JP2715216B2 - Immune enhancer for aquaculture and production method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the use of a licorice warm or hot water extract after glycyrrhizin extraction as an immunopotentiator.

[0002]

2. Description of the Related Art We have previously found that a fraction extracted from licorice under acetic acid acidity, especially a fraction obtained by further extracting lactic acid from Afghanistan with acetic acid, has a strong immunopotentiating effect ( JP-A-2-300136) and found that these fractions have an antitumor activity presumably due to an immunopotentiating effect, and have been found to be used as an antitumor agent (JP-A-3-255032). No.).

Conventionally, when producing this substance, a hot water extract of untreated licorice root is adjusted to be acidic, and a supernatant obtained by removing a formed precipitate is adjusted to be neutral. A precipitate formed by adding an equal amount of alcohol to the supernatant from which the substance has been removed is taken. Further, an equal amount of alcohol is added to a 1N acetic acid extract of the precipitate, and salts are removed from the formed precipitate.

[0004]

However, this method is not suitable for mass production due to the complicated process, and it is difficult to obtain other useful components in licorice such as glycyrrhizin. Is not economically favorable.

Further, feeds and feed additives for livestock and livestock containing licorice extract (JP-A-3-173827, JP-A-3-175934, JP-A-2-250832) have already been used. However, its effectiveness is mainly due to the detoxification and antibacterial effects of glycyrrhizin and the like, and those containing an immunopotentiating substance in licorice other than glycyrrhizin as an active ingredient have not been known until now.

An object of the present invention is to provide a method for producing a novel immunopotentiator which overcomes the disadvantages of the conventional methods.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have extracted glycyrrhizin with water or an aqueous alkaline solution, and then extracted the licorice with hot or hot water. In addition, a strong immunopotentiating activity was found in the high molecular substance fraction, and the present invention was completed. On the other hand, the licorice hot water extract which was not subjected to the water or alkali aqueous solution extraction operation had weak immunoenhancing activity. This is considered to be due to the immunosuppressive action of low molecular substances such as flavonoids extracted together with glycyrrhizin. Therefore, this extraction operation is essential to obtain the substance of the present invention. The immunopotentiating activity is specifically shown in Examples.

That is, the immunopotentiator for aquatic livestock of the present invention is a licorice extract obtained by extracting glycyrrhizin with water or an aqueous alkali solution, a hot water or hot water extract, a concentrate thereof, a dried product thereof, or a polymer substance thereof. , As an active ingredient.

[0009] The licorice used in the present invention is not limited to the place of origin and species, but in particular from Afghanistan (Glycyrrhiza).
It is preferable to use licorice from glabla or Xinjiang (Glycyrrhiza glandulifera). Glycyrrhizin is extracted with water or an aqueous alkaline solution, preferably 0.1 to 0.1 g.
It is desirable to use 5% ammonia water under the conditions of an extraction temperature of 5 to 40 ° C. and an extraction time of 1 to 24 hours.

Further, the conditions for extracting licorice with glycyrrhizin after hot or hot water extraction are as follows: 40 to 120 ° C., 0.5 to 24 ° C.
Time, preferably in the range of 50 to 95 ° C. for 5 to 16 hours. The pressure at the time of extraction is usually in a slightly pressurized state. However, normal pressure may be used. If desired, the pressure may be reduced.

After completion of the extraction, the supernatant and the residue are separated, and the supernatant is concentrated or dried. Although it shows sufficient immunopotentiating activity even when used in this state, it will have a higher effect by concentrating or drying the polymer substance fraction separated by membrane separation or alcohol precipitation. Can be obtained. These immunopotentiating activities were measured using the mitogenic activity on mouse lymphocytes and the activity of promoting antibody production as indices.

[0012] The immune enhancer for aquaculture of the present invention can be used for various applications. The first use is an activator of immunity for aquatic and livestock making use of its immunity enhancing action as it is. Second
Is a feed for aquatic animals that is formulated with the expectation of its immunopotentiating effect. The amount used depends on the individual or species of the aquatic animal product, but can be appropriately determined based on the body weight per 1 kg / day.

[0013]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

(Example 1) Acquisition of an immunopotentiator: 100 g of licorice root powder was added with 100 ml of 0.3% ammonia water, extracted for 16 hours, separated into a supernatant and a residue by a filtration operation, and the residue was treated with 50 ml of distilled water. Wash and filter to obtain residue. 100 ml of distilled water is added to the residue, and 5
After extraction at 0 ° C. for 12 hours and at 95 ° C. for 1 hour, the supernatant was freeze-dried to obtain a brown powder in a yield of 3 to 5%.
These are referred to as samples A and B, respectively. 95 ° C
An equal amount of methanol was added to the supernatant of the 1-hour extraction, and the resulting precipitate was freeze-dried to obtain a brown-gray powder in a yield of 1 to 2%. This is designated as Sample C. The ammonia-extracted supernatant was neutralized to pH 7 and freeze-dried to obtain Sample D.

Separately from the above, 100 g of licorice root powder was added with 100 ml of distilled water, extracted for 16 hours, separated into a supernatant and a residue by a filtration operation, and 100 ml of distilled water was added to the residue to add another 16 ml.
After extraction for a time, the residue is obtained by filtration. After adding 100 ml of distilled water to the residue and extracting at 50 ° C. for 12 hours and at 95 ° C. for 1 hour, the supernatant was freeze-dried to remove brown powder from 5 to 7 hours.
% Yield. These are referred to as samples E and F, respectively. In addition, an equal amount of methanol was added to the supernatant of the extraction at 95 ° C. for 1 hour, and the resulting precipitate was freeze-dried to remove a brown powder from 1 to 1.
Obtained in 3% yield. This is designated as Sample G. A sample H was obtained by freeze-drying the above-mentioned water-extracted supernatant.

(Example 2) Measurement of mitogen activity: Mouse spleen cells and a sample were added to a 96-well plate, and cultured for 48 hours. Then, live cells were stained with propidium iodide (PI) and the fluorescence intensity was measured. By doing so, the amount of cell proliferation was quantified. The results are shown in FIG.

From the results shown in FIG. 1, samples A, B,
C, E, F, and G were all found to have strong mitogenic activity. Samples D and H had no activity and tended to be suppressed.

(Example 3) Antibody production promoting activity test: Mouse spleen cells and a sample are added to a 96-well plate, and cultured for 48 hours, and the culture supernatant is collected. Then, the antibody (IgM) in the culture supernatant
Was determined by quantification by a fluorescent antibody method. The results are shown in FIG.

From the results shown in FIG. 2, samples A, B, C,
It was found that E, F, and G all had an effect of promoting antibody production. On the other hand, Samples D and H tended to suppress.

[0020]

As shown in the above examples, it has been confirmed that the substance of the present invention has a strong immunopotentiating activity and can be produced at low cost by a simple operation without impairing useful components in licorice such as glycyrrhizin. .

[Brief description of the drawings]

FIG. 1 is an actual measurement showing the mitogen activity of the immunopotentiator of the present invention.

FIG. 2 is an actual measurement showing the antibody production promoting activity of the immunopotentiator of the present invention.

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-304024 (JP, A) JP-A-4-76002 (JP, A) JP-A-2-300136 (JP, A) JP-A 48-48 88213 (JP, A) JP-A-1-135723 (JP, A)

Claims (4)

(57) [Claims] 1. An immunopotentiator for aquatic animals, comprising a hot or hot water extract of licorice after glycyrrhizin extraction. 2. The immunity enhancer for aquatic animals according to claim 1, which is obtained by concentrating or drying a hot water or hot water extract. 3. The immunopotentiator for aquatic livestock according to claim 1, comprising a high molecular substance fraction in hot water or hot water extract. 4. A method for producing an immunopotentiator for aquatic livestock, comprising extracting licorice after glycyrrhizin extraction with hot or hot water.