JPS59176264A - Physiologically active substance - Google Patents

Abstract

NEW MATERIAL:The compound of formula (R1 is H, lower alkyl or acetyl; R2 is OH, lower alkoxy, NH2, amino, amino acid residue or peptide group: R3 is H, lower alkyl, acetyl, amino acid residue or peptide group). EXAMPLE:2-Amino-9-(4-imidazolyl)-7-azanonanoic acid (named as giselocin). USE:A physiologically active substance causing a disease (GE) having the erosion and ulcer of gastric muscle as main symptoms to broilers and useful as a reagnet for the GE-development test. PREPARATION:Fish meal having GE-developing activity, e.g. meal of red-meat fish such as mackerel, sardine, mackerel pike, tuna, etc. is hydrolyzed by conventional process, and the hydrolyzed product is purified by treatment with activated carbon or decoloring resin, chromatography with a strongly acidic ion exchange resin, and chromatography with octadecyl silica gel, etc. to obtain the objective giselocin.

Description

[Detailed description of the invention] Ru.

In recent years, a disease mainly characterized by muscle gastric erosion and ulceration (hereinafter referred to as GE) has been occurring frequently in broilers, and research on this disease is being actively conducted both domestically and overseas. It has been announced overseas that there is a correlation between the occurrence of GE and the high intake of certain types of fishmeal, and the present inventors also
It has been confirmed that the cause of this outbreak is due to a specific fishmeal contained in feed, and the results have already been published (Journal of the Japanese Poultry Society, Vol. 17, No. 6, pp. 351-357 (1980), etc.) ).

However, the causative agent and mechanism of action are still unknown, and appropriate preventive measures have not yet been determined.

Therefore, the present inventors conducted further research and, after repeated trial and error, finally discovered the causative substance. and,
We have discovered that this substance is novel and exhibits GE expression activity whether alone or in a peptide-bound state, and based on this finding, we have completed the present invention.

The compound of the present invention has the general formula (wherein R1 represents hydrogen, a lower alkyl group, or an acetyl group, R2 represents an OH group, a lower alkoxy group, an amine group, an amino acid residue, or a peptide, and R3 represents hydrogen, a lower represents an alkyl group, acetyl group, amino acid residue, or bezotide).

Among the compounds represented by this general formula, those obtained by hydrolyzing fishmeal are 2-amino-9-(4
-imidazolyl)-7-adetunanoic acid (this compound was named gizerocin).

However, as shown in the general formula above, R in this general formula
Even if one or both of 1 and R5 is a C1-4 lower alkyl group such as a methyl group, ethyl group, isopropyl group, or an acetyl group, the compound has GE expression activity,
Even if R2 is a C7-4 lower alkoxy group such as a methoxy group, ethoxy group, or inleuci group, or an amino group, it has GE expression activity. Furthermore, R2 or R
One or both of 3 is an amino acid residue or 1l-lnie'-2
It may be petite. Amino acid residues exist in nature, such as glycine, alanine,
These include nogulin, inleucine, serine, phenylalanine, soroline, tryptophan, cysteine, asijragine, glutamic acid, arginine, lysine, ornithine, and histidine. The peptide is a combination of these amino acids, and its molecular weight may be, for example, about Orico+Veptide, or about 100,000.

Moreover, this pezotide may be a glycoprotein, a lipoprotein, or the like.

The acidic moiety or basic moiety of such a compound may form a salt.

Among the compounds of the present invention, gizerocine can be obtained from fishmeal having GE expression activity, for example, fishmeal from red fish such as mackerel, sardine, swordfish, and tuna. For example, 1
The yield of Gizerocin can be increased by heat treatment at 30°C for about 5 hours.

Fishmeal is first hydrolyzed. Hydrolysis may be carried out in accordance with the conventional method of hydrolyzing proteins to produce amino acids, for example, 3-
What is necessary is to add about 6N hydrochloric acid and heat at 80 to 120°C for about 6 to 48 hours. In addition, it goes without saying that enzymatic decomposition using protease may be performed.

To separate gizerocin from the hydrolyzate, first remove pigments and aromatic compounds using activated carbon, decolorizing resin, etc., remove nonionic substances using chromatography using an ion exchange resin, and separate gizerocin and amino acids. Then, chromatography using octyl silica gel, chromatography using cellulose, and chromatography using octadecyl silica gel may be sequentially performed. Among these methods, treatment with activated carbon or decolorizing resin, chromatography using strongly acidic ion exchange resin, and chromatography using octadecyl silica gel have particularly great purification effects. Only strong basic substances are collected in the first two steps, but chromatography using octadecyl silica gel is extremely effective for separating these strong basic substances and isolating gizerocine. This achievement is largely due to chromatography.

In these chromatography methods, the gizerosin fraction can be detected by measuring GE expression activity, but thin layer chromatography is a simple method. Details of this method will be described in the Examples.

Needless to say, Gizerocin can be obtained by a synthetic method rather than by hydrolysis of such natural products.

Among the compounds of the present invention, those in which one or both of R1 and R3 in the general formula are lower alkyl groups may be introduced using a known method of introducing an alkyl group into an amino group using gizerocine as a raw material, For example, it may be alkylated by the action of a rhodanide such as methyl bromide or isozolopyl bromide, and if necessary purified by chromatography using a strongly acidic ion exchange resin.

Furthermore, in the case where one or both of R1 and R3 in the general formula is an acetyl group, the amine group may be acetylated by reacting acetic anhydride or acetyl chloride with gizerocin, and if necessary, the product may be purified by distillation or the like.

In the case where II2 is a lower alkoxy group, it is sufficient to use a known method of esterifying a carboxyl group using gizerocin as a raw material, for example, by reacting with an alcohol in the presence of an acid catalyst.
Those in which R2 is an amine group may also be synthesized by a known method of amidating a carboxyl group using gizerocine as a raw material.

Those in which one or both of R2 and R3 are amino acid residues or 4-ptedones may be synthesized by a known method of bonding amino acids to each other through peptide bonds. It can also be separated and obtained by hydrolysis and chromatography using DEAE-cellulose.

The compound of the present invention has extremely high GE expression activity in broiler chickens. For example, when about 50 μg of gizerocin is administered to broiler chicks per day, GE symptoms will develop within one week. The GE expression activity of the compounds of the present invention is much stronger than that of histamine, since it is necessary to administer about 50 doses of histamine daily to induce the same symptoms using histamine.

The compound of the present invention is extremely useful as a reagent for GE expression testing in research on countermeasures to eradicate GE, and since its physiological activity is extremely high, the development of new medicines can be expected.

GE expression activity in the following examples was measured as follows.

A fixed amount of feed was added to commercially available feed, and 4 to 7 3-day-old broiler chicks were given free access to this feed.

The rearing temperature was 20°C, and the animals were dissected one week later and the degree of injury to the muscular gastric mucosa was visually observed. The muscular gastric mucosa of chicks that died during rearing was also observed.

The degree of injury to the muscular gastric mucosa is determined by GE intensity O: no abnormality is observed in the keratinoid layer, and the degree of roughening and erosion is extremely mild.

GEE grade 1: Minor changes such as roughness and irregularities in the running of pleats are observed.

GEE grade 2: A clear defect in the keratinoid layer is observed.

GE intensity 3: Severe ulceration of the muscular stomach and perforation that has penetrated into the abdominal cavity.

For each GE intensity, the number of GE intensity times the number of affected birds was calculated, and the value obtained by adding these together was used as the GE score.

Control tests were conducted on 10 controls and 1 control, + control was G.
Tests were conducted with 20% fishmeal with E activity and one control with 20% fishmeal without GE activity.

Example 1) Hydrolyzed mackerel fishmeal 1 kl? Add 20A of 6N hydrochloric acid and heat to 110°C.
Hydrolysis was carried out by heating for 24 hours.

2) Separation Add activated carbon 130.9 to this hydrolyzate to give 50 to 6
Stirred at 0°C for 1 hour. Activated carbon was distributed from house to house, and the p liquid was concentrated under reduced pressure to remove hydrochloric acid and water.

Water was added to the concentrate, and this liquid was passed through a strongly acidic cation exchange resin DOWEX 50WX8 (H type) 5A.

Water 13A, 0.5N hydrochloric acid 13A, 2N hydrochloric acid 67〃, and 4N hydrochloric acid 137A were sequentially passed through this column for stepwise elution, and the effluent eluted with water, 0.5N hydrochloric acid, and 2N hydrochloric acid is 13! When the effluent eluted with 4N hydrochloric acid was fractionated into 45j fractions, GE expression activity was observed in the first two fractions eluted with 4N hydrochloric acid. Among the results of measuring GE expression activity using 7 chicks for each group,
A portion of the 3rd fraction eluted with 2N hydrochloric acid is shown in the table below, 217 g.

4th fraction 212 1 5th fraction 198 2 4N hydrochloric acid elution 1st fraction 174 12
2nd fraction 189 0 3rd fraction 2
06 l (+control) 140 (
12) Concentrate and dry the first volume of 4N hydrochloric acid elution, and remove this dry volume.
□ Added 8 ml of water to the mixture. This solution is mixed with a weakly acidic cation exchange resin Amberly) IRCG-50 (N-ethylmorpholine type) 170mA! Then, 500 ml each of IMN-ethylmorpholine, 2MN-ethylmorpholine, and 3M aqueous ammonia were sequentially passed through the column for stepwise elution, and the GE-expressing active substance was eluted with the 3M aqueous ammonia. G using 4 chicks for each elution fraction
The results of measuring E expression activity are shown in the table below.

IMN-ethylmorpholine elution fraction 155,9
12MN-ethylmorpholine elution fraction 170.9
13M ammonia water elution fraction 138,9 1
1 (10 controls 134.9 8) (1 control 162.!iJ 1) The 3M ammonia water elution fraction was concentrated to dryness and then dissolved in about 2 ml of triethylamine-acetate buffer (pH 7.0). This solution was mixed with octyl silica dal (Lichroprep RP).
-8, manufactured by Merck & Co., Ltd.) was applied to a column packed with 15.7 mJ, and the same buffer solution was mixed with isopropanol at 0%, 15%, and 3.
When the solutions added at 0%, 45%, 6o, 75 and 100% were sequentially eluted at 100mA each, the GE-expressing active substance was found in the elution fraction of the buffer only. discovered. G using 4 chicks for each elution fraction
The results of measuring E expression activity are shown in the table below.

Inglopanol 0chi 125,9 131
5chi 168 3 30% 175 1 45% 179 0 60chi 178 1 75% 154 1 100% 181 3 (+ control 140 13 (= control 1633 Chromatogram sheet with cellulose as adsorbent (product name, Eastman Kodak Company) Each eluted fraction was spotted on a tube made by Ingropanol-28% NH40)T-
Thin layer chromatography was performed by developing with a developing solvent of H2O (16: 3.5: 4), and color development with a diazosulfanilic acid reagent revealed that the Rf of the GE-expressing active substance
The value was 0.58.

The elution fraction containing only the buffer solution was dried under reduced pressure and dissolved by adding a small amount of water. Add this solution in advance to isopropanol-28 NH
It was applied to a cellulose column equilibrated with a solvent of 40H-H2O (16:1:4) (packing i: 24.0 ml), developed with the same solvent, and the effluent was fractionated into 81 nl portions. When both of these fractions were analyzed by the thin layer chromatography described above, the active substance was eluted in both fractions at 0.24 to 0.40 times the column volume. Of this active fraction, we had confirmed through thin layer chromatography that other diazosulfanilic acid reagent color-forming substances were mixed in both the frontmost fraction and the last fraction. These colored substances were separated and removed using chromatography.

The final stage of purification was performed by reverse phase partition high performance liquid chromatography using octadecyl silica gel. First, the active fractions from the previous step were collected and dried under reduced pressure, and a small amount of 0.05M
) Dissolved in ethyl ammonium-acetate buffer (pH 7.0). Dispense this solution at a time equivalent to 50g of fishmeal into Nucleosil NC,-18 (trade name,
When the solution was run at a rate of 3 m/:/min through an 8 x 250 column packed with 12.6 rnl (manufactured by Nagle) and then the same buffer was run at the same rate for elution development, the chromatogram shown in the figure was obtained. . Using five chicks, G
The results of measuring E expression activity are shown in the table below.

1 140g 2 2 95 12 3 134 1 4 1, 42 0 5 131 1 6 123 0 (+Control 114 8) (One comparison 154 0) The results of the above purification steps are summarized in the table below.

Fish meal 1 kg440U
2300IncJ/U Dowex 50WX8
Fl 380 13 Amber Light IRC
G-5012g 110 11 octyl silica gel 0.29 55 3.6 Cellulose 10■ 5002 octadecyl silica gel 0.7m9 28 0.03
*11 chicks [The activity to express GE within 7 days was defined as 1 unit.

Cellulose and silicadal were used as adsorbents for both of this second peak, and inzolopanol-28% was used as the developing solvent.
NH40H-H20 (16:3.5:4), n-butanol-HCoOH-H20 (17:4:5) and inzolopanol-HCOOI (-H20 (16:5)
．． A total of 6 types of thin layer chromaburography were performed using 3 types of 5:6), and when color was developed with both diazosulfanilic acid reagent and fluorescamine, only one spot of gold was shown in each case. The object turned out to be a single object, and was named Gizeroshin.

When the UV absorption of the aqueous Gizerocin solution was measured, λm was found to be 209 nm.

3) Structure determination Gizerosinch E/D mass spectrum was measured.
m/Z 241 (M+H”), 263 (M+Na”
) and 279 (M+K'') were observed. As a result, it was determined that the molecular weight of Gizerocin was 240.

Add acetic anhydride and methanol to Gizerocin and stir at room temperature for 4 hours.
The reaction was allowed to proceed for 8 hours. As a result of measuring this Gizerocin derivative using EI bulk resolution mass spectrometry, mA was 338.19.
518 (C16H2604N4, theoretical value 338.195
40) was observed.

This revealed that Gizerocin has two amino or imino groups and one carboxyl group that can be acetylated, and that the molecular formula of Gizerocin is C11H2oO2N4.

Dissolve Gizerocin in heavy water and generate 400 MHz 'H-N
When the MR spectrum was measured, two proton peaks at δH732 and 8.43 derived from the imidazole ring were observed. In addition, due to spin decoupling, -N-(CH2)4-CH-N- and -C-cT(2
It was found that -CH2-N- was present, suggesting that there were lysine and histamine residues in the molecule. Therefore, 400MT of lysine and histamine were added under the same conditions as Gizerocin.
(Z'H-NMR spectrum was measured and compared with the spectrum of Gizerocine, confirming that Gizerocine has these residues. Furthermore, this comparison shows that the ε-methylene of lysine is the same as that of the imidazole ring. It was suggested that the methylene of the amine group of histamine was not bonded to N and was not an amide bond.

Furthermore, as a result of measuring the above-mentioned Gizerocin derivative by the linked scan method of EI mass spectrum, m/Z 338
m7z-poor a-g and 245 derived directly from the parent ion of
A fragment ion peak was observed. and,
By high-resolution mass coverage trometry, m/Z 2
The molecular formula of the fragment ion of 45 is C11H21N2
04 (actual value 245.152) 3, theoretical value 245.15
0'12). This fragment ion is considered to be the imidazole moiety removed from the parent ion. Further, high-resolution mass spectrometry revealed that the molecular formula of the fragment ion with m/Z 208 was C11H8N30 (actual value 208.14472, theoretical value 208.14498). This fragment ion is derived from the parent ion)ic (NI(COC
H3) It is thought that COOCH6 was desorbed. As a result, Gizerocin is thought to have a monosubstituted imidazole ring and an α-aminocardexyl group in its molecule.

As a result of the above, the structure of Gizerocine is It was decided.

The fact that Gizerocin has this structure is further evidenced by the 'H-NMR spectrum of the synthesized product obtained by chemically synthesizing it.
It was also confirmed that the cuttle was identical to that of gizerocin obtained from fishmeal. This synthetic gizerocin also showed strong GE expression activity.

[Brief explanation of drawings]

The drawing shows the results of chromatography using octadecyl silica dal in the purification process for obtaining the compound of the present invention from fishmeal hydrolyzate. Patent applicant: Itochu Feed Co., Ltd. Representative: Masahiro Tanaka, patent attorney

Claims (1)

[Claims] General formula (wherein R1 represents hydrogen, a lower alkyl group, or an acetyl group, R2 represents an OH group, a lower alkoxy group, an amine group, an amino acid residue, or a peptide, and R3 represents hydrogen, a lower Represents an alkyl group, acetyl group, amino acid residue or peptide)