JP4470152B2 - Novel tripeptide and method for producing the same - Google Patents

Description

  The present invention relates to a novel tripeptide and a method for producing the same. More specifically, the present invention relates to a tripeptide composed of one molecule of β-alanine and two molecules of ornithine, and also relates to a method for producing the tripeptide from a shijimi extract.

The inventors of the present invention have been continuously researching the active ingredients of shijimi.
Shijimi has been used by the public since ancient times as a nourishing tonic food. In recent years, research on the active ingredients has progressed, and the elucidation of the ingredients has also progressed. Corbicula in particular including various vitamins such as vitamin B ₁₂ is useful components indicating the important physiological activities, for example, alpha with lipid metabolism improving action, beta-unsaturated fatty acids, activate the urea cycle, liver function It contains amino acids such as ornithine that have a dieting action by improving action and growth hormone secretion promoting action. In addition, there is a report that amino acids such as methionine and cystine contained in swordfish also show liver function enhancing action.
Among the useful components in these similar extracts, ornithine is attracting particular attention. Shijimi has long been known to be effective for so-called hangovers or to reduce the symptoms thereof, and it is considered that the physiologically active action of ornithine is involved in the action. The present inventor has also already proposed a method for increasing the in vivo content by promoting and promoting the useful action of ornithine in the living body (Patent Documents 1 and 2).
JP 2001-204432 A JP 2003-274907 A

However, it seems that there are still unknown useful substances in Shijimi, and the elucidation of these substances is awaited.
Under such circumstances, an object of the present invention is to provide a specific tripeptide which is a novel useful substance and a method for producing the same.

（２） シジミエキスを水で希釈し、分画することを特徴とする前記（１）記載のＮ末端からβ−アラニン、オルニチン、オルニチンの順でペプチド結合したトリペプチドを製造する方法、
（３） α−アミノ基とδ−アミノ基を保護したオルニチン及びβ−アミノ基を保護したβ−アラニンを用いて化学的に合成することを特徴とする前記（１）記載のＮ−末端からβ−アラニン、オルニチン、オルニチンの順でペプチド結合したトリペプチドを製造する方法、
に関する。
As a result of advancing research on useful components in shijimi extract, the present inventors are well known that β-alanine and ornithine are contained in shijimi extract as described above, but these amino acids are free. In addition to the amino acid form, it was also found that the peptide form exists. As a result of further research, it was found that this peptide is a novel tripeptide in which the two amino acids form a peptide with a specific binding structure, and the present invention has been achieved.
That is, the present invention
(1) A tripeptide in which β-alanine, ornithine and ornithine are peptide-bonded in this order from the N-terminus represented by the following formula :

(2) A method for producing a tripeptide having peptide bonds in the order of β-alanine, ornithine, ornithine from the N-terminus according to (1), wherein the swordfish extract is diluted with water and fractionated,
(3) chemically synthesizing using ornithine in which α-amino group and δ-amino group are protected and β-alanine in which β-amino group is protected, from the N-terminal according to (1) above a method for producing a tripeptide having peptide bonds in the order of β-alanine, ornithine, ornithine,
About.

  The tripeptide composed of one molecule of β-alanine and two molecules of ornithine according to the present invention is a novel peptide. And this peptide can be easily manufactured from the swordfish extract which is a food, has high safety, and can expect at least the action similar to the bioactive action exhibited by ornithine from its constituent amino acids. Furthermore, it is useful as a raw material for producing at least its constituent amino acids.

この新規トリペプチドは、シジミエキスから製造することができる。シジミエキスの製造法は、一般的には生シジミ、あるいは冷凍シジミに水を加えて沸騰させ、エキス分を抽出し、濾過し、必要により濃縮し、あるいは凍結乾燥などの手段で粉末化するというものである。その他、すでに種々の方法が開発されているが、本発明に使用するシジミエキスは何れの方法によって得られた物でも構わない。しかし、その取扱の易さなどから粉末化したエキスを使用することが好ましい。 The novel tripeptide of the present invention is composed of one molecule of β-alanine and two molecules of ornithine, and its structure is represented by the following formula in which β-alanine, ornithine and ornithine are peptide-bonded in this order from the N-terminus. It is a tripeptide.

This novel tripeptide can be produced from a swordfish extract. The production method of shijimi extract is generally said to be boiled by adding water to fresh or frozen shijimi, extract the extract, filter, concentrate if necessary, or powder by means such as freeze-drying Is. In addition, various methods have already been developed, but the swordfish extract used in the present invention may be obtained by any method. However, it is preferable to use a powdered extract because of its ease of handling.

The tripeptide of the present invention can be produced by diluting a concentrate of a rainbow trout extract or a dried powder thereof in water and fractionating (purifying) it by chromatography such as gel filtration or ion exchange.
That is, the swordfish extract can be diluted with water to about 20 to 50 times the solid content and fractionated by gel filtration or ion exchange. However, although the composition of the shijimi extract varies depending on the kind and season of shijimi, it is roughly about 50% glycogen-based carbohydrate, about 35% protein, about 10% ash, and about 5%. Among them, the tripeptide of the present invention has a low molecular weight of 317, and its content in the solid content of the swordfish extract is small. The high molecular weight carbohydrates and proteins are removed by performing ultrafiltration and dialysis using a membrane of about 10,000 to 10,000, and the resulting low molecular fraction is further purified by gel filtration or ion exchange. It is preferable to obtain the target tripeptide by fractionation.

As described above, the tripeptide of the present invention can be produced by fractionation from a shijimi extract, but can also be chemically synthesized in the same manner as other peptides.
From the viewpoint of ease of operation, it is preferable to synthesize by a solid phase method using a peptide synthesizer commercially available from each company. That is, the peptide chain is sequentially extended from the C-terminal by the Fmoc method according to the program of the apparatus to be used. Ornithine [Fmoc-0rn (Boc)] in which the α-amino group and the δ-amino group were previously protected with Fmoc and Boc in order to use the carrier as a support for solid phase peptide synthesis and prevent side reactions. An Fmoc amino derivative of β-alanine [Fmoc-β-Ala] in which the β-amino group is protected with Fmoc is used. Fmoc-Orn (Boc) is bound to the carrier, the α-amino group is deprotected using piperidine or the like, and then the C-terminus of Fmoc-Orn (Boc) is coupled using a coupling agent, and again with piperidine or the like. The α-amino group of the second ornithine is deprotected and the C-terminus of Fmoc-β-Ala is coupled in the same manner. Finally, all the deprotection and carrier removal are carried out using an acid such as trifluoroacetic acid, and the tripeptide of the present invention is produced by lyophilizing the one extracted with 0.1% aqueous trifluoroacetic acid. It can be purified by reverse-phase high performance liquid chromatography if necessary.

The tripeptide of the present invention is composed of β-alanine and ornithine. Therefore, the tripeptide of the present invention can be expected to have physiological functions similar to those of peptides containing these amino acids.
For example, carnosine (β-alanine-histidine) and anserine (β-alanine-methylhistidine) are known as peptides containing β-alanine. These are the main peptides contained in many vertebrate skeletal muscle tissues, and are known to have strong intracellular pH buffering, antioxidant activity, enzyme activation, and physiological functions as neurotransmitters. Since the tripeptide of the present invention has three amino groups in the molecule and is extremely characteristic, it is considered that the tripeptide has a structure sufficient to express physiological functions like carnosine and anserine.
In addition, ornithine has the physiological function as described above, and the tripeptide of the present invention can be expected to have a similar liver function enhancing action.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples.
(1) Elucidation of the free amino acid composition of the shijimi extract and the amino acid composition of the hydrolyzate The hydrolysis of the shijimi extract is carried out by taking 1 mg of the shijimi extract powder in a test tube in accordance with a method generally used for the measurement of protein constituent amino acids. 1 ml of 20% hydrochloric acid was added, and the mixture was hydrolyzed at 110 ° C. for 20 hours after sealed under reduced pressure. Thereafter, the mixture was dried under reduced pressure, and various amino acid contents including ornithine were measured under free amino acid analysis conditions using a fully automatic amino acid analyzer (JEOL, JLC-500 / V). The results of the free amino acid composition of the shijimi extract and the amino acid composition of the hydrolyzate are shown in Table 1.

The ornithine content was 5.9 (nmol / mg) before hydrolysis, that is, in the free state, and 66.4 (nmol / mg) in the hydrolyzate. The value (B)-(A) obtained by subtracting the value of the free amino acid (A) from the value of the hydrolyzate (B) is considered to be the amount of amino acids constituting the protein or peptide. Therefore, it was revealed that ornithine that is not free but constitutes a protein or peptide is present in the extract at 60.5 (nmol / mg).
The amino acids other than ornithine and β-alanine shown in Table 1 are amino acids that constitute the protein, so that the protein contained in the extract is decomposed into hydrolyzed amino acids by hydrolysis, and the amino acid content of the hydrolyzate increases. Of course.
However, in general, ornithine and β-alanine are not amino acids that constitute proteins, so it was very interesting to see what structure they are.

(2) Structure elucidation of ornithine-containing substance contained in shijimi extract Therefore, in order to obtain knowledge about the molecular weight of the ornithine-containing substance contained in shijimi extract, the present inventors used an ultrafiltration membrane of shijimi extract. Tried fractionation. The shijimi extract solution was sufficiently dialyzed against water using a dialysis membrane having a fractional molecular weight of 10,000 (manufactured by PIERCE, Slide-A-Lyzer), and the dialysis internal solution was a polymer fraction having a molecular weight of 10,000 or more, The dialyzed external solution was recovered as a low molecular fraction having a molecular weight of 10,000 or less, concentrated under reduced pressure, and then powdered by lyophilization.

  About these fractions, the amino acid composition before and after hydrolysis was compared in the same manner as described above, and it was examined in which fraction the ornithine-containing substance was fractionated. As a result, it was found that the ornithine-containing substance was fractionated into a low molecular fraction having a molecular weight of 10,000 or less. Furthermore, as a result of examining in the same manner using a dialysis membrane having a molecular weight cut off of 3,500 (manufactured by PIERCE, Slide-A-Lyzer), the ornithine-containing substance was a fraction of the outer liquid of dialysis, that is, a low molecular weight having a molecular weight of 3,500 or less. It was found that it was fractionated into fractions. From this, the ornithine-containing substance was considered to be a peptide having a molecular weight of 3,500 or less.

  Next, using a reverse phase column such as Sep-Pac tC18 (manufactured by Waters) and “Develosil RPAQUEOUS (manufactured by Nomura Chemical Co., Ltd.), which is often used for peptide research, the mobile phase was 0.1% trifluoroacetic acid. Attempts were made to purify the ornithine-containing substance using a concentration gradient of nitrite and acetonitrile, but the ornithine-containing substance was eluted in the non-adsorbed fraction and could not be purified. After removing salts and free amino acids from the sample, purification by gel filtration using HPLC was attempted.

The HPLC conditions were Superdex Peptide HR 10/30 (manufactured by Amersham Pharmacia Biotech), the column was 20 mM phosphate buffer (pH 7.2) containing 250 mM NaCl, the flow rate was 0.25 ml / min, and the detection was Absorbance at 205 nm was measured.
And 500 microliters was fractionated using the fraction collector.
20 μl from each fraction was placed in a test tube, freeze-dried, 1 ml of 20% hydrochloric acid was added, and the tube was sealed under reduced pressure and hydrolyzed at 110 ° C. for 20 hours. Thereafter, the mixture was dried under reduced pressure, and various amino acid contents including ornithine were measured under free amino acid analysis conditions using a fully automatic amino acid analyzer (JEOL, JLC-500 / V).

FIG. 1 shows the absorbance at 205 nm obtained by HPLC and the ornithine content after hydrolysis of each fraction. As a result, ornithine was detected as a symmetrical peak in fraction numbers 30 to 36 and was considered to be a single compound.
The retention volume of the ornithine-containing substance was 17 ml, but in gel filtration, it is possible to estimate the molecular weight of the target substance from comparison with the retention volume of a standard preparation whose molecular weight is known. Thus, when the molecular weight of the ornithine-containing substance was estimated from the elution position (Table 2) of the sample in the gel filtration column used this time, it was found to be about 300 (FIG. 2).

  Moreover, only β-alanine and ornithine were detected from the amino acid analysis of the hydrolyzate of fraction numbers 30 to 36, and the ratio was 1: 2. Since the molecular weight of β-alanine is 89 and the molecular weight of ornithine is 132, the molecular weight of a compound in which one molecule of β-alanine and two molecules of ornithine are peptide-bonded is 317, which is almost the same as the molecular weight estimated from gel filtration. That is, the ornithine-containing substance contained in the shijimi extract was considered to be a compound in which one molecule of β-alanine and two molecules of ornithine were peptide-bonded. Hereinafter, the ornithine-containing substance contained in the shijimi extract is referred to as an ornithine-containing compound.

(3) Structure elucidation of ornithine-containing compound contained in shijimi extract Next, fraction numbers 30 to 36 obtained by HPLC were collected, desalted, and then substituted with heavy water, and ¹ H-NMR was measured. As a result, all protons bonded to carbon were assigned. Signals around 4.15 ppm and 4.00 ppm are equivalent to two protons bound to the alpha carbon of two ornithine molecules, and signals around 2.56 and 3.07 ppm are on the alpha and beta carbons of β-alanine. The signal near 2.83 ppm for every 4 protons bound is 4 signals for every 2 protons bound to the delta carbon of 2 ornithine molecules, the signal around 1.5 to 1.7 ppm is Two ornithine molecules are derived from eight protons bound to the beta and gamma carbons of every four protons, all 18 protons bound to carbon are assigned, and one β-alanine molecule and two ornithine molecules are peptide-bonded. (Fig. 3).

  From the above results, there are three possible structures of ornithine-containing compounds from the N-terminus: β-alanine-ornithine-ornithine, ornithine-β-alanine-ornithine, ornithine-ornithine-β-alanine. Therefore, hydrazine degradation was attempted to determine the C-terminus. When the peptide is reacted with anhydrous hydrazine, the peptide bond is cleaved and all amino acids except the C-terminal amine acid become amino acid hydrazide. Therefore, the ornithine-containing compound was reacted with anhydrous hydrazine at 100 ° C. for 2 hours, and then subjected to an amino acid analyzer. As a result, ornithine was detected and β-alanine was not detected. From this, the C-terminal amino acid is ornithine, and the structure of the ornithine-containing compound is either β-alanine-ornithine-ornithine or ornithine-β-alanine-ornithine from the N-terminus.

  Next, the present inventors measured changes with time of β-alanine and ornithine released by hydrolysis. That is, about 50 μg of ornithine-containing compound that has been freeze-dried is added with 500 μl of 20% hydrochloric acid, sealed under reduced pressure, hydrolyzed at 110 ° C. for 0, 1, 2, 4, 8, 20 hours, and released using an amino acid analyzer. Crude β-alanine and ornithine were measured. As a result, it was found that β-alanine was almost completely released after 1 hour of hydrolysis treatment, while ornithine gradually hydrolyzed until 20 hours later (FIG. 4). This cannot occur in the structure of ornithine-β-alanine-ornithine, and it is essential that β-alanine is at the end. That is, the N-terminal was β-alanine, and the structure of the ornithine-containing compound was determined to be β-alanine-ornithine-ornithine having the structure shown below.

なお、上記式で表される化合物を別途化学的に合成し、^１Ｈ−ＮＭＲを測定して前述の各シグナルを確認した。

In addition, the compound represented by the said formula was synthesize | combined chemically separately, and < ¹ > H-NMR was measured and each above-mentioned signal was confirmed.

The graph which shows the gel filtration chromatography of a low-molecular-weight extract of a shijimi extract. The graph which shows the relationship between the retention volume of the sample used for estimation of the molecular weight of an ornithine containing substance in gel filtration chromatography, and molecular weight. Graph showing the ¹ H-NMR of ornithine-containing compounds of the present invention. The graph which shows the time-dependent change of the ornithine and (beta) -alanine liberated by the hydrolysis of the ornithine containing compound of this invention.

Claims (3)

A tripeptide in which β-alanine, ornithine, and ornithine are peptide-bonded in this order from the N-terminus represented by the following formula .

The method for producing a tripeptide having peptide bonds in the order of β-alanine, ornithine and ornithine from the N-terminal according to claim 1 , wherein the swordfish extract is diluted with water and fractionated. The β-alanine from the N-terminal according to claim 1, which is chemically synthesized using ornithine in which an α-amino group and a δ-amino group are protected and β-alanine in which a β-amino group is protected. A method for producing a tripeptide having peptide bonds in the order of ornithine and ornithine.

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