JPH03284626A - Immunoactivator - Google Patents

Abstract

PURPOSE:To obtain an immunoactivator comprising an extract from marine algae belonging to the genus Porphyra with an aqueous solvent. CONSTITUTION:Marine algae belonging to the genus Prophyra are treated with an organic solvent, an oil component, oligosaccharides, an oil-soluble dyestuff, etc., are dissolved in the organic solution, the organic solvent is removed by filtration or centrifugal separation and the prepared residue is extracted with water, an acidic aqueous solution or a basic aqueous solution. The pH of the aqueous solvent is properly 2-12, treatment at pH<=3.5, especially pH<=3 provides an immunoactivator having stronger immunoactivating action than treatment at a higher pH and in higher yield. The immunoactivator is administered orally or parenterally, a dose thereof is 0.5-1,000mg, preferably 1-300mg calculated as solid content of active ingredient per adult daily per kg weight and administered once or dividedly three times.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunostimulant containing a seaweed extract as an active ingredient.

[Problem B to be solved by the conventional technology and invention] Conventionally,
Several pharmaceutical compositions are known that contain as an active ingredient ingredients obtained by extraction or the like from red algae. For example, JP-A-63-31
No. 6732 describes an antiviral agent containing components obtained by hot-water extraction of various red algae, including the order Ceramicales, and alkali extraction of the residue. An anti-obesity drug using a water extract as an active ingredient has been described, and in JP-A-64-66126, an anti-obesity drug using a water extract as an active ingredient mainly of seaweeds of the Amanitaceae or Agonidae family (Amanitaceae is not mentioned) is described. Tumor agent (
This ingredient also has an immunostimulatory effect).

Also, Shiraki et al., Cancer Letters, 26 (
1985) 241251 and Shiraki et al., Cancer L
etters, 35 (1987) 109118, it is described that an edible seaweed powder containing Porphyra chinensis exhibits antitumor activity.

However, the immunostimulatory effect of extracts of seaweeds of the genus Porphyra is not known.

[Means to solve the problem]

The present invention relates to an immunostimulant containing as an active ingredient an extract of a seaweed belonging to the genus Porphyra in water, an acidic aqueous solution, or a basic aqueous solution (hereinafter, for convenience, these are collectively referred to as aqueous solvents).

The present invention will be explained in detail below. Examples of the seaweeds of the genus Lalerium borbiculata used in the present invention include H. borbiculata (H.b. uborbiculata), La.

dentata), Kosujinori (P, average and IL)
, Usoburuinori (P, Tenra), Asakusanori (P, tenera), Susabinori (P,
B Mata 411 Lee, Marubachi Macronori (P, umb
ilicalis), Ononori (P, onoi)
, Furitasa (P, ■sha■lniji), etc.

Active ingredients can be obtained from these seaweeds by extraction with an aqueous solvent from the beginning, but in order to remove oils, oligosaccharides, oil-soluble pigments, etc., the seaweeds are first treated with an organic solvent that can dissolve them. It is better.

As such an organic solvent, for example, lower alkanols such as methanol, ethanol, n-propanol and isopropanol, lower alkanones such as acetone, etc. can be used. Also, these solvents and a small amount of water (80:20v
/v) may be used. Methanol, ethanol, and a mixed solvent of these and water are preferred.

The amount of such an organic solvent (including a mixed solvent with water) to be used is not particularly limited, but an appropriate amount is about 0.5 to 101 parts per 100 g of seaweed (dry basis). There are no particular restrictions on the temperature and time of organic solvent extraction;
C to the boiling point of the organic solvent for 5 minutes or more, for example 15 minutes to 1 hour.

Next, the organic solvent is removed by decantation, filtration, centrifugation, etc., and the resulting residue is subjected to extraction with an aqueous solvent. As the aqueous solvent, water, an acidic aqueous solution or a basic aqueous solution is used, but a small amount of a hydrophilic organic solvent, for example, 10% by volume or less, may be further contained. "Acidity" and "basicity" are usually provided by acids and bases, respectively, but commonly used buffers may also be used. There are no particular restrictions on the acid, but
Inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid,
Organic acids such as citric acid can be used.

The base is not particularly limited, but may include ammonia, alkali or alkaline earth metal hydroxides, carbonates or bicarbonates, such as sodium hydroxide, potassium hydroxide,
Calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc. are commonly used. The base may further be a hydrophilic organic base such as pyridine. As the buffer, commonly used buffers such as Tris-based, phosphoric acid-based, citric acid-based, and boric acid-based buffers can be used.

The pH of the aqueous solvent is not particularly limited, but is usually 1-13, particularly 2-12. In addition, pH 3.5 or lower, especially p
) Active ingredients obtained at a pH of 13 or less generally have a stronger immunostimulatory effect than active ingredients obtained at a pH higher than that. In addition, when processing at pH 3.5 or below, especially below pH 3, the yield of the active ingredient in -C is superior to when processing at pH 3 or below. There is no particular restriction on the amount of the aqueous solvent used. is usually 1 to 10 per 100g of raw seaweed (dry basis)
About 1 is appropriate. There is no particular restriction on the temperature and time for extraction with an aqueous solvent, but it is appropriate to use a temperature of 4° C. to the boiling temperature of the system, usually room temperature to the boiling temperature of the system, for 5 minutes or more, for example, 30 minutes to 20 hours.

Note that two or more extractions using water, an acidic aqueous solution, and a basic aqueous solution may be performed in combination in series (for example, a water extraction residue is extracted with an acidic aqueous solution).

The extract can be used as an immunostimulant as it is or by neutralizing, desalting, and concentrating as necessary, but it may be necessary to further remove low molecular weight substances, increase the specific activity of the active ingredient, and for convenience in formulation. It is usually further subjected to general purification treatment. As such a purification treatment, precipitation with an organic solvent, salting out, dialysis, ultrafiltration, reverse osmosis treatment, gel filtration, etc. can be used, and two or more of these treatments may be performed in combination.

Precipitation with organic solvents is usually carried out by precipitating the active ingredient by adding a hydrophilic organic solvent that does not dissolve or only slightly dissolves the active ingredient. Is it possible to take it? As the g-medium, lower alkanols such as methanol, ethanol, chloropropanol, isopropanol, and lower alkanones such as acetone can be used. Lower alkanols such as methanol, ethanol, n-propanol, and isopropanol are preferred. In addition, extraction with acidic aqueous solution,
In the case of extraction with a basic aqueous solution, it is preferable to perform organic solvent precipitation after neutralization (the base and acid used for neutralization may be those used for basic and acidic aqueous solutions, respectively).

Salting-out agents used in the salting-out step include ammonium sulfate, common salt, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferably used. Further, as a post-treatment after the salting-out step, any one of dialysis, ultrafiltration, gel filtration, reverse osmosis treatment, etc. or a combination of two or more of these steps is usually performed.

Dialysis is usually performed using a semipermeable membrane such as a cellophane membrane or a collodion membrane. Gel filtration is performed using a column filled with dextran or polyacrylamide gel. Fillers sold under the names of Sephadex and Biogel are commonly used.

Ultrafiltration and reverse osmosis are both methods of fractionation using a membrane under pressure. The former is 0°5~5 kg/cm”
The latter is usually carried out at a pressure of 20 to 35 kg/cm2.

Further, in addition to the above operations, ion exchange treatment may be performed as necessary.

The active ingredient solid obtained by the above purification procedure can be used as it is as an immunostimulant or its active ingredient, but it is usually further dried (spray drying, freeze drying, hot air drying, etc.)
do.

The aqueous solvent extracts of seaweed belonging to the genus Porphyra obtained as described above (including those that have been subjected to treatments such as purification and drying) can be used as such or in various forms mixed with various pharmaceutically acceptable carriers. It can be used as an immunostimulant in dosage form.

In the case of oral administration, tablets, granules,
Powders, capsules, etc. usually contain binders, inclusion agents, excipients, lubricants, etc. commonly used in the formulation in their composition.
Contains additives such as disintegrants and wetting agents. When used as an oral liquid preparation, it may be in the form of an oral solution, a shaken mixture, a suspension, an emulsion, or a syrup, or it may be in the form of a dry product that is redissolved before use. Good too. Furthermore, such liquid preparations may contain any of commonly used additives and preservatives.

When used for injection, the composition typically includes stabilizers, buffers,
They contain additives such as preservatives and tonicity agents, and are usually presented in the form of unit-dose ampoules or multi-dose containers. It is noted that the compositions may be in the form of aqueous solutions, suspensions, solutions, emulsions in oily or aqueous vehicles, wherein the active ingredient is dissolved in a suitable vehicle, e.g., a pyrogen-free, sterile vehicle, before use. It may also be a powder that is redissolved in water.

The immunostimulant of the present invention is administered orally or parenterally to humans and animals. Oral administration includes sublingual administration.

Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous, infusion, and the like.

The dose of the immunostimulant of the present invention depends on whether it is an animal or a human; it is also influenced by age, individual differences, medical conditions, etc., so in some cases, doses outside the range below may be administered, but in general, it is intended for humans. In this case, the oral dosage is 0.5 to 1000 mg/kg body weight per day for adults as solid amount of active ingredient.
, preferably 1 to 300 mg, administered in 1 to 3 divided doses.

The acute toxicity of this extract (specifically, the dried product obtained in each example) is t, OS. (ICR mouse,
oral administration) >3 g/kg.

〔Example〕

Crush 50g of Futane Masudo Susabinori (dried) and store in 1 container! into a beaker, add 500 d of 85% methanol, and add 4
Stirred at 5-50°C for 15 minutes, then cooled to room temperature. The slurry was filtered through Nutze and separated into a filtrate and a residue. The residue was treated with 85% methanol again in the same manner.

750°C of water was added to the residue, stirred at 90-100°C for 30 minutes, then cooled to room temperature, and the slurry was separated into an extract and a residue by centrifugation. Continue the main water extraction operation two more times.
This was repeated several times, and the resulting extracts were combined.

Ethanol was added to the extract to give a concentration of 80% by volume, and the resulting precipitate was separated by centrifugation and freeze-dried to obtain 2.1 g of a dried product (dried product A).

On the other hand, 100M water was added to the water extraction residue, and the pH was adjusted with concentrated hydrochloric acid.
2, stirred and extracted at room temperature for 20 hours, and centrifuged the slurry to obtain an extract. This acid extract was neutralized with 2N sodium hydroxide, ethanol was added to the solution to give a concentration of 80% by volume, and the resulting precipitate was separated by centrifugation and freeze-dried to obtain 3.8 g of a dry product ( Dry product B).

Practical Lieutenant I 2000 ml of water was added to the extraction residue obtained by treating Japanese laver with methanol in the same manner as in Example 1, the mixture was adjusted to P) 110 with 5N sodium hydroxide, and the mixture was stirred at room temperature for 20 hours. The slurry was centrifuged to obtain an extract.

After neutralizing this base extract with 2N hydrochloric acid, ethanol was added to give a concentration of 80% by volume, and the resulting precipitate was separated by centrifugation and dried under reduced pressure at 60°C for 2 hours to obtain a dried product of 4.
．． 1 g was obtained (dry product C).

Fruit...Crush 50g of Susabinori (dried), put it in a beaker with a capacity of 31, add 2000d of water, and adjust the pH to 2 with concentrated hydrochloric acid.
After stirring and extracting at room temperature for 30 hours, the slurry was centrifuged to obtain an extract. Add this acid extract to 2N
The mixture was neutralized with sodium hydroxide, dialyzed against water using a cellophane membrane, and then freeze-dried to obtain 8.1 g of a dried product (dried product D).

Ethanol was added to the sodium hydroxide neutralized solution obtained by treating the disaster relief soil in the same manner as in Example 3 to give a concentration of 80% by volume, and the resulting precipitate was separated by centrifugation and freeze-dried. 6.2 g of dried product was obtained (dried product E).

Tengandan i The results of various analyzes of the dried products obtained in Examples 1 to 4 are shown in the table below.

Table 1 Each analysis was based on the following method.

Molecular weight analysis: Kashi column HODX OHP for GPC
HPLC conditions are as follows: Column temperature: 40"C Eluent: Water flow rate = 0.5 Detector: Differential refractometer Sugar content: Phenol Sulfuric acid method. Converted value assuming all galactan.

Protein content: 2 Lowry sulfate esters: 8 na1. Biochem,, 4
1.471-476 (1971) Galactose: Phenol sulfuric acid method Constituent sugars: A sample was hydrolyzed with 2N sulfuric acid at 110°C for 2 hours and then analyzed by HPLC.

The HPLC conditions are as follows: Column: 5hodex Sugar 5Z-5532 Column temperature: near 0°C Eluent: Niacetonitrile:water (85:15) Flow rate: 0
．． 7 d/min Detector: Differential refractometer (immunostimulatory effect) The activation effect of the reticuloendothelial system in mice was investigated using the carbon clearance method.

Mice (C57BL/6) J, male, 6 weeks old) were given dry matter A to E dissolved in phosphate buffered saline (PBS) to a predetermined concentration, 5 times every 24 hours, and administered in g.
0.03 m/day was administered intraperitoneally, and carbon clearance was measured 24 hours after the final administration. That is, 0.01 d/g of body weight of carbon ink (Pelican Font India) diluted 6.25 times with PBS containing 1% gelatin was administered into the tail vein of a mouse, and 5 minutes after administration, 10μ! Blood was collected, dissolved in 2 ml of 0.1% sodium carbonate, and
Absorbance at 60 nm was measured. There were 7 animals in each group, and the reticuloendothelial system activation ratio was determined as the OD value of the test group relative to the OD value of the control group (PBS administration group). The results are shown in the table below.

Table 2 Sample dosage (mg/kg/day) Carbon concentration ratio Dry matter A300 Dry matter B 500 50 Dry matter C500 Dry matter D 500 Dry matter E 500 Umbrella to Zymosan 100 0.549 0.230 0.467 0.509 0.457 0.306 0.710 *Commercially available immunostimulant, cell wall polysaccharide component derived from Satucharomyces cerevisiae (manufactured by Sigma) Chiseiyoi Capsule dry product A 200g corn starch 150g talc
80g Magnesium stearate 30g The above ingredients are thoroughly mixed, passed through a 60 sieve wire gauze to adjust the particle size, and then filled into 100 Seratin capsules.

1 ship, half-dried product B, 140g cacao butter
1200g of cacao butter was heated to 50°C to melt it, and the dry matter was added to it to make it homogeneous.Then, it was poured into a container, cooled and solidified, and half-opened.
Manufacture 0 pieces.

Dried injections 0. 400g polyoxyethylene hardening and mustard oil 500g distilled water for injection
An injection is prepared by a conventional method using a total amount of 10 ff of the above ingredients, and 5 injections are filled into each ampoule.

[Effects of the Invention] An immunostimulant containing an extract of seaweed belonging to the genus Porphyra as an active ingredient is provided.

Claims (1)

[Claims] An immunostimulant containing as an active ingredient an extract of seaweed belonging to the genus Porphyra in the form of water, acidic aqueous solution, or basic aqueous solution.