JPH0476002A - Polysaccharide and immunoputentiator containing said polysaccharide as effective component - Google Patents

Abstract

PURPOSE:To obtain an immunopotentiator having an excellent action of proliferating lymphocites by using a polysaccharide having specified physical and chemical properties as an effective component. CONSTITUTION:An immunopotentiator containing a polysaccharide having the following physical and chemical properties as an effective component: (A) appearance; light-brown fiber, (B) molecular weight: 390000 or above (gel permeation chromato-graphy), (C) constituent sugars: rhamnose, arabinose, glucose, glucuronic acid, etc., (D) monodispersity: being monodisperse according to electro-phoresis, gel permeation chromatography, etc., and (E) bonding manner of constituent sugars: consisting of main bonds such as 3-bonded glucose, 4-bonded glucose and 3-bonded galactose and bonds such as terminal arabinofuranose, 4-or 5-bonded arabinose, terminal rhamnose, 2-bonded rhamnose, 3-bonded rhamnose and terminal glucose. This polysaccharide has an action of proliferating lymphocites and is useful as a pharmaceutical such as an immunoputentiator. It can be prepared by treating a water extract of crude drug Glycyrrhizae radix or its plant Glycyrrhiza glabra var. glandulifera.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a polysaccharide that has a lymphocyte proliferation effect and is useful as a pharmaceutical agent such as an immune enhancer.

[Prior Art and Problem 1 The main cause of immunodeficiency diseases is a decrease in immunocompetent cells. Among these, a decrease in lymphocytes leads to a decline in the function of cellular immunity, which is a defense mechanism against infectious diseases, and eventually leads to immunodeficiency.

Therefore, by proliferating these lymphocytes, it is possible not only to suppress the severity of the disease, but also to have a therapeutic effect.

Currently, as a treatment for immunodeficiency, drugs are administered to patients as antiviral and antibacterial agents when their immune system is weakened, but there is no fundamental treatment for immunodeficiency. This is the current situation, and its appearance has been eagerly awaited.

[Means for Solving the Problems 1 The present inventors have conducted intensive studies to solve the above problems, and have discovered that polysaccharides having the following properties have an excellent lymphocyte proliferation effect, and have developed the present invention. The invention was completed.

That is, the present invention has the following physical and chemical properties: (1) Properties: Light brown fibrous, (2) Molecular weight: 390,000 or more (gel filtration chromatography), (2) Constituent sugars: rhamnose, fucose, arabinose, mannose, glucose, galactose. , galacturonic acid, glucuronic acid, ■Uniformity: Uniformity is shown by electrophoresis, gel filtration chromatography, high performance liquid chromatography, ■Constitutional sugar binding mode: 3-bond glucose, 4-bond glucose, 3-bond galactose, 3 , 4-branched galactose is the main bonding mode, and in addition to this, terminal arabinofuranose, 4- or 5-bonded arabinose, terminal rhamnose, 2-bonded rhamnose, 3-bonded rhamnose, 4-bonded rhamnose, 2,3-branched rhamnose, 4-bonded fucose, Terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
A polysaccharide composed of branched glucuronic acid (hereinafter referred to as polysaccharide A), (2) The following physical and chemical properties ■Property: light brown fibrous ■Molecular weight: 160,000 (gel filtration chromatography) ), ■ Constituent sugar: rhamnose, fucose, arabinose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, ■ Unity: Unity is shown by electrophoresis, gel filtration chromatography, high performance liquid chromatography, ■ Composition Sugar bonding mode: 3-bond glucose, 4-bond glucose, 3-bond galactose, 3,4-branched galactose are the main bonding modes, and in addition to these, terminal arabinofuranose, 4- or 5-bond arabinose, terminal rhamnose, 2-bond rhamnose, 3-bond Linked rhamnose, 4-linked rhamnose, 2,3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,
6-branched glucose, terminal galactose, 4-branched galactose, 4,6-branched galactose, 3,6-branched galactose, 4-branched mannose, 3,6-branched mannose, 4,6
Branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronic acid, 4-linked glucuronic acid, 2,
A polysaccharide composed of 4-branched glucuronic acid (hereinafter referred to as polysaccharide B), (3) Physical and chemical properties mentioned above.・-), ■ Constituent sugars: Ral\nose, fucose, arabinose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, ■ Uniformity: Uniformity determined by electrophoresis, gel filtration chromatography, and high performance liquid chromatography ■ Constituent sugar bonding mode: 3-bond glucose, 4-bond glucose, 3-bond galactose, 3,4-branched galactose are the main bonding modes, and in addition to these, terminal arabinofuranose, 4- or 5-bond arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, 2.3-branched rhamnose
-su, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,
6-branched glucose, terminal galactose, 4-branched galactose, 4,6-branched galactose, 3,6-branched galactose, 4-branched mannose, 3,6-branched mannose, 4,6
Branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronic acid, 4-linked glucuronic acid, 2,
This is an immune enhancer containing a polysaccharide composed of four-branched glucuronic acid (hereinafter referred to as polysaccharide C), and polysaccharides A, B, and C as active ingredients.

Hereinafter, polysaccharides A, B and C will be collectively referred to as the polysaccharide of the present invention.

The polysaccharide of the present invention has the properties shown above, and since there have been no publications regarding such a polysaccharide, the polysaccharide of the present invention is recognized as a novel polysaccharide. It will be done.

Further, Table 1 shows the molar ratio of the constituent sugars of the polysaccharide of the present invention, with galactose being 100.

Table 1 The polysaccharide of the present invention is obtained, for example, from the root of Glycyrrhiza radix, a leguminous family. There is.

In addition, the present inventors have revealed that the polysaccharide component of licorice plays an important role in the expression of anti-complement activity and lymphocyte rejuvenation activity of Juzentaihoto [Yamada Yo Shiro et al., Abstracts of the 36th Annual Meeting of the Japanese Society of Herbal Pharmaceutical Sciences (1989), p.
, April, Its active ingredients have not yet been clarified, and were clarified through further research by the present inventors.

The polysaccharide of the present invention can be obtained, for example, as follows.

Herbal medicine Licorice, its original material Licorice or other congenerous plants are crushed, extracted with about 10 times the volume of an aqueous solvent, and the filtrate obtained by filtering the extract is subjected to operations such as ethanol precipitation and dialysis. Obtain the phase polysaccharide fraction.

This crude polysaccharide fraction is dissolved in purified water, mixed with quaternary ammonium such as cetyl-trimethylammonium bromide, allowed to stand, and then centrifuged to obtain a precipitate.

The precipitate was dissolved in a 5-30% sodium chloride aqueous solution, ethanol was added, and centrifuged. The precipitate obtained was further dissolved in purified water, dialyzed, and the non-dialysed material was freeze-dried to obtain a polysaccharide-containing fraction. get a minute.

The polysaccharide of the present invention can be obtained by subjecting this polysaccharide-containing fraction to commonly used ion exchange chromatography one or more times.

The aqueous solvent for extraction is preferably water, especially purified water, and hot extraction is desirable for extraction.

In addition, during dialysis, 2 to 5
It can be done for about a day.

Ion exchange chromatography - A specific example of a gel is DEAE-Sepharose C.
Examples include L-6B.

Next, the fact that the polysaccharide of the present invention has an immunocompetent cell proliferation activity and is useful for treating immune diseases such as immunodeficiency diseases will be explained using experimental examples.

Experimental Example The spleen of a 6- to 8-week-old BALB/c female mouse was removed, and a single cell suspension was prepared using a culture medium (RPM11640 culture medium containing 10% tallow calf serum and 5 x 10 M 2-mercaptoethanol). Prepared. 3 cells per hole
Dispense into a 96-well plate so that there are 10 cells,
The cells were cultured for 3 days at 37°C under 5% carbon dioxide. 6 hours before the end of culture, add MTT reagent (manufactured by Sigma) and culture.
10% 5DS (sodium dodecyl sulfate) at the end of culture
was added to stop the reaction, and cell proliferation ability was measured by colorimetry.

The results are shown in Table 2 as the cell proliferation rate (%) relative to the control.

As is clear from Table 2, it was confirmed that the polysaccharide of the present invention has lymphocyte priming activity and is useful for the treatment of immune diseases.

Next, the dosage and formulation of the polysaccharide of the present invention will be explained.

The polysaccharide of the present invention can be administered to animals and humans as it is or together with a conventional pharmaceutical carrier. The dosage form is not particularly limited and may be selected and used as necessary, and may include oral dosage forms such as tablets, capsules, granules, fine granules, and powders, and parenteral dosage forms such as injections and suppositories. It will be done.

In order to exert the desired effect as an oral agent, although it varies depending on the age, weight, and severity of the disease of the patient, the polysaccharide of the present invention is usually administered in an amount of 500 mg to 10 g in divided doses several times a day for adults. It seems appropriate to take .

Oral agents include, for example, starch, lactose, sucrose, mannitol,
It is manufactured using conventional methods using carboxymethyl cellulose, corn starch, inorganic salts, etc.

In addition to the excipients mentioned above, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, and the like can be used in this type of preparation as appropriate. Specific examples of each are shown below.

[Binder 1 Starch, dextrin, gum arabic powder, gelatin,
Hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

[Degraded starch, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl cellulose, low substituted hydroxypropyl cellulose.

[Surface active Saizuki sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 8o0 [Namezawa Saizuki talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Fluidity promoter] Light gaic acid anhydride, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

Furthermore, the polysaccharide of the present invention can be administered as a suspension, emulsion, syrup, or elixir.
These various dosage forms may contain flavoring agents and coloring agents.

In order to exert the desired effect as a parenteral agent, the polysaccharide of the present invention should be administered at a daily dose of 0.1 mg to 1 g per day for adults, although it varies depending on the age, weight, and severity of the disease of the patient. Injection, intravenous drip, subcutaneous injection, and intramuscular injection are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and diluents generally include distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, etc. Soybeans? Derived from domorococcin, propylene glycol, polyethylene glycol, etc. can be used. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, the water removed by ordinary freeze-drying techniques12, and a liquid preparation prepared from the freeze-dried product immediately after use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, suppositories for rectal administration, and the like, which are manufactured according to conventional methods.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Example 1 61 g of water was added to 500 g of licorice, extracted by heating at around 100° C. until the liquid volume was reduced to half, the extract was filtered, and 7 g of a crude polysaccharide fraction was obtained by ethanol precipitation and dialysis. This crude polysaccharide fraction was fractionated by the setablon fractionation method, and 0.8 g of both fractions were precipitated by adding setablon.
Using EAE-8epharose CL-6B column, fraction 1~, both fractions 36m eluted with 0.5M saline.
I got g. 36 mg of this fraction was collected from Asahi
i-pak) GS-510 and Asahi Backpack (Asahi)
-pak) Using a column bound with GS-320 as a carrier, the polysaccharide A'1.2 of the present invention was fractionated by high performance liquid chromatography, starting from the earliest elution order.
mg, polysaccharide B 9.72 mg and polysaccharide C10,8
mg of each were obtained.

Example 2 ■Corn starch 44g ■Crystalline cellulose 40g ■Carboxymethyl cellulose calcium 5g ■Light silicic anhydride 0.5g ■Magnesium stearate 0.5g ■Polysaccharide A obtained in Example 1
1.0 total 100 g According to the above recipe, ① to ② were mixed uniformly and compressed using a tablet machine to obtain tablets each weighing 200 mg.

These tablets contain 20 mg of the polysaccharide A obtained in Example 1, and are taken by adults in 10 to 25 tablets a day in several doses.

Example 3 ■ Crystalline cellulose 84.5 g ■ Magnesium stearate 0.5 g ■ Calcium carboxymethyl cellulose 5 g ■ Sugar B1 in Example 1 Total 100 g According to the above recipe, ■, ■, and part of ■ were uniformly mixed and compressed. After molding, the mixture was pulverized, the remaining amounts of (1) and (2) were added and mixed, and the mixture was compressed using a tablet machine to obtain 200 mg tablets.

These tablets contain 20 mg of the polysaccharide B obtained in Example 1, and are taken by adults in 10 to 25 tablets a day in several doses.

Example 4 ■ Crystalline cellulose 49.5 g ■ 10% hydroxypropyl cellulose ethanol solution 35 g ■ Carboxymethyl cellulose calcium 5 g ■ Magnesium stearate 0.5 g ■ Total 100 g of glycoside C1 obtained in Example 1 ■, ■, and Mix (■) uniformly, make a netting using a conventional method, granulate it using an extrusion granulator, dry and crush it, then mix (■) and ■ and compress it using a tablet machine to make one tablet of 200 mg. Got the tablets.

These tablets contain 020 mg of the polysaccharide obtained in Example 1, and are taken by adults in 10 to 25 tablets divided into several doses per day.

Example 5 ■Corn starch 34.5g ■Magnesium stearate 50g ■Carboxymethyl cellulose calcium 5g ■Light silicic anhydride 0.5g■ Total of 100 g of sugar A obtained in Example 1 Mix ■~■ uniformly according to the above recipe. After compression molding using a compression molding machine, the mixture was crushed using a crusher and sieved to obtain granules.

1 g of this granule contains polysaccharide Al00m obtained in Example 1.
It contains 2 to 5 g per day for adults, divided into several doses.

Example 6 ■ Crystalline cellulose 55 g ■ 10% hydroxypropyl cellulose ethanol solution 35 g ■ Yusu sugar B obtained in Example 1 10 total 100 g According to the above recipe, ■ to ■ were mixed uniformly and made into a paste. After granulation using an extrusion granulator, the mixture was dried and sieved to obtain granules.

1 g of this granule contains 100 m of polysaccharide B obtained in Example 1.
It contains 2 to 5 g per day for adults, divided into several doses.

Example 7 ■ Corn starch 89.5 g ■ Light anhydrous silicic acid 0.5 g ■ Glycoside C1 obtained in Example 1 Total 100 g Mix ■ to ■ uniformly according to the above recipe and give 200 mg
was filled into a No. 2 capsule.

One capsule of this preparation contains 020 mg of the polysaccharide obtained in Example 1, and adults should take JO to 25 capsules a day in several doses.

Example 8 ■ Large W Tomari 5g ■ Retained water on the side for injection 89.5g ■ Thick bean paste],・
Fat 2.5g ■ Glycerin
2g ■ Polysaccharide A1 obtained in Example 1 Total amount 100g Dissolve ■ in ■ and ■ according to the above recipe, and add ■
Add the solutions of and ■ to emulsify and obtain an injection 1.

Claims (1)

[Scope of Claims] [1] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 390,000 or more (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose,
3,6-branched glucose, 4,6-branched glucose, terminal galactose, 4-bonded galactose, 4,6-branched galactose, 3,6-branched galactose, 4-bonded mannose, 3
, 6-branched mannose, 4,6-branched mannose, 4-bond galacturonic acid, terminal glucuronic acid, 3-bond glucuronic acid, 4-bond glucuronic acid, and 2,4-branched glucuronic acid. [2] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 160,000 (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
A polysaccharide consisting of branched glucuronic acid. [3] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 60,000 (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
A polysaccharide consisting of branched glucuronic acid. [4] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 390,000 or more (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
An immune enhancer containing as an active ingredient a polysaccharide composed of branched glucuronic acid. [5] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 160,000 (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
An immune enhancer containing as an active ingredient a polysaccharide composed of branched glucuronic acid. [6] The following physical and chemical properties (1) Properties: Light brown fibrous, (2) Molecular weight: 60,000 (gel filtration chromatography), (3) Constituent sugars: rhamnose, fucose, arabinose,
Mannose, glucose, galactose, galacturonic acid, glucuronic acid, (4) Unity: Shows unity by electrophoresis, gel filtration chromatography, high performance liquid chromatography, (5) Constituent sugar binding mode: 3-linked glucose, The main bonding modes are 4-linked glucose, 3-linked galactose, and 3,4-branched galactose, and in addition to these, terminal arabinofuranose, 4- or 5-linked arabinose, terminal rhamnose, 2-linked rhamnose, 3-linked rhamnose, 4-linked rhamnose, and 2-linked rhamnose. , 3-branched rhamnose, 4-linked fucose, terminal glucose, 2-linked glucose, 6-linked glucose, 3,6-branched glucose, 4,6
Branched glucose, terminal galactose, 4-linked galactose, 4,6-branched galactose, 3,6-branched galactose, 4-linked mannose, 3,6-branched mannose, 4,6-branched mannose, 4-linked galacturonic acid, terminal glucuronic acid, 3-linked glucuronide acid, 4-linked glucuronic acid, 2,4
An immune enhancer containing as an active ingredient a polysaccharide composed of branched glucuronic acid.