JPH02300136A - Immunological enhancing agent - Google Patents

Abstract

PURPOSE:To obtain an immunological enhancing agent containing acidic polysaccharide in Glycyrrhiza glabla or fraction containing said polysaccharide as main comprising element. CONSTITUTION:The aimed agent contains an extract with acetic acid of a substance selected from Glycyrrhiza glabla (especially Glycyrrhiza glabla made in Afghanistan), water-extract of Glycyrrhiza glabla and water-containing polar solvent-extract of Glycyrrhiza glabla as a main comprising element. For instance, Glycyrrhiza glabla made in Afghanistan is extracted with hot water, subjected to deproteinization, precipitated with alcohol and resultant precipitate is further extracted with acetic acid to afford a fraction having strong mitogen activity. Said fraction mainly contains heteroacidic polysaccharide containing galacturonic acid as a principal component and rhamnose, arabinose, galactose and several other saccharides, and glucan and coloring component other than said heteroacidic polysaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to the use of acidic polysaccharide in Lili grass as an immune enhancer.

[Prior Art and Problems to be Solved by the Invention] Living organisms are protected from attack by microorganisms such as bacteria and viruses, or by parasites, mainly by the action of the immune system.

Furthermore, spontaneous regression of cancer is not a rare phenomenon, but rather a common phenomenon in everyday life. This finding suggests the existence of an immune response to cancer.

Therefore, by activating or strengthening the immune system,
It is believed that host resistance will be improved, and that it may also be possible to prevent and treat infectious diseases and cancer.

Against this background, three types of polysaccharide systemic disease agents (immunotherapy agents), Krestin, lentinan, and schizophyllan, were developed and have grown into major drugs today, but to date, these drugs have not been effective as anticancer agents. It's just sanctioned.

However, there are a considerable number of reports that polysaccharides have immune-enhancing effects other than anti-cancer effects, and it seems possible to use them for the following purposes, such as increasing resistance and preventing and treating infectious diseases. .

Meanwhile, in recent years, AIDS (acquired immunodeficiency syndrome) has become a worldwide problem. To date, no effective therapeutic drug has been found, but due to the nature of AIDS, immune enhancers such as polysaccharides are thought to be effective to some extent in preventing and treating the disease.

[Means to solve the problem]

Therefore, in order to obtain new and more effective immunostimulants, we investigated the effects of polysaccharide fractions extracted from various yeasts and crude drugs on the immune system using mitogen activity (lymphocyte blastogenesis) as an indicator. went.

The results showed that the components extracted from grass under acetic acid acidity, especially licorice from Afghanistan (hereinafter afghan abbreviated as grass; G1)
A strong mitodiene activity was observed in both extracts (hereinafter abbreviated as AL-4) obtained by removing protein from a hot water extract of 7c7r+hirx glxbl and precipitating with alcohol, and then further extracting with acetic acid. Furthermore, it was discovered that AL-4 has an effect of promoting antibody production and an effect of activating macrophages.

As a result of analysis, AL-4 has an acidic polysaccharide composed of uronic acid and glucose with a concentration of -1.1.

In addition, Kumagai et al. from Chiba have previously reported that neutral polysaccharide extracted from licorice has an immune-enhancing effect. 9 (+97811, AL-, l
This is clearly different.

From the above results, it has been found that the acidic polysaccharide of licorice, especially silver lily, has a strong effect of activating the immune system, and the present invention has been completed.

[Action and effect of the invention]

The methods and results for testing the mitogen activity, antibody production promoting activity, and macrophage activation of AL-4 in the present invention are shown above.

(1) Mitogen activity test Add mouse liver cells and samples to a 96-well plate,
After culturing for an hour, tritium thymidine is added and the culture is further cultured for 4 hours. Then, the cells were collected onto a filter paper, and the mitogen activity was determined by measuring the amount of tritium incorporated into the cells using a liquid scintillation counter. In addition, ConA is used as a mitogen active substance.
(concanavalin A) and LPS (lipopolysaccharide).

The analytical method is the carbazole/sulfuric acid method for uronic acid;
Neutral sugars such as glucose were quantified by liquid chromatography after treatment with trifluoroacetic acid.

Mito-diene activity is expressed as tritiated thymidine incorporation relative to control.

The results are shown in Table 1.

Table 1 Sample (pg/*l) Mito-diene activity AL-/
l 1 2-4 1010-20 100 30-50 The new species is grass polysaccharide 1 1-2 10 5-10 IQo 10-20 ConA 2 2G-40L
P 3 5 15-30 From the results shown in Table 1, it became clear that AL-4 had strong mitoneene activity. Moreover, its activity is ConA
'? ? It was estimated that this activity was superior to the optimal activity of LPS.

It also showed more than twice the activity as samples from 11 new varieties of Chinese plants prepared under similar conditions.

(1) Antibody Production Promotion Activity Test Add mouse 11I visceral cells and a sample to a 96-well plate, culture for 48 hours, and then collect the culture supernatant. and,
The determination was made by quantifying the antibody (rgM) in the culture supernatant using the ELISA method.

The results are shown in Table 2.

Table 2 AL-4r gM Nail 020-60 150-100 10 200-400 +00 > 500 n=3 From the results shown in Table 2, it was found that AL-4 has the effect of promoting antibody production.

(3) Macrophage activation test Macrophage activation was tested using promotion of glucose consumption as an indicator. That is, macrophage activation was determined by adding a sample to mouse intraperitoneal exudate cells induced with thioglycollate medium, quantifying glucose in the culture supernatant after 24 hours, and determining its consumption rate.

The results are shown in Table 3.

Table 3 AL-4 glucose consumption rate 0 5-10 t IQ ~20 1010-20 From the results shown in Table 3, ΔL-4, whose main component is acidic polysaccharide extracted from Afghan grass, has the effect of activating macrophages. It turns out that there is.

As is clear from the above experimental results, the AL-/
I was found to have various immunoenhancing effects.

〔Example〕

A part of the present invention will be illustrated below using Example F1, but the present invention is not limited thereto.

Example 1 Afghani was prepared by adjusting the pH of a hot water extract of grass roots to 2.6, centrifuging it, taking the supernatant, adjusting the pH to 5.2, centrifuging to obtain the supernatant, and adding methanol to it. Add to the solution, centrifuge, and remove the precipitate. IN of that precipitate
Methanol was added to the acetic acid extract, centrifuged, and the precipitate was dissolved in water and subjected to dialysis. A Δl5-4 fraction was obtained from the dialyzed fluid at a yield of 0.3%.

This mitogen activity is due to its concentration of 1.10°10
[When the concentration was 1 pg/*l, the values were 2.11.34 (incorporation of tritium thymidine when the control was set to 1).

Regarding the production amount of IgM, its concentration is 1°10,
When tooμg/ml, respectively, 71.303. ＞
500ng/ml (control was 45ng/ml).
ml).

The glucose concentration of macrophages was +3 when the concentration was 1, 10, and 100 μg/wl, respectively.
．． 15. It was 18% (control: 8%).

Procedural restraint +' to be corrected: (spontaneous) 1. Indication of the case 1999 Patent application No. 119497 2, Name of the invention Immune enhancer 3, Person making the amendment Relationship to the case Patent applicant address Chiyo, Tokyo [4-5 Marunouchi-chome, IJ-ku Name ( 234) Sanyo Kokusaku Valve Co., Ltd. 4, Agent Address: 16, Kami 10 Kita-Jimono-cho, Chiyoda-ku, Tokyo
101 3rd floor of Ei Building Description ■1 Name of the invention Immune enhancer 2. Claims (1) Licorice, an aqueous extract of licorice; Immune enhancer as the main component.

(2) An immune enhancer whose main component is acidic polysaccharide of licorice or both components thereof.

(3) Licorice is 11 grasses from Afghanistan (Glyc7r
+hi-xs glabls).

3. Detailed Description of the Invention [Industrial Field of Application] The present invention relates to the use of acidic polysaccharides in the palm of the hand or compounds containing the same as immune enhancers.

[Prior Art and Problems to be Solved by the Invention] Living organisms are protected from attack by microorganisms such as bacteria and viruses, or by parasites, mainly by the action of the immune system.

Furthermore, spontaneous regression of cancer is not a rare phenomenon, but rather a common phenomenon in everyday life. This finding suggests the existence of an immune response to cancer.

Therefore, by activating or strengthening the immune system,
It is believed that host resistance will be improved, and that it may also be possible to prevent and treat infectious diseases and cancer.

Against this background, polysaccharide-based cancer drugs (immunotherapy drugs) such as Krestin, Lentinan, and Schizophyllan were developed and have grown into major drugs today.
These drugs are only approved as anticancer agents.

However, there are a considerable number of reports on polysaccharides having immune-enhancing effects other than anticancer effects, and it seems possible to use them for purposes such as increasing resistance and preventing and treating infectious diseases.

On the other hand, in recent years, AIDS (acquired immunodeficiency syndrome 1 (1)) has become a worldwide problem. To date, no effective treatment has been found, but due to the nature of AIDS, polysaccharide Immune-enhancing agents such as these seem to be effective to some extent in preventing and treating the onset of the disease.

[Means to solve the problem]

Therefore, in order to obtain a new and more effective immune enhancer, we investigated the effects of polysaccharide fractions extracted from various yeasts and crude drugs on the immune system using mitogen activity (lymphocyte blastogenesis) as an indicator. Ta.

The results showed that the components extracted from grass under acetic acid acidity, especially licorice from Afghanistan (hereinafter afghan abbreviated as grass; G1)
7c7+rhixa glabla) was deproteinized and precipitated with alcohol, and then extracted with acetic acid. Strong mitodiene activity was observed in both fractions (hereinafter abbreviated as AL-4). Furthermore, it was discovered that AL-4 has an effect of promoting antibody production and an effect of activating macrophages.

As a result of analysis, AL-4 is mainly composed of heteroacidic polysaccharide containing galacturonic acid and several other sugars such as rhamnose, arabinose, and galactose, and also contains glucan and coloring components (Figure 1). , Figure 2).

Previously, Kumagai et al. of Chiba University reported that neutral polysaccharides extracted from 1R grass exhibit immune-enhancing effects.
Japanese and Chinese Medicine, I+, 79 (1978)], AL-4 as D
It is clearly different from the fact that a strong immunoenhancing activity is observed even when the acidic polysaccharide alone is fractionated and purified using an EAE Cellulofine column.

From the above results, it has been found that the acidic polysaccharide of licorice, especially afghan grass, and the components thereof have a strong effect of activating the immune system, and the present invention has been completed. AL-4 for reference
and an infrared chart of the fractionated and purified acidic polysaccharide.

[Action and effect of the invention]

The test methods and results for the mitogen activity, antibody production promoting activity, and macrophage activation of AL-4 in the present invention are shown below.

(1) Mytone 4 Enactivity Test Add mouse liver cells and samples to a 96-well plate,
After culturing for an hour, tritium thymidine is added and the culture is further cultured for 4 hours. Then, the cells were collected on a filter paper, and the tritium incorporated into the cells was measured using a liquid scintillation counter to determine the mitogen activity. In addition, ConA (
Concativalin A) and LPS (lipopolysaccharide) were compared.

Sugars were identified by liquid chromatography after treatment with trifluoroacetic acid. Also about things that haven't been disassembled! 'C-NMR was measured and used as a reference for analysis.

Mito-diene activity is expressed as tritiated thymidine incorporation relative to control.

The results are shown in Table 1.

Table 1 Sample (μg/ml) Mito-diene activity AL-4
12-4 1010-20 100 30-50 New species is grass polysaccharide 1 1-2 10 5-10 100 10-20 ConA 2 211-40LP8
5 15-30 From the results shown in Table 1, it became clear that AL-4 had strong mitonene activity. Moreover, its activity is
It was estimated that this activity was superior to the optimal activity of ConA and LPS.

It also showed more than twice the activity as a sample from a new type of Chinese licorice prepared under similar conditions.

(1) Antibody production promotion activity test Add mouse liver cells and samples to a 96-well plate,
After culturing for an hour, collect the culture supernatant. The determination was made by quantifying the antibody (IgM) in the culture supernatant using the ELISA method.

The results are shown in Table 2.

Table 2 AL-41gM amount (Ig/wl) (ng/gl) 020-60 150-100 10 200-400 +00 > 500 From the results shown in Table 2, AL-4 has the effect of promoting antibody production. I understand.

(3) Macrophage activation test Macrophage activation was tested using promotion of glucose consumption as an indicator. That is, macrophage activation was determined by adding a sample to mouse intraperitoneal exudate cells induced with thioglycollate medium, limiting glucose in the culture supernatant after 24 hours, and determining its consumption rate. .

The results are shown in Table 3.

Table 3 AL-4 glucose consumption rate (8g/sl) (%) 0
5-10 1 10-20 1010-20 100 15-30 From the results shown in Table 3, it was found that AL-4, whose main component is acidic polysaccharide extracted from Afghan grass, has the effect of activating macrophages. Ta.

As is clear from the above experimental results, the AL-4 of the present invention
was found to have various immune-enhancing effects.

〔Example〕

Hereinafter, a part of the present invention will be illustrated by examples, but the present invention is not limited thereto.

Example 1 A hot water extract of Afghan IJ grass roots was adjusted to pH 2.6, centrifuged, the supernatant was taken, and the pH was adjusted to 5.2.
After centrifugation to obtain a supernatant, add an equal amount of methanol to it, centrifuge, and collect the precipitate. An equal volume of methanol was added to an IN acetic acid extract of the precipitate, centrifuged, and the precipitate was dissolved in water and subjected to dialysis. Then, an AL-4 fraction was obtained from the dialyzed fluid at a yield of 0.3%.

This mitogen activity is due to its concentration of 1.10°10
At 0 pg/*l, they were 2.11.34, respectively (tritiated thymidine uptake when the control was set to 1).

Regarding the amount of IgM produced, its concentration is 1°10, 1
00 pg/sl, respectively 71.303. ＞
500ng/ml (control was 45ng/ml).
ml).

Regarding glucose consumption by macrophages, when its concentration is 1, 10, and 100 ag/device, respectively, 13
．． It was 15.18% (control: 8%).

Example 2 ΔL-41g was added to DEAE-Cellulofine 300ml
(0.05M T r i 5-HC1 gentle sanitation solution pH 7 equilibration). Both fractions eluted with IMNaCl were collected and desalted by dialysis to obtain about 500 mg of acidic polysaccharide.

This is a DEAE-Sephadex column 0-0.5
MNa(l) gradient elution showed a single peak.

Further, the content of galacturonic acid was 53.2% as determined by the carbazole sulfuric acid method using galacturonic acid as a standard.

The mitodiene activity of this is that its concentration is 0°100.
ag/sl was 13.35 (incorporation of tritiated thymidine when the control was set to 1).

In addition, the amount of IgM produced is determined by its concentration at 10°IQ [l o#
350. >500 ng/ml.

[Brief explanation of the drawing]

FIG. 1 is a chart showing the IR of the AL-4 fraction of Example 1, and FIG. 2 is a chart showing the IR of the fractionated and purified acidic polysaccharide of Example 2.
It is a chart showing R.

Claims (4)

[Claims] (1) An immune enhancer whose main component is an acetic acid extract of licorice. (2) Glycyrrhiz from Afghanistan
The immune enhancer according to claim 1, comprising an acetic acid extract of A. agl-abla) as a main component. (3) An immune enhancer whose main component is acidic polysaccharide of licorice. (4) Glycyrrhiz from Afghanistan
4. The immune enhancer according to claim 3, which contains an acidic polysaccharide of Agl-Abla) as a main component.