JPH0331689B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel gastric juice secretion suppressant, and more particularly to a gastric juice secretion suppressant containing a melanin substance obtained from squid or octopus ink. Conventionally, various useful medicines have been extracted from animal organs, and some are currently in practical use. On the other hand, squid and octopus are eaten, but the large amount of ink produced during this process poses a major problem in their disposal. The present inventor predicted that the black color of squid and octopus ink may be derived from melanin-based substances, and if so, it may have some physiological activity. We conducted research on the ingredients and physiological activities. Specifically, we homogenized squid or octopus ink, filtered it through a cloth, and dialyzed the resulting fluid, and examined the physiological activity of the internal fluid obtained.
The present invention was completed based on the discovery that this compound has excellent gastric juice secretion suppressing and anti-inflammatory effects. Many of the drugs conventionally used as anti-inflammatory agents were associated with side effects related to gastrointestinal disorders. However, the present invention is characterized by the discovery of a physiologically active substance that not only has an anti-inflammatory effect but also actively suppresses gastrointestinal disorders. The melanin-based substance of the present invention is produced, for example, as follows. First, an alkaline agent such as caustic soda or caustic potash is added to squid or octopus ink to adjust the pH to 6 to 12, and the mixture is sufficiently homogenized using a homogenizer or the like. Next, the solid content is removed by passing it through a cloth or by centrifugation. Melanin-based substances can be obtained by placing the solution in a cellulose tube, dialyzing it against water or physiological saline, and freeze-drying the internal solution. The melanin-based substance of the present invention thus obtained has the following physical properties and physiological activities. (1) Physicochemical properties Shape Black amorphous powder Solubility Slightly soluble in water, insoluble in ethanol, ethyl acetate, chloroform, benzene, dimethylformamide, dimethyl sulfoxide Melting point 300℃ or higher Elemental analysis value C: 47-50% , H: 3-4%, O: 38-41%,
N: 8-9%, ash: 0.06-0.08% Molecular weight From the gel filtration of Cephadex G-100, it is a mixture of a substance with a molecular weight of approximately 25,000 and a substance with a molecular weight of over 160,000 (Figure 1) Ultraviolet absorption spectrum 2 As shown in the figure Amino acid composition (in 50ml of dialysis fluid)

【table】

[Table] Sugar 1.16% (phenol sulfuric acid method) Stability Deactivated at 121℃ for 30 minutes Stable with N-hydrochloric acid at 100℃ for 30 minutes Stable with N-caustic soda at 100℃ for 30 minutes Based on the above physical properties, the present invention The melanin-based substance was confirmed to be a melanin-based substance containing protein and sugar. (2) Acute toxicity (LD ₅₀ ) 6-week-old dd-N mouse (body weight: male 20-27 g,
(Female 20-26g) Intraperitoneal administration 1g/Kg or more Oral administration 5g/Kg or more (3) Physiological activity Gastric juice secretion suppressive effect The gastric juice secretion suppressive effect was measured using Shay's pyloric ligation method [Gastroenterology 5 , 43 (1945)]. I went there. B. Intraperitoneal administration: 24 male Wistar rats weighing 200-250 g.
After an hourly fast, the abdomen was opened under ether anesthesia, and the pylorus was ligated. Immediately after ligation, Example 1 (Test A)
Alternatively, the physiological saline solution of the melanin substance obtained in Example 2 (Test B) was administered intraperitoneally. Four hours after administration, the stomach was removed, and the amount of gastric juice was measured.
Total acidity and pepsin activity were measured. The total acidity is 0.05 N using phenolphthalein as an indicator.
- Titrated with NaOH and expressed as hydrochloric acid in μEq/100g body weight. Pepsin activity was measured by the casein hydrolysis ability of gastric juice. The activity was determined by measuring the absorbance of tyrosine liberated by the decomposition of casein at 280 nm, and expressed in terms of mg tyrosine/100 g body weight. In addition, as a control group, physiological saline was administered,
Similarly, gastric juice volume, total acidity, and pepsin activity were measured. The results are shown in Table 2.

[Table] b. Intraduodenal administration The inhibitory effect on gastric juice secretion during intraduodenal administration was tested using the same procedure as for intraperitoneal administration. The test solution was prepared by dissolving the melanin substance obtained in Example 1 in physiological saline, and directly administering it into the duodenum through a 1/4 injection needle immediately after pyloric ligation. The results are shown in Table 3.

[Table] Anti-ulcer effect The anti-ulcer effect was tested using the pylorus ligation ulcer method. 48 male Wistar rats weighing 200-250g
After an hourly fast, the pylorus was ligated in the same manner as in the assay for gastric secretion, and the stomach was removed 18 hours after ligation. This is incised from the greater curvature side and ulcerative changes that occur in the forestomach are examined using the method of Adami et al. [Arch.int.
Pharmacodyn., 147, 113 (1964)]. As a test substance, the substance obtained in Example 1 dissolved in physiological saline was administered twice, immediately after ligation and 8 hours after ligation. The results are shown in Table 4.

[Table] As is clear from the above experiments, the melanin-based substance of the present invention significantly suppresses gastric juice secretion when administered intraperitoneally at 5 mg/Kg and intraduodenally at 50 mg/Kg.
Shows anti-ulcer effect when administered twice intraperitoneally at 25 mg/Kg. Anti-inflammatory effect (a) Rat footpad carrageenan edema suppression test Van
Arman [J.Pharmacol.Exp.Ther., 150 , 328
(1965)] and Winter [Proc.Soc.Exp.Biol.
Med., 111 , 544 (1962)].
That is, the melanin substance obtained in Example 1 as a specimen was dissolved in physiological saline, and this was administered intraperitoneally 30 minutes before carrageenan injection. Carrageenin was added to each footpad as a 1.5% saline solution.
0.1 ml was administered. Male Wistar rats weighing 130 to 150 g were used, and the edema rate relative to the paw volume immediately after carrageenan injection was determined over time. At this time, as a control group, physiological saline was administered intraperitoneally, footpad edema was created by the above method, and the edema rate was determined over time. The results are shown in Table 5.

[Table] As is clear from Table 5, the edema rate in the sample administration group was significantly lower than that in the control group, and edema caused by carrageenan was suppressed. In addition, in the sample administration group, the edema rate in the 5 mg/Kg administration group was lower than the edema rate in the 1 mg/Kg administration group, and dose dependence was observed. (b) Examination of irritation When examining anti-inflammatory effects, when the test substance is irritating, the apparent edema rate is suppressed. Therefore, we investigated the irritating properties of melanin-based substances obtained from squid ink. Effect on squirming The physiological saline solution of the melanin-based substance obtained in Example 1 was intraperitoneally administered to male ddy mice (body weight 20-22 g), and 10 minutes after administration, a 10-minute period of
The number of squirming was measured. As a positive control, a 0.7% acetic acid solution was intraperitoneally administered to mice, and the number of squirmings was measured in the same manner. The results are shown in Table 6.

[Table] As is clear from Table 6, there was no difference in the number of squirming in the melanin substance obtained from squid ink administration group compared to the physiological saline administration group, and no irritation was observed. Effect on vascular permeability Whittle et al. [Brit.J.Pharmacol., 22 , 246
(1946)] according to the method of ddY male mice (body weight
0.1 ml of 4% trypan blue was administered to the mice (20 to 22 g) through the tail vein, and 10 minutes after administration, the specimen was intraperitoneally administered. After another 20 minutes, the abdomen was opened under ether anesthesia, and the abdominal cavity was washed out with 5 ml of water. Trypan blue exuded into the washing solution was determined by absorbance at 590 nm, and the effect of the specimen on vascular permeability was measured. The results are shown in Table 7.

[Table] As is clear from Table 7, the exudation amount of trypan blue in the sample administration group was not different from that in the physiological saline administration group, and no vascular permeability enhancing effect was observed. As a result of the above, the melanin substance obtained from squid ink has no irritating effect, and this confirms that the carrageenan edema suppressing effect is based on the anti-inflammatory effect of this substance. The melanin-based substance used in the present invention is administered orally or parenterally in the form of powder, granules, tablets, capsules, injections, etc., either as is or mixed with appropriate pharmaceutical carriers, excipients, diluents, etc. be able to. For oral administration, the dosage is, for example, for adults.
The dosage is preferably 50 to 500 mg/day, but it can be increased or decreased depending on symptoms, age, body weight, etc. Next, an example will be given and explained. Example 1 Purple squid (Ommastrephes bartrami)
The contents of 80 pieces (243 g) of Sumibukuro (Lesueur) were washed out with 350 ml of physiological saline. The pH was adjusted to 8 by adding 4N aqueous sodium hydroxide solution, homogenized with a grinder mirror, and filtered through double gauze to obtain 300 ml of liquid. The solution was placed in a cellulose tube, dialyzed against water for 72 hours, and the dialyzed solution was freeze-dried to remove melanin-based substances.
26.4g was obtained. Example 2 The contents of 5 pieces (2.5 g) of Octopus vulgaris Cuvier were washed out with 14 ml of physiological saline. A 4N aqueous sodium hydroxide solution was added to the mixture to adjust the pH to 9, the mixture was homogenized using a glass potter type homogenizer, and filtered through double gauze to obtain 8 ml of a solution. The solution was dialyzed in the same manner as in Example 1 and freeze-dried to obtain 0.7 g of melanin-based substance. Example 3 Using melanin-based substances isolated from squid ink,
Tablets were prepared, each having the following composition. Squid ink melanin substance (Example 1) 100 mg Corn starch 50 Lactose 27.5 Magnesium stearate 2.5 Total amount 185 mg Example 4 Using a melanin substance separated from squid ink,
Capsules were prepared by filling No. 1 capsules with a mixture having the following composition. Squid ink melanin substance (Example 1) 50mg Lactose 393 Magnesium stearate 7 Total amount 450mg

[Brief explanation of drawings]

Figure 1 shows the elution pattern of the melanin-based substance of the present invention when gelled through Cephadex G-100, and Figure 2 shows the ultraviolet absorption spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. A gastric juice secretion suppressant characterized by containing a melanin substance containing protein and sugar obtained from squid or octopus ink and exhibiting the following physical properties. Shape: Black amorphous powder Solubility: Slightly soluble in water, insoluble in ethanol, ethyl acetate, chloroform, benzene, dimethylformamide, dimethyl sulfoxide Melting point: 300℃ or higher Elemental analysis: C47-50%, H3-4% , O38~
41%, N8-9%, ash 0.06-0.08% Molecular weight: A mixture of a substance with a molecular weight of approximately 25,000 and a substance with a molecular weight of 160,000 or more (Sephadex G-100 gel filtration) Ultraviolet absorption spectrum: Figure 2 Stability 121℃, Inactivated in 30 minutes, stable with N-hydrochloric acid at 100℃ for 30 minutes, stabilized with N-caustic soda at 100℃ for 30 minutes.