JPS6410519B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing a melanin-based substance, and more particularly to a method for producing a melanin-based substance obtained from squid or octopus ink that has gastric juice secretion suppressing and anti-inflammatory effects. 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. Then, we homogenized squid or octopus ink, passed it through a cloth, etc., and dialyzed this solution.We investigated 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. That is, the present invention provides a method for producing a melanin-based substance containing protein and sugar exhibiting the following physical properties, which is characterized by homogenizing squid or octopus ink and dialysis of a solution from which coarse solids have been removed. It is something to do. 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. 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 present invention can be carried out, 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, ethanol, ethyl acetate,
Insoluble in chloroform, benzene, dimethylformamide, dimethyl sulfoxide Melting point 300℃ or higher Elemental analysis values C: 47-50%, H: 3-4%, O: 38-41
%, N: 8-9%, Ash content: 0.06-0.08% Molecular weight From gel filtration of Sephadex 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 (Fig. 1) Ultraviolet absorption spectrum As shown in Fig. 2 Amino acid composition (in 50 ml of dialysis fluid)

[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 mice (body weight: male 20-27
g, female 20-26 g) Intraperitoneal administration 1 g/Kg or more Oral administration 5 g/Kg or more (3) Physiological activity Gastric juice secretion suppressive effect The gastric juice secretion suppressive effect was measured using Shay's pylorus ligation method [Gastroenterology 5 , 43 (1945)] I went there by B. Intraperitoneal administration After fasting male Wistar rats weighing 200 to 250 g for 24 hours, the abdomen was opened under ether anesthesia, and the pylorus was ligated. Immediately after the ligation, a physiological saline solution of the melanin substance obtained in Example 1 (Test A) or Example 2 (Test B) was administered intraperitoneally. Four hours after administration, the stomachs were removed, and gastric juice volume, total acidity, and pepsin activity were measured. Total acidity is determined by using phenolphthalein as an indicator.
It was titrated with 0.05N-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 released by the decomposition of casein at 280 nm.
It was expressed in terms of tyrosine amount mg/100g body weight. Physiological saline was also administered as a control group, and gastric juice volume, total acidity, and pepsin activity were measured in the same manner. 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 pyloric ligation ulcer method. After fasting male Wistar rats weighing 200 to 250 g for 48 hours, the pylorus was ligated in the same manner as in the assay for gastric juice secretion, and the stomach was removed 18 hours after the ligation. This is incised from the greater curvature side,
Ulcerative changes that occur in the forestomach were evaluated using the method of Adami et al. [Arch.int.Pharmacodyn., 147, 113]
(1964)] to an ulcer index. 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 was administered intraperitoneally at a dose of 5 mg/Kg and
Intraduodenal administration of mg/Kg significantly suppresses gastric juice secretion, and two intraperitoneal administrations of 25 mg/Kg exhibits anti-ulcer effects. Anti-inflammatory effect (a) Rat footpad lacagenin edema suppression test Van
Arman [J.Pharmacol.Exp.Ther., 150 ,
328 (1965)] and Winter [Proc.Soc.Exp.
Biol. Med., 111 , 544 (1962)]. That is, Example 1 as a specimen
The melanin-based substance obtained was dissolved in physiological saline, and this was administered intraperitoneally 30 minutes before carrageenan injection. Carrageenin was administered as a 1.5% physiological saline solution in an amount of 0.1 ml to each footpad. Rats are Wistar males, weight
Using a sample of 130 to 150 g, the edema rate relative to the paw volume immediately after carrageenin 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.
Suppressed edema caused by carrageenan. 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 ddY male mice (body weight 20-22 g).
After 10 minutes, the number of squirmings for 10 minutes was measured. As a positive control, 0.7% acetic acid solution was intraperitoneally administered to mice, and the same procedure was performed.
The number of squirming was measured. The results are shown in Table 6.

[Table] As is clear from Table 6, in the group administered with melanin-based substances obtained from squid ink,
There was no difference in the number of squirmings compared to the physiological saline administration group, and no irritation was observed. Effect on vascular permeability Whittle et al. [Brit.J.Pharmacol., 22 ,
246 (1964)], 0.1 ml of 4% trypan blue was administered via the tail vein to ddY male mice (body weight 20-22 g).
The specimen was administered intraperitoneally 10 minutes after administration. 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 detected at 590nm.
The effect on vascular permeability of the specimen 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. 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.

[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)

[Claims] 1. A method for producing a melanin-based substance containing protein and sugar exhibiting the following physical properties, which comprises homogenizing squid or octopus ink and dialysis of a solution obtained by removing coarse solids therefrom. . 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.