having a protein content of 80% or more, casein, total milk protein (TMP), or reduced milk can be cited as advantageous raw material candidates. What has been illustrated above is not meant to be limiting.
There are many reports [for example, Yu Kuwata: Monthly Food Chemical, February, 68 (1991); US Patent 5,420,249, etc.] on the method of fractionating β-Lg and α-La from WPI. Commercial products can also be used.
[0021]
Total milk protein (TMP) is obtained by removing lactose and minerals from skim milk powder and contains both casein and whey as protein components. In general, there are precipitation methods and membrane treatment methods. However, only those produced by the precipitation method are sometimes referred to as TMP or co-precipitate, and those produced by UF membrane treatment may be distinguished as milk protein concentrate (MPC).
It is considered that the TNF-α and / or IL-6 production inhibitory activity of the hydrolyzate depends on the degree of hydrolysis (DH). Below, various well-known techniques (related literatures, patents, etc.) relating to protein hydrolysis are listed. A person skilled in the art can screen the peptides of the present invention from milk proteins by using these known techniques alone or in combination.
[0022]
Food grade protein hydrolases (proteases) include endo-type and exo-type proteases. The basic enzyme is often selected from proteases with high endo-type activity. Endo-type protease is, for example, Alcalase ^R (From Bacillus licheniformis), esperase (from B. lentus), Neutrase ^R Examples of exo-type peptidase / endo-type protease complex enzymes (derived from B. subtilis), protamex (derived from bacteria), PTN6.0S (pig pancreatic trypsin) and the like include, for example, flavorzyme (derived from Aspergillus oryzae) ( Novo) In addition, as endo-type proteases, for example, chymotrypsin (Novo, Boehringer), protease N Amano (from Bacillus subtilis, Amano Pharmaceutical), biolase (from Bacillus subtilis, Nagase Sangyo), papain W-40 (Amano Pharmaceutical), Examples of the exo-type protease include carboxypeptidase derived from porcine or bovine viscera. These enzymes are not meant to be limiting.
Enzymatic degradation of allergic milk proteins with reduced antigenicity, in addition to relatively highly specific animal-derived trypsin and chymotrypsin, plant-derived papain and bromelain, nonspecifically lowering the milk protein Subtilisin-derived enzymes derived from microorganisms are used. Research reports using plant-derived papain, proteases from bacteria and fungi (Food Technol., 48: 68-71, 1994; Trends Food Sci. Technol., 7: 120-125, 1996; Food Proteins and Their Applications: pp. 443-472, 1997). Hydrolysis is carried out alone or in combination of these basic enzymes.
A summary report on hydrolysis of whey protein (Trends Food Sci. Technol., 7: 307-319, 1998) is given below.
[0023]
Enzymatic activity to hydrolyze whey protein varies greatly. Pepsin degrades α-La and modified α-La, but does not degrade native β-Lg (Neth. Milk dairy J., 47: 15-22, 1993). Trypsin hydrolyzes α-La slowly, but β-Lg remains almost undegraded (Neth. Milk dairy J., 45: 225-240, 1991). Chymotrypsin degrades α-La quickly while β-Lg degrades slowly. Papain hydrolyzed bovine serum albumin (BSA) and β-Lg, while α-La was resistant (Int. Dairy Journal 6: 13-31, 1996a). However, α-La not bound to Ca was completely degraded by papain at acidic pH (J. Dairy Sci., 76: 311-320, 1993). Enzyme and Chemical Modification of protein's in Food proteins and their Applications can be achieved by controlling the enzymatic degradation of milk proteins to modify the protein over a wide range of pH and processing conditions. pp. 393-423, 1997 Marcel Dekker, Inc., New York, 1997; Food Technol., 48: 68-71, 1994). Hydrolysis of peptide bonds results in increased number of charged groups and hydrophobicity, lower molecular weight, and modification of the molecular configuration (J. Dairy Sci., 76: 311-320, 1993). The change in functional properties is largely dependent on the degree of hydrolysis. The biggest changes commonly seen in whey protein functionality are increased solubility and decreased viscosity. When the degree of hydrolysis is high, the hydrolyzate often does not precipitate upon heating and is highly soluble at pH 3.5-4.0. The hydrolyzate is also much less viscous than the intact protein. This difference is particularly noticeable when the protein concentration is high. Other effects are changes in gel properties, increased thermal stability, enhanced emulsification and foaming, reduced emulsification and foam stability (Int. Dairy journal, 6: 13-31, 1996a; Dairy Chemistry 4, pp. 347-376, 1989; J. Dairy Sci., 79: 782-790, 1996).
[0024]
There have been reports on a number of peptides with potential angiotensin converting enzyme I (ACE) inhibitory activity deduced from in vitro activity measurements (eg J. Dairy Res., 67: 53-64, 2000; Br. J. Nutr., 84: S33-S37, 2000). Research reports have been made to purify and identify ACE inhibitory peptides from hydrolysates using various chromatographic techniques (eg, Maruyama, S., & Suzuki, H .: Agricultural and Biological Chemistry, 46: 1393 -1394, 1982; Miyoshi S. et al .: Agri. Biol. Chem., 55: 1313-1318, 1991; Food Science and Biotechnology, 8: 172-178, 1999; Biosci. Biotech. Biochem., 57: 922 -925, 1993). These reports suggest that ACE inhibitory activity is present in many fractions obtained by column operations based on various separation principles, indicating that the molecular properties of ACE inhibitors are quite diverse. ing. The fact that ACE inhibition is found in hydrolysates produced by various proteins, proteases and hydrolysis conditions suggests that various peptides with diverse amino acid sequences may also have ACE inhibitory activity is doing. Due to the chemical diversity of such peptides, chromatographic purification of the hydrolyzate will always be accompanied by a partial loss of active peptide. During hydrolysis, ACE inhibitory activity is continuously formed while being degraded. The maximum activity of the hydrolyzate is the result of optimization of these two processes. The overall peptide composition of the hydrolyzate determines the ACE inhibitory activity, which depends on the specificity of the hydrolase and the process conditions.
[0025]
Therefore, there is a report on the optimization of whey protein hydrolysis using response surface methodlogy (International Dairy Journal 12: 813-820, 2002) in order to minimize the required hydrolysis and maximize the ACE inhibitory activity. Has been made.
[0026]
Referring to the literature on peptides with ACE inhibitory activity, the shortest peptides are IPP and VPP (Masaaki Yoshikawa: Advanced functions of milk, edited by Masaaki Yoshikawa et al., P. 188, Kogaku Publishing (1998)). .
In addition, α-La and β-Lg are converted into pepsin or trypsin alone, or hydrolyzates obtained by combining pepsin and trypsin, pepsin, trypsin and chymotrypsin, and this hydrolyzate is further divided into two stages. The ACE inhibitory activity of each retentate and permeate after treatment with UF filtration (cutoff value 30 kDa → 1 kDa) was measured (J. Dairy Res., 67: 53- 64, 2000). According to it, α-La and β-Lg ACE inhibitory activity (IC ₅₀ ) Both have the highest <1 kDa fraction of trypsin hydrolyzate (3 h) (α-La: IC ₅₀ = 109 μg / mL, β-Lg = 237 μg / mL). At this time, DH of α-La was 6.2%, and molecular weight distribution was> 5 kDa: 52%, 2-5 kDa: 15%, 1-2 kDa: 20%, <1 kDa: 13%, and β-Lg DH is 6.7% and the molecular weight distribution is> 5 kDa: 23%, 2-5 kDa: 27%, 1-2 kDa: 28%, <1 kDa: 24%.
[0027]
In addition, a peptide consisting of amino acid residues 5 to 12 has been reported as an ACE inhibitory peptide derived from milk casein (Agric Biol Chem., 49: 1405-1409, 1985).
On the other hand, in the examples described later, the peptide having TNF-α and / or IL-6 production inhibitory activity of the present invention has a chain length (number of amino acid residues) of 5 to 14 and a molecular weight in the range of 500 to 1800. Is in.
Taking these into consideration, the peptide having TNF-α and / or IL-6 inhibitory activity of the present invention has a chain length of 3 to 16 and a molecular weight distribution of 400 to 2100 kDa (the molecular weight of one amino acid is 130). Estimated.
Regarding the effect of peptides derived from milk proteins on cytokine production, a report that bovine casein-derived peptides increase LPS-induced TNF-α and IL-6 production from murine bone marrow macrophages (J. Sci. Food Agric., 81: 300-304, 2000) and the report that there is a peptide that induces IL-6 production by LPS stimulation in the supernatant of fermented milk by probiotic lactic acid bacteria (Milchwissenschaft, 57 (2): 66-70, 2002) There is.
[0028]
Peptides having the action of suppressing LPS-induced TNF-α and IL-6 production are also present in many fractions obtained by column operation based on various separation principles, such as the peptides having the ACE inhibitory activity described above. We cannot deny the possibility of being.
Therefore, optimization of milk protein hydrolysis conditions (denaturation temperature, pH, temperature, hydrolysis time, and enzyme / substrate ratio) is described above using the inhibitory effect of LPS-induced TNF-α and / or IL-6 production as an index. You can try referring to the literature (International Dairy Journal 12: 813-820, 2002). Therefore, the resulting hydrolysis optimization conditions are encompassed by the present invention.
According to the examples of the present invention, it has been found for the first time that a peptide derived from whey protein has TNF-α and / or IL-6 production inhibitory activity. Therefore, by further expanding the hydrolysis target and changing the hydrolysis conditions in various ways, using TNF-α and / or IL-6 production inhibitory activity as an index, the work of screening a new peptide having the activity is as follows: For those skilled in the art, it has become a routine task that does not require any inventive effort.
[0029]
That is, methods for measuring the amount of TNF-α and IL-6 are publicly known (eg, experimental medicine separate volume, biomanual UP experiment series, cytokine experiment method, Atsushi Miyajima, Masaru Yamamoto, Yodosha Co., Ltd., (1997)). In order to screen for peptides that suppress the production of these inflammatory cytokines, those skilled in the art can set various hydrolysis conditions for milk proteins by referring to the above-mentioned literature or the following related literature. Became possible.
Hydrolysis can in principle also be acid or base hydrolysis, but this method involves uncontrollable processes. That is, protein degradation occurs irregularly and some of the amino acids are degraded (eg Leu, Val and / or Ile can be destroyed). Therefore, enzymatic hydrolysis is usually selected.
The functionality of the enzymatic hydrolyzate depends on DH as described above. DH can be monitored by methods known to those skilled in the art, for example, increase in osmotic pressure measured from freezing point drop, measurement of free amino acid by OPA or TNBS method, measurement of free amino acid by pH stat method, and the like. DH can be controlled by raw material, enzyme properties and reaction conditions (pH, temperature and time).
[0030]
In addition, there is a literature (Food and Development, vol. 31 (2): 20-22) on typical changes in DH when casein is hydrolyzed with various enzymes. That is, casein is converted to Alcalase ^R And flavorzyme combination, flavorzyme, protamex, Neutrase ^R , Alcalase ^R Or when hydrolyzed with esperase ^R And Neutrase ^R The time course of DH changes in exactly the same manner, DE increases rapidly (about 14%) until 1 hour after hydrolysis, and gradually increases after that (approximately 18% after 4 hours). Protamex's DE is also at about the same value. DE has the highest value for flavorzyme alone or Alcalase ^R And flavorzyme, both of which have remained at the same value. The DE in the combination increased rapidly (approximately 34%) until 1 hour after decomposition and gradually increased thereafter (approximately 53% after 4 hours).
[0031]
From the above literature, it is estimated that the maximum DE value can be obtained with a decomposition time of 6 hours.
Patent 3167723 discloses a method of hydrolyzing whey protein to DE 15 to 35% by a non-pH stat method using a protease derived from B. licheniformis and a protease derived from B. subtilis.
JP 10-507641 discloses a method for hydrolyzing milk protein using a combination of neutral and alkaline proteases derived from B. licheniformis and endo / exoproteases derived from A. oryzae.
On the other hand, there is Patent 3222638 relating to oligopeptide mixtures useful for nutritional management of allergic patients and preoperative and postoperative patients. The oligopeptide mixture contains whey protein, a protease derived from the genus Bacillus [Biolase (Nagase Seikagaku), Alcalase (Novo), etc.] and a protease derived from actinomycetes [Actinase (Kaken Pharma) ), Pronase (Sigma)]]] and after removing the enzyme and insoluble hydrolyzate, the average chain length is 3-5, the maximum molecular weight is 1500 or less, and the antigenicity is β-Lg It is characterized by very little bitterness and umami at 1 / 10,000 or less.
[0032]
The hydrolysis temperature, pH, reaction time, enzyme addition amount, etc. can be determined with reference to the manufacturer's instructions, etc., but the temperature is 30 to 65 ° C., the pH is 5 to 9, and the reaction time is less than 20 hours. It can be selected with reference to the above-mentioned documents.
Enzyme deactivation depends on the heat resistance of the enzyme. The hydrolyzate is filtered through a UF membrane with a molecular weight cut-off of 5000-100,000 Daltons, preferably a UF membrane with 1000-100,000 Daltons. The cutoff value in this case is not critical. The method of measuring the cut-off value varies depending on the membrane manufacturer, and there is no standardized method yet. Membrane modules include P & F (plate and frame) type, TU (tubular) type, HF (hollow fiber) type, SW (spiral wound) type, and PL (pleated) type. UF filtration largely eliminates hydrolysates with molecular weights in the permeate that greatly exceed 2000 daltons.
The permeate can be concentrated, spray dried or freeze dried by a known method.
[0033]
α-La trypsin hydrolyzate, β-Lg hydrolyzate, or Lf hydrolyzate UF permeate, for example, is active in reversed-phase HPLC (ODS Prep, 20 × 300 mm, Toso, Tokyo, Japan) The components can be fractionated. Elute with a linear gradient of acetonitrile (1% / min) containing 0.1% TFA (10 mL / min). Elution is monitored at 210 nm. Each fraction is collected and TNF-α inhibitory activity or IL-6 inhibitory activity is measured. The active fraction is present at a molecular weight <2,000 daltons.
[0034]
The primary structures of α-La, β-Lg and bovine Lf are known (Kunio Yamauchi, Ken Yokoyama, edited by Milk General Encyclopedia, Asakura Shoten, 1997).
From α-La, WLAHK (amino acid residues 104-108), LAHKAL (amino acid residues 105-110), and CEVFR (amino acid residues 6-10) and LDQWLCEK (amino acid residues 115-122) A peptide LDQWLC (-CEVFR) EK in which the C residues of SS are bound is obtained. From β-Lg, ALPMH (amino acid residues 142 to 146), ALPMHIR (amino acid residues 142 to 148) and FKIDALNE (amino acids Residues 83-89) and Lf from WQWR (amino acid residues 41-44), EDLIWK (amino acid residues 283-288), ETAEEVK (amino acid residues 352-358) and LGAPSITCVR A peptide LGAPSITC (-CR) VR in which (residues 48 to 57) and the C residue of CR are SS-linked can be obtained.
Accordingly, the permeate TNF-α inhibitory active components of the α-La hydrolyzate having a UF cutoff value of 10,000 are the peptides WLAHK, LAHKAL and LDQWLC (-CEVFR) EK. An α-La hydrolyzate containing at least one of the three peptides has activity. Those of β-Lg are ALPMH, ALPMHIR and FKIDALNE, and β-Lg hydrolysates containing at least one of the three peptides are active. From Lf, there are WQWR, EDLIWK, ETAEEVK and LGAPSITC (-CR) VR, and an Lf hydrolyzate containing at least one of these three peptides has activity.
By varying the trypsin hydrolysis conditions of α-La, β-Lg and bovine Lf, the active peptide and its composition can be altered. Therefore, the present invention is not limited to the above active peptide. The same can be said for other enzymes other than trypsin.
Trypsin acts as an endopeptidase that selectively cleaves the peptide bond on the carboxyl side of Arg (R) and Lys (K) (Biochemical Dictionary 3rd edition, Tokyo Dojin Co., Ltd., 1998). However, α-La-derived LAHKAL and β-Lg-derived ALPMH and FKIDALNE are cleaved non-specifically.
Therefore, several new trypsin recognition peptides were chemically synthesized and examined for TNF-α inhibitory activity (hereinafter also referred to as “activity”).
4 residues of IPAV between K (amino acid residue 77) and FKIDALNE near the N-terminus of FKIDALNE (amino acid residues 82 to 89) and N-terminal of the trypsin recognition site of FKIDALNE A peptide IPAVFK (amino acid residues 78 to 83) linked to FK2 residue was synthesized. This IPAVFK was examined for its TNF-α inhibitory activity (Table 1, ED). ₅₀ = 7.1 μM). IDALNENK (amino acid residues 84 to 91), in which FKIDALNE trypsin recognition site K and subsequent IDALNE was linked to NK adjacent to C-terminal of FKIDALNE (amino acid residues 84 to 91) was also recognized (Table 1, ED). ₅₀ = 3.2 μM). In addition, the activity of the peptide IPAVFKIDALNENK (amino acid residues 78 to 91) connecting IPAVFK and IDALNENK was also observed (Table 1, ED). ₅₀ = 0.8 μM).
The peptide IPAVFKIDALNE (amino acid residues 78 to 89) excluding the C-terminal NK of IPAVFKIDALNENK was a peptide whose C-terminal was non-specifically cleaved by trypsin, but activity was observed (Table 1, ED). ₅₀ = 1.8 μM).
As described above, the four newly synthesized peptides IDALNENK, IPAVFK, IPAVFKIDALNENK and IPAVFKIDALNE can be obtained by setting the conditions for hydrolysis with trypsin.
By the way, all three peptides except IPAVFK share IDALNE (amino acid residues 84 to 89). In addition, comparing the TNF-α inhibitory activities of the four peptides, the ED of the three peptides ₅₀ Is 0.8-3.2 μM. On the other hand, IPAVFK ED ₅₀ Is extremely weak at 7.1 μM, but those linked to IDALNE or IDALNENK are 3.9 to 8.9 times more active. Considering these facts, it is suggested that the consensus sequence IDALNE is essential for the expression of TNF-α inhibitory activity. Therefore, it is possible to determine the minimum amino acid sequence (core sequence) essential for the expression of activity by measuring the TNF-α inhibitory activity of IDALNE and, when the activity is observed, by sequentially removing the N-terminal or C-terminal. Therefore, the peptide having the activity thus obtained is included in the present invention.
Furthermore, a trypsin recognition site exists in addition to the above peptides on the primary structure of α-La, β-Lg, or Lf. Therefore, new active peptides can be screened by variously changing the trypsin hydrolysis conditions. In addition, various cleaved fragment peptides recognized by trypsin can be chemically synthesized and their activities can be examined. Therefore, the peptide having the activity thus obtained is included in the present invention.
On the other hand, when LDQWLCEK and CEVFR obtained by cleaving the SS bond of LDQWLC (-CEVFR) EK derived from α-La were examined for TNF-α production inhibitory activity, the former was found to be active but the latter was I couldn't. Moreover, when the activity of LGAPSITCVR and CR obtained by cleaving the SS bond of LGAPSILC (-CR) VR derived from bovine Lf was examined, the activity was observed in the former, but not in the latter. It was.
The above results are summarized as follows. As a peptide having TNF-α production inhibitory activity derived from trypsin-specific hydrolyzate or non-specific hydrolyzate of milk protein, α-La has WLAHK (SEQ ID NO: 1), LAHKAL ( SEQ ID NO: 2) and LDQWLCEK (SEQ ID NO: 3), from β-Lg, ALPMH (SEQ ID NO: 4), ALPMHIR (SEQ ID NO: 5), FKIDALNE (SEQ ID NO: 6), IDALNENK (SEQ ID NO: 7), IPAVFK (SEQ ID NO: 7) 8), IPAVFKIDALNENK (SEQ ID NO: 9), IPAVFKIDALNE (SEQ ID NO: 10), and from bovine LF, WQWR (SEQ ID NO: 11), EDLIWK (SEQ ID NO: 12), ETAEEVK (SEQ ID NO: 13), LGAPSILCVR (SEQ ID NO: 14) Can be obtained. From the molecular weight of these peptides or the molecular weight distribution of peptides having biological activity derived from milk proteins, it is suggested that the TNF-α production-inhibiting fraction of milk proteins exists at a molecular weight <2,000 daltons.
Here, α-La-derived WLAHK and LAHKAL share LAHK. Therefore, in addition to these peptides, the presence of 4 peptides of LAHK, WLAHKA, WLAHKAL and LAHKA can be considered, and the presence or absence of TNF-α production inhibitory activity can be confirmed. Therefore, peptides whose activity has been confirmed are included in the present invention.
[0035]
In any of the above peptides, the N-terminal or C-terminal amino acid residues are sequentially deleted, and the activity of these deleted peptides can be confirmed. Therefore, when activity is confirmed, it is included by this invention.
Furthermore, although IPAVFKIDALNE and IPAVFKIDALNENK peptides are weak, IPAVFK has activity. Therefore, peptides obtained by sequentially deleting amino acid residues after IPAVFK of these two peptides are very likely to have activity and are active. Each peptide in which is confirmed is included in the present invention.
Furthermore, peptides of which the activity has been confirmed or peptide derivatives in which the amino acid residues of these peptides are substituted with other amino acid residues (including D-amino acids) are also encompassed in the present invention. Report that D-amino acid decapeptide (PDP-58) inhibited TNF-α translation in LPS-stimulated RAW264.7 cells and murine colitis but did not affect mRNA synthesis (Inflamm. Res., 51: 479-482, 2002) supports this.
In addition, based on the primary structure of α-La, β-Lg, or bovine Lf, the entire primary structure is covered, with several residues (about 3-5 residues) and overlapping peptides (about 5-10 residues). It is also possible to chemically synthesize and screen the target peptide using TNF-α production inhibitory activity or IL-6 activity as an index. Methods for chemical synthesis of peptides are known. Solid phase method developed by Merrifield, RB (Biochemistry 1964, 3, pp. 1385; The Peptide 1979, 2, pp. 1-284, by ed E. Gross & J. Meienhofer). Alternatively, the same solid phase method can be synthesized using Fmoc-amino acids, where the flow method is applied and the side chain is protected with an acid labile group (Atherton, E. & Sheppard RC “ Solid Phase peptide synthesis—a Practical approach ”, IRL PRESS, Oxford, 1989). Therefore, all peptides whose activity has been confirmed are included in the present invention.
[0036]
Acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, benzoate, citrate And organic acid salts such as oxalate, maleate, fumarate, succinate, tartrate, lactate, methanesulfonate, toluenesulfonate, aspartate, and glutamate.
The milk protein hydrolyzate having the action of suppressing the production of TNF-α or IL-6 of the present invention, the peptide having the activity roughly purified from the hydrolyzate, or one containing two or more of the purified peptides It is well suited for parenteral and peripheral parenteral administration for alleviating inflammation in SIRS by appropriate mixing of carbohydrates, lipids, vitamins, minerals, etc.
[0037]
That is, the milk protein hydrolyzate of the present invention, a peptide roughly purified from the hydrolyzate or a purified peptide is used for the production of oral and enteral nutrients useful for the control of SIRS and CARS for the prevention of organ failure due to invasiveness. Expectation to use for. It can also be used for preoperative nutritional management. The effective amount of peptide is estimated to be 3 g (in terms of active peptide) or more in 100 kcal, but the effective amount should be determined by human experiments. Specifically, it is an oral and enteral nutrient for surgery, trauma, burns, acute pancreatitis, infections, peritonitis, malignant tumors and the like.
In addition, the peptide of the present invention is expected to be effective for treatment, prevention or improvement of diseases involving TNF-α. Diseases involving TNF-α are caused by overproduction of TNF-α. Examples of such diseases include diabetic complications such as inflammatory diseases (retinopathy, nephropathy, neuropathy, macrovascular disorders, etc.) Rheumatoid arthritis, osteoarthritis, rheumatoid myelitis, arthritis such as gouty arthritis, periosteitis, etc .; low back pain; inflammation after surgery / traumatic injury; remission of swelling; neuralgia; pharyngitis; cystitis; Atopic dermatitis; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; meningitis; inflammatory eye diseases; pneumonia; inflammatory lung diseases such as silicosis, pulmonary sarcoidosis and pulmonary tuberculosis), 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, autoimmune hemolytic anemia, psoriasis, neurodegeneration Disease (eg, Alzheimer's disease, Kinson disease, amyotrophic lateral sclerosis, AIDS encephalopathy, etc.), central nervous system disorders (eg, cerebrovascular disorders such as cerebral hemorrhage and cerebral infarction, head trauma, spinal injury, cerebral edema, multiple sclerosis, etc.), poison (Eg, sepsis, septic shock, endotoxin shock, gram-negative bacterial sepsis, toxin shock syndrome), Addison's disease, Creutzfeldt-Jakob disease, viral infections (cytomegalovirus, influenza virus, herpes virus, etc.) Infection), transplant rejection, dialysis hypotension, osteoporosis and the like.
[0038]
The peptides of the invention can also be used as pharmaceutical preparations containing a suitable carrier. That is, the oral dosage form is in a dosage form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules, and powders are conventional formulation adjuvants such as binders (syrup, gum arabic, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose, sugar) Corn starch, calcium phosphate, sorbit, glycine, etc.), lubricants (eg, magnesium stearate, talc, polyethylene glycol, silica, etc.), wetting agents (sodium lauryl sulfate, etc.). Tablets can be coated by conventional methods. The oral solution can be made into an aqueous solution or a dry product. Such oral solutions include conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).
The effective amount of the peptide of the present invention should be determined in clinical trials, but refer to a report of 0.5 ppm, preferably 5-50 ppm (Japanese Patent Laid-Open No. 8-165248).
[0039]
【Example】
The present invention will be specifically described with reference to examples and test examples, but the present invention is not construed as being limited to these specific examples.
Example 1 Preparation of Whey Protein Hydrolyzate
100 mg / ml WPI (protein content 90%, Davisco), β-Lg (β-Lg content 90%, Davisco) or α-La (α-La content 90%, Davisco) in phosphate buffer Was dissolved at a concentration of As a control, sodium caseinate (purity 95% or more, Wako Pure Chemical Industries) was dissolved in phosphate buffer at the same concentration. 50 mg of trypsin (derived from porcine pancreas, activity: 25 USP chymotrypsin Units / mg or less, Wako Pure Chemical Industries, Ltd.) per 1 g of protein was added and reacted at 37 ° C. for 6 hours. After the reaction, the mixture was centrifuged at 20,000 × g for 10 minutes to precipitate and remove insoluble components, and then subjected to UF membrane treatment (Millipore Ultrafree-MC) having a molecular weight cut-off of 10,000. The permeate was subjected to a 0.22 μm MF membrane (Millipore Mirex-GV) and then subjected to experiments.
[0040]
[Example 2] Preparation of WPI hydrolyzate
WPI (protein content 90%, Davisco) was dissolved in phosphate buffer at a concentration of 100 mg / ml and divided into the following four reaction categories.
(1) Trypsin (same as in Example 1, the same applies hereinafter) was added and reacted at 50 ° C. for 5 hours.
(2) 50 mg of protease A Amano G was added and reacted at 50 ° C. for 5 hours.
(3) After trypsin was added and reacted at 50 ° C. for 5 hours, 50 mg of protease A Amano G was further added and reacted at 50 ° C. for 5 hours.
(4) After adding 50 mg of protease A Amano G and reacting at 50 ° C. for 5 hours, 50 mg of trypsin was further added and reacted at 50 ° C. for 5 hours.
After completion of the reaction, it was boiled for 15 minutes. After centrifuging (20,000 × g, 10 minutes), UF membrane treatment (Millipore Ultra Free-MC) with a molecular weight cut off of 10,000 was performed. Permeate was subjected to the experiment after 0.22 μm MF membrane treatment.
[0041]
[Example 3] Identification of peptide having TNF-α production inhibitory activity derived from β-Lg and α-La
A trypsin hydrolyzate of β-Lg (β-Lg content 90%, Davisco) or α-La (α-La content 90%, Sigma) was prepared according to Example 1.
The active components of these trypsin hydrolysates were fractionated by reverse phase HPLC. The analytical column was C18 SG120 (Shiseido) 4.6 mmφ x 250 mm, and the mobile phase was a linear concentration gradient with a solvent of acetonitrile / 0.1% TFA. The preparative column was C18 SG120 (Shiseido) 15 mmφ x 250 mm, and the same solvent was used. FIG. 4 shows a reverse phase HPLC chromatogram of a peptide derived from β-Lg, and FIG. 6 shows a reverse phase HPLC chromatogram of a peptide derived from α-La.
[0042]
[Test Example 1] TNF-α production inhibitory action
The inhibitory effect on TNF-α production was examined for WPI, β-Lg, α-La and casein before hydrolysis, or their hydrolysates in Example 1.
THP-1 cells (derived from patients with acute monocytic leukemia, RIKEN), RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (FBS: Mitsubishi Chemical), phorbol ester (TPA, Wako Pure Chemical Industries) 200 nM ), 5% CO ₂ The cells were cultured at 37 ° C. for 72 hours under the above conditions to induce differentiation into monocytes / macrophages.
After differentiation induction, WPI, β-Lg, α-La, and casein before degradation 0 mg / mL (control), 0.1 mg / mL, 1.0 mg / mL, or hydrolyzate 0 mg / mL (control) The culture medium was replaced with RPMI 1640 medium containing 0.02 mg / mL and 0.2 mg / mL and cultured for 2 hours. Subsequently, LPS (List Biol. Lab. Inc.) was added to the medium at a final concentration of 100 ng / ml and cultured for 4 hours. The amount of TNF-α in the culture supernatant was measured using a commercially available ELISA kit for TNF-α measurement (Amersham Bioscience). FIG. 1 shows the TNF-α production inhibitory action of WPI, β-Lg, α-La, and casein before degradation, and FIGS. 2, 8 and 9 show the TNF-α production inhibitory action of these hydrolysates.
Each whey protein before degradation and trypsin degradation products of each whey protein showed TNF-α production inhibitory effect. Casein had no inhibitory effect on TNF-α production before or after degradation. Trypsin degradation product of whey protein has high heat stability and suppresses TNF-α production even when heat-treated, so oral and enteral nutrition for patients with systemic inflammatory response syndrome (SIRS) to alleviate the inflammatory response It can be added to the agent.
[0043]
[Test Example 2] TNF-α production inhibitory action of hydrolyzate by combination of proteases
For each protease hydrolyzate of WPI obtained in Example 2, the amount of TNF-α produced in the culture supernatant was measured according to the method of Test Example 1. The results are shown in FIG.
The inhibitory effect of WPI hydrolyzate on TNF-α production by trypsin was stronger than that of hydrolyzate by A-Amano G.
On the other hand, when WPI is decomposed with trypsin and further decomposed with A-Amano G (trypsin → A-Amano G), or conversely, WPI is decomposed with A-Amano G and then decomposed with trypsin (A-Amano G → GPTN6.0S) In either case, the TNF-α production-suppressing effect was almost the same, showing a slightly stronger tendency than that of trypsin alone. This suggests that a degradation product with higher TNF-α production inhibitory activity can be obtained by the combination of the two enzymes.
[0044]
[Test Example 3] Isolation and purification of peptide having TNF-α production inhibitory activity
Β-Lg, α-La prepared by the same method as in Example 1 and bovine Lf (Wako) UF permeate similarly prepared by the method of Example 1 were fractionated by reverse phase HPLC (FIGS. 4 and 6). And 11). Each peak was collected, and the amount of TNF-α production (TNF-α production inhibitory activity) was measured according to the method of Test Example 1. As a result, TNF-α production inhibitory activity was observed at each of three peaks for β-Lg and α-La, and four peaks for Lf. Therefore, each peak was purified, and molecular weight measurement (liquid chromatograph / mass spectrometer (LC-MSD: HP1100 series) and amino acid sequence were determined. From β-Lg, ALPMH, ALPMHIR and FKIDALNE, from α-La, LAHKAL, WLAHK, and peptides with C-residues of CEVFR and LDQWLCEK SS-linked, and Lf yielded peptides with SS-bonded C-residues of ETAEEVK, LGAPSITCVR and CR, WQWR and EDLIWK. Based on the primary structure, IPAVFK, which joins two residues of IPAV4 and N-terminal FK adjacent to the N-terminal of FKIDALNEN, except for FK at the N-terminal of FKIDALNEN, and IDALNENK with the addition of NK at the C-terminal, N-terminal of FKIDALNEN IPAVFKIDALNE with 4 residues of IPAV adjacent to the N-terminus, and IPAVFKIDALNENK with NK adjacent to the C-terminus of FKIDALNEN were chemically synthesized, and the α-La cleaved SS bond between CEVFR and LDQWLCEK. CNFFR and LDQWLCEK obtained were examined for their TNF-α production inhibitory activity As a result, CEVFR showed no activity.
In addition, as for the bovine Lf, the activity of inhibiting the TNF-α production of LGAPSITCVR and CR obtained by cleaving the SS bond between LGAPSILCVR and CR was examined. As a result, no activity was observed in CR. To summarize the above results, as a peptide having a milk protein-derived TNF-α production inhibitory activity, α-La has WLAHK (SEQ ID NO: 1), LAHKAL (SEQ ID NO: 2) and LDQWLCEK (SEQ ID NO: 3), β -Lg includes ALPMH (SEQ ID NO: 4), ALPMHIR (SEQ ID NO: 5), FKIDALNE (SEQ ID NO: 6), IDALNENK (SEQ ID NO: 7), IPAVFK (SEQ ID NO: 8), IPAVFKIDALNENK (SEQ ID NO: 9), IPAVFKIDALNE (SEQ ID NO: 10), and from LF, WQWR (SEQ ID NO: 11), EDLIWK (SEQ ID NO: 12), ETAEEVK (SEQ ID NO: 13), and LGAPSILCVR (SEQ ID NO: 14) were obtained.
[0045]
As a result of the homology search, SEQ ID NOs: 3, 6, 7, 8, 9, 10, 13 and 14 were found to be novel substances. SEQ ID NOs: 1, 2, 4 and 5 are reported to have angiotensin converting enzyme inhibitory activity (Journal of Dairy Research, 67: 65-71, 2000; British Journal of Nutrition, 84: Suppl. 1: S33-37, 2000) Although there is no report on TNF-α production inhibitory activity. WQWR has anti-herpetic activity, and EDLIWK has anti-cancer, anti-apoptosis, anti-ulcer agent, wound treatment agent, cardiotonic agent, anti-heparin, anti-ichemia, and antioxidant agent.
Based on the amino acid sequences of these peptides, the corresponding peptides were synthesized, and the TNF-α production inhibitory action of the synthesized peptides was examined according to the method of Test Example 1. As a result, these peptides all showed TNF-α production inhibitory activity in a concentration-dependent manner at 1 to 10 μg / mL (FIGS. 5, 7, 10 and 11). ED of these peptides ₅₀ (50% effective dose) is summarized in Table 1.
[0046]
[Table 1]

[0047]
[Test Example 4] IL-6 production inhibitory action
Six-week-old ICR mice (male) were purchased, and allowed to acclimatize with AIN-93M feed and drinking water for one week. As a group of 7 mice, the casein degradation product administration group (5 mg / mouse), β-Lg trypsin degradation product administration group (5 mg / mouse), α-La trypsin degradation product administration group ( 5 mg / mouse), FKIDALNE (SEQ ID NO: 6) administration group (1 mg / mouse), FKIDALNE (SEQ ID NO: 6) administration group (5 mg / mouse), LDQWLCEK (SEQ ID NO: 3) administration group (1 mg / mouse), LDQWLCEK (sequence) Number 3) Divided into 7 groups of administration group (5 mg / mouse). Samples were administered orally using a sonde for 3 consecutive days (13:00). 24 hours after the final administration, LPS was intraperitoneally administered at 50 μg / mouse. After 90 minutes, blood was collected from the orbit and serum was obtained by centrifugation. Serum IL-6 concentration was measured by ELISA (FIG. 13). The statistical difference by Fisher PSLD was tested for significant difference from the casein administration group.
The IL-6 concentration in the β-Lg trypsin degradation product administration group, FKIDALNE (SEQ ID NO: 6) administration group, and LDQWLCEK (SEQ ID NO: 3) administration group significantly decreased in a dose-dependent manner with respect to casein (** p The decrease in the latter two groups was concentration-dependent.
[0048]
【The invention's effect】
According to the present invention, it was possible to provide an orally ingestible peptide having an action of stimulating immunity while suppressing the production of inflammatory cytokines such as TNF and IL-6 without stimulating it. . Moreover, the peptide used for manufacture of the oral and enteral nutrient which prevents the metabolic abnormality and organ disorder at the time of a living body invasion, and enhances immunity could be provided. Furthermore, the peptide having an anti-inflammatory action could be provided.
[0049]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the LPS-induced TNF-α production inhibitory effect of undegraded WPI, β-lactoglobulin, α-lactalbumin or casein after induction of differentiation of THP-1 cells into monocytes and macrophages.
FIG. 2 shows the TNF-α production inhibitory effect of trypsin hydrolyzate of WPI, β-lactoglobulin, α-lactalbumin and casein.
FIG. 3 WPI trypsin (from porcine pancreas, Wako) hydrolyzate, WPI A amano G hydrolyzate, WPI trypsin and A amano G combined hydrolyzate, and A amano G and trypsin hydrate It shows the TNF-α production inhibitory effect of the degradation product.
FIG. 4 shows a reverse phase HPLC chromatogram of UF permeate of tryptic hydrolyzate of β-lactoglobulin.
FIG. 5 shows the TNF-α production inhibitory action of β-lactoglobulin derived peptides ALPMH, ALPMHIR and FKIDALNE.
FIG. 6 shows a reverse phase HPLC chromatogram of UF permeate of tryptic hydrolyzate of α-lactalbumin.
FIG. 7 shows the TNF-α production inhibitory action of α-lactalbumin derived peptides WLAHK, LAHKAL, CEVFR and LDQWLCEK.
FIG. 8 shows the TNF-α production inhibitory effect of each of the concentrations of trypsin hydrolyzate of β-lactoglobulin.
FIG. 9 shows TNF-α production inhibitory action at various concentrations of α-lactalbumin trypsin hydrolyzate.
FIG. 10 shows the TNF-α production inhibitory action of the peptides IDALNENK, IPAVFK, IPAVFKIDALNE and IPAVFKIDALNENK derived from β-lactoglobulin.
FIG. 11 shows a reverse phase HPLC chromatogram of tryptic hydrolyzate of bovine lactoferrin.
FIG. 12 shows TNF-α production inhibitory action at each concentration of bovine lactoferrin-derived peptides WQWR, EDLIWK, ETAEEVK, and LGAPSILCVR.
FIG. 13 Casein trypsin hydrolyzate, β-Lg trypsin hydrolyzate, α-La trypsin hydrolyzate, β-Lg derived peptide FKIDALNE, or LPS in serum after oral ingestion in α-La derived LDQWLCEK mice Inhibition of induced IL-6 production.

Claims (3)

WLAHK (SEQ ID NO: 1), LAHKAL (SEQ ID NO: 2), LDQWLCEK (SEQ ID NO: 3), ALPMH (SEQ ID NO: 4), ALPMHIR (SEQ ID NO: 5), FKIDALNE (SEQ ID NO: 6), IDALNENK (SEQ ID NO: 7), IPAVFK (SEQ ID NO: 8), IPAVFKIDALNENK (SEQ ID NO: 9), IPAVFKIDALNE (SEQ ID NO: 10) , EDLIWK (SEQ ID NO: 12), ETAEEVK (SEQ ID NO: 13), a peptide selected from the group consisting of LGAPSILCVR (SEQ ID NO: 14) or an acid thereof An anti-inflammatory agent comprising an addition salt and a carrier. WLAHK (SEQ ID NO: 1), LAHKAL (SEQ ID NO: 2), LDQWLCEK (SEQ ID NO: 3), ALPMH (SEQ ID NO: 4), ALPMHIR (SEQ ID NO: 5), FKIDALNE (SEQ ID NO: 6), IDALNENK (SEQ ID NO: 7), IPAVFK (SEQ ID NO: 8), IPAVFKIDALNENK (SEQ ID NO: 9), IPAVFKIDALNE (SEQ ID NO: 10) , EDLIWK (SEQ ID NO: 12), ETAEEVK (SEQ ID NO: 13), a peptide selected from the group consisting of LGAPSILCVR (SEQ ID NO: 14) or an acid thereof An oral and enteral nutritional agent for alleviating inflammatory response and nutritional treatment in systemic inflammatory response syndrome (SIRS) associated with biological invasion, comprising an addition salt and a carrier. The oral / enteral nutritional agent according to claim 2, wherein the biological invasion is surgery, trauma, burn, infection, acute pancreatitis, liver failure, peritonitis, or malignant tumor.

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