JPH0154330B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel aqueous extract component B- of oyster shells. Oyster shells are used as Chinese medicine for antacid, sedative, and
It is an important herbal medicine that is prescribed in Annachusan, Saikokaryukotsu Kakito, etc. for the purpose of tonicity, and is frequently used in herbal medicine. It has been reported that oysters contain calcium carbonate, a small amount of calcium phosphate, and the water-insoluble protein component conhyorine. The present inventors succeeded in isolating a novel aqueous extract component having an immune-enhancing effect from oyster shells, and named it B-. That is, the present invention provides an aqueous extract component of oyster shell B-, which has an effect of enhancing antibody production, an effect of activating macrophages, an effect of enhancing infection resistance, etc. and is useful as an immunostimulant, and a method for producing B-. It is. B- of the present invention is produced by extracting oyster shells with water, concentrating the extract, adding an organic solvent to it, dissolving the resulting precipitate in water, dialysis, and drying the inner solution. Can be done. The oyster shells used as extraction raw materials are oysters belonging to the family Osteridae,
The shells of Ostrea oysters, Ostrea oysters, Ostrea oysters, etc. are listed.
gigas Thunb.) shells are advantageously used.
Natural or cultured shells that have been appropriately processed by washing, drying, heating, chopping, pulverizing, etc. can be used. When extracting water from oyster shells, the amount of water varies depending on the particle size of the oyster shells, but is usually 2-20 ml per 1 kg of shells. Regarding the extraction temperature and extraction time, it is convenient to heat for a long time to improve the extraction efficiency, but generally it is desirable to extract at 50-120°C for 0.5-5 hours. The extraction operation may be performed at once, or may be performed several times. The extract thus obtained is reduced to 1/50-
Concentration under reduced pressure to 1/200 yields a concentrated extract.
becomes. The concentrated extract thus obtained has an immune-enhancing effect even as it is, and can be used as a medicine. Next, an organic solvent that is easily miscible with water is added to the extract to precipitate hydrophilic substances. In this case, suitable organic solvents include acetone, lower alkanols (e.g., ethanol, methanol), etc.
It is usually preferable to add 3 to 6 times the amount of the concentrated extract. Then, collect the resulting precipitate, add 10-20 times the volume of water, stir at 30-50℃ for about 30 minutes, and remove substances that are poorly soluble in water (such as conhyorin and inorganic substances).
is removed, for example, by centrifugation. Centrifugation is 1000
-3000 rpm for 5-20 minutes is preferred. B- is obtained by removing low molecular weight substances from the supernatant using a dialysis membrane, ultraviolet filtration, molecular sieve, etc. and drying it. Aqueous extract component B- of oyster shells thus obtained
has the following properties. (b) It has an immune-enhancing effect (b) It is easily soluble in water (according to the Japanese Bureau 9 General Rules 21) (c) It does not pass through a cellulose dialysis membrane (d) It elutes near ferritin in column chromatography using Sepharose 4B ( e) Color reaction: positive for phenol sulfuric acid reaction, negative for Lieberman-Bruhhardt reaction, negative for ferric chloride reaction (f) In the infrared absorption spectrum, 3370,
It shows a maximum value around 2910, 1635 cm ^-1 (G) It does not show a maximum value in the ultraviolet absorption spectrum Furthermore, B- is hydrolyzed to produce sugars such as glucose, galactose, mannose, and lysine.
Aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine,
Provides amino acids such as methionine, leucine, and isoleucine. Furthermore, B- is almost insoluble in ethanol, ethyl ether, benzene, and chloroform. B- of the present invention has an excellent immunostimulatory effect and low toxicity as shown in the following experimental examples. Experimental Example 1 Adjuvant activity of B- SRBC1× to 1 group of 5 male 8-week-old CF# ¹ mice
After sensitization by intravenously administering ¹⁰⁷ B-
Alternatively, physiological saline (0.2 ml/10 g body weight) was intraperitoneally administered, and antibody-producing cells in the spleen were collected on the 4th day of administration using the Cunningham method [Immunology,
Vol. 14, p. 599 (1968)] (Table -
1).

[Table] As is clear from the above results, B- acts as an adjuvant in the antibody production system. Furthermore, as shown in Table 2, the adjuvant activity of B- was also observed in many other mouse strains.

[Table] Experimental Example 2 Macrophage activating effect of B- B-25 to 1 group of 5 female 5-week-old CF# ¹ mice
mg/Kg was administered once intravenously or intraperitoneally, and the carbon clearance ability was examined 3 and 5 days later. i.e. carbon (Pelican C ₁₁ /1431a250Å)
was prepared at 20 mg/ml with 1% gelatin-supplemented saline, kept at approximately 37°C, and injected at 200 mg/ml from the tail vein of a mouse.
After administering Kg, blood was collected from the mouse orbital capillaries over time, 20 μl of which was hemolyzed in 2 ml of 0.1% Na ₂ CO ₃ solution, and the carbon concentration in the blood was measured using an automatic spectrophotometer (Table 3).

[Table] As is clear from the above results, B- significantly enhances the carbon clearance ability after 3 and 5 days when administered once at 25 mg/Kg intravenously or intraperitoneally to mice. At the same time, the weight of the spleen, which is a major organ of the reticuloendothelial system (RES), also increases significantly. These experimental results indicate that B- is an immune modulator that has a wide range of action points on the RES system. Experimental Example 3 Immune Competence Restoration Effect of B- After intravenously administering ¹ ×10 ⁷ SRBC to male 8-week-old mice to sensitize them, 6-mercaptopurine (6-mercaptopurine) was administered as a drug that exhibits immunosuppressive effects the next day. 6MP)
Under experimental conditions, 70 mg/Kg was administered intraperitoneally once to reduce humoral antibody production ability, and from the next day, B- or physiological saline was intraperitoneally administered for 2 days. Antibody-producing cells in the cells were examined by the Cunningham method (Table 4).

[Table] As is clear from the above table, B- has the ability to quickly recover from the suppressed state caused by the administration of drugs that exhibit immunosuppressive effects such as 6MP. Experimental Example 4 Infection resistance enhancing effect of B- Using male 4-week old Slc:ICR mice, 25
Pretreatment was performed by intraperitoneally administering 100 mg/Kg every other day for 2 days, and 3 or 5 days later, human Klebsiella Pneumoniae (DT strain) was tested.
By injecting 0.2 ml of TSB medium (manufactured by BBL) 18-hour culture (diluted 10 ^-8 with 5% mucin) once into the abdominal cavity of mice, the effect of clearly increasing infection resistance against K. pneumoniae was demonstrated. It was found that (Table 5).

[Table] Experimental Example 5 Acute toxicity and subacute toxicity Male 4-week-old CF# ¹ mouse, weight 18-20g, group 1
As a result of examining acute toxicity using 10 animals, B
- LD ₅₀ value is 3000mg/Kg for oral administration.
The above amounts were 1000 mg/Kg or more when administered intravenously. Furthermore, 10, 50, and 100 mg/Kg of B- were administered to each group of 4-week-old male CF# ¹ mice.
It was administered as a daily dose in the diet for 4 weeks, and subacute toxicity was investigated. As a result, there were no abnormalities in weight gain rate or general symptoms in each administration group. At autopsy, there were no noteworthy findings except for an increase in the weight of the spleen due to the immune-enhancing effect. General blood tests and plasma biochemical tests showed no noticeable side effects except for a slight increase in the number of white blood cells and an increase in the amount of β and γ-globulin in the serum due to the immune-enhancing effect of B-.
That is, B- is an extremely low toxicity immunostimulant. Novel physiologically active substance B- obtained by the present invention
It has an excellent immune-enhancing effect and an infection-resistance enhancing effect, and is effective in mammals (e.g. dogs, dogs, etc.).
Used as an infection resistance enhancer or immune enhancer when antibiotics are used in pet animals such as cats, laboratory animals such as rats and mice, and humans, etc., or when immune function is weakened before or after surgery, and as an agent to improve weak constitution. Useful. When the substance obtained according to the present invention is used as an immunostimulant, it can be prepared by itself or mixed with appropriate pharmacologically acceptable carriers, excipients, and diluents to form powders, granules, tablets, capsules, and syrups. It can be safely administered orally or parenterally in the form of injections, suppositories, etc. The dosage depends on the target disease,
Although it varies depending on the symptoms, subject of administration, administration method, etc., for example, when orally administered as an immune enhancer for adult patients with decreased immune function during surgery, it is preferable to use about 100 mg of B-5 per dose. EXAMPLES The present invention will be explained in more detail below based on Examples, but these Examples do not limit the scope of the present invention. Example 1 40 kg of oyster shell powder (natural oyster, produced in Maizuru) and water
500 was added, heated at 100°C for 1 hour, filtered, the filtrate was concentrated under reduced pressure to 4, and then 16 of ethanol was added. The resulting precipitate (52 g) was dissolved in 1.5 g of water,
The insoluble portion was removed by centrifugation at 3000 rpm for 10 minutes, and then dialyzed against purified water for 4 days in an ice chamber (4°C), replacing the water once a day, using a dialysis tube (Visking 18/32). About 1/20 of the liquid under reduced pressure
After concentration and freeze-drying, 4.5 g of B-2 was obtained as a pale colored powder. Figure 1 shows the elution curve of B- in column chromatography [Column: Sepharose 4B (φ25 x 450 mm), solvent: 0.1M
Phosphate buffer (PH7.0), solid line: B- (phenol sulfuric acid coloring, absorbance at 490 nm), dotted line: ferritin (absorbance at 280 nm)]. Example 2 Add 500 kg of water to 40 kg of oyster powder (Osaka market product),
After heating at 100°C for 1 hour, filter, concentrate the filtrate to 5%, and add 20% of ethanol. The resulting precipitate (43g) was dissolved in 1.3ml of water, and the insoluble part was heated at 3000 rpm.
After removing it by centrifugation for 10 minutes, use a dialysis tube (Visking 18/32) to remove purified water.
After dialysis in an ice chamber (4° C.) for 4 days while changing the water once a day, the internal solution was collected, concentrated under reduced pressure, and freeze-dried to obtain 2.2 g of B- as a pale colored powder. Example 3 When the novel aqueous extract component B- of the present invention is used, for example, as an immune enhancer, it can be used in the following formulation. 1 Tablet (1) B- 10mg (2) Lactose 64.5mg (3) Corn starch 20mg (4) Hydroxypropyl cellulose 5mg (5) Magnesium stearate 0.5mg 1 tablet 100mg By a pharmaceutical method known per se (1), ( After mixing all the amounts of 2), (3), and (4), granulate it. Add (5) to this and pressure mold it into a tablet. 2 Syrup (1) B- 500mg (2) Methylparaben 100mg (3) Propylparaben 25mg (4) Citric acid 80mg (5) Sodium citrate 520mg (6) Cane sugar 25g (7) Purified water (required amount) Total volume: 100ml (1), (2), (3), (4), (5)
Mix and (6) and add (7) to make a total volume of 100ml. 3 Injection (1) B- 500mg (2) 0.9% saline (required amount) Total volume: 100ml Add (1) and (2) using a known pharmaceutical method to make a total volume of 100ml, sterilize under pressure, filter with a filter,
Seal in a 1 ml brown ampoule and replace with nitrogen gas. All steps are performed under sterile conditions.

[Brief explanation of drawings]

FIG. 1 shows elution curves of physiologically active substance B- and ferritin in column chromatography.

Claims (1)

[Claims] 1. Manufactured by extracting oyster shells with water, concentrating the extract, adding an organic solvent to it, dissolving the resulting precipitate in water, dialysis, and drying the inner solution, Aqueous extract component B- of oyster shells having the following properties. (b) Has an immune-enhancing effect (b) Easily soluble in water (c) Does not pass through a cellulose dialysis membrane (d) Elutes near ferritin in column chromatography using Sepharose 4B (e) Color reaction: phenol sulfate Positive reaction, negative Lieberman-Burhard reaction, negative ferric chloride reaction (F) In the infrared absorption spectrum, 3370,
Shows a maximum value around 2910, 1635 cm ^-1 (g) Does not show a maximum value in the ultraviolet absorption spectrum 2 Extract oyster shells with water, concentrate the extract, add an organic solvent to it, and dissolve the resulting precipitate in water. It is characterized by subjecting it to dialysis treatment and drying the internal fluid.
Oyster shell aqueous extract component B- having the following properties:
manufacturing method. (b) Has an immune-enhancing effect (b) Easily soluble in water (c) Does not pass through a cellulose dialysis membrane (d) Elutes near ferritin in column chromatography using Sepharose 4B (e) Color reaction: phenol sulfate Positive reaction, negative Lieberman-Burhard reaction, negative ferric chloride reaction (F) In the infrared absorption spectrum, 3370,
Shows a maximum value around 2910, 1635 cm ^-1 (g) Does not show a maximum value in the ultraviolet absorption spectrum