JPH0474128A - Immuno-enhancing agent - Google Patents

Abstract

PURPOSE:To obtain an essentially non-toxic immuno-enhancing agent administrable at a high dose without causing side effects by using squeezed juice of premature green leaves of wheat as an active component. CONSTITUTION:Green leaves of what in premature stage, preferably stalk and leaf of wheat of a period from the tiller-starting stage to the full heading time are squeezed preferably by mechanical means without applying excessive thermal denaturation and coarse solid materials are removed from the juice. The obtained green juice is centrifuged and the supernatant liquid is disinfected and filtered to obtain a squeezed juice component. The green juice may be used as the squeezed juice component as it is. The squeezed juice component can be administered as it is, however, it is preferably spray-dried or freeze-dried and the obtained powder, its solution or an extract is used as the objective agent. The daily does of the active component is 0.1-5 mg, preferably 0.5-2 mg per 1 kg of body weight.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunopotentiator, and more particularly to a substantially non-toxic and highly safe immunopotentiator whose active ingredient is a juice component of natural young wheat leaves.

Knowing that the green juice component of the green leaves of barley before maturity contains a wide variety of useful natural ingredients, we developed this green juice component into a stable powder while maintaining the original state of the green juice. Having succeeded in obtaining the patent, the present inventors previously obtained Japanese Patent No. 645378 (Japanese Patent Publication No. 46-38548) [corresponding U.S. Patent No. 3787591, British Patent No. 13580].
In No. 52, etc. 1, we proposed a method for producing wheat green leaf powder.

According to this patent, the pH of green juice obtained by separating and removing coarse solids from mechanically crushed green leaves of barley before the ripening stage is 6.
By spray-drying or freeze-drying the neutralized product in steps 9 to 9, a stable powder of the green juice component of young barley leaves can be obtained. It also describes that green juice powder is useful in a wide range of fields, including foods including luxury goods, and pharmaceuticals including health drugs and cosmetics. An example is shown in which tablets were prepared using Bofutsu extract powder, starch, lactose, talc, magnesium stearate, and ethyl alcohol, and it is disclosed that this agent can be taken for the prevention and treatment of arteriosclerosis. However, the patent does not mention any findings that suggest that the above-mentioned powder containing green juice components of green leaves of barley before the ripening stage has an immune-enhancing effect.

In the process of conducting research focusing on the physiological activity of the above-mentioned green juice ingredients, the present inventors happened to discover that there is a phenomenon in which people who take green juice powder are less susceptible to wind. As a result of further investigation into the cause, it was discovered that the green juice contains components that have an immune-enhancing effect, leading to the completion of the present invention.

Thus, the present invention provides an immune enhancer characterized by containing as an active ingredient a juice component of green leaves of barley before the ripening stage.

The active ingredient in the agent of the present invention is a juice component of green leaves of barley before the ripening stage, such as the above-mentioned Japanese Patent Publication No. 46-385
It can be obtained according to the method described in Japanese Patent No. 48. Green leaves of natural source wheat before maturity, preferably wheat from the beginning of tillering to the heading stage, such as barley, naked wheat, oats,
Furthermore, the green leaves (the general term for stems and leaves) of Norito barley are squeezed, preferably by mechanical means, without undue heat denaturation, and coarse solids are removed to obtain green juice. , preferably after further centrifugation, the superfluous liquid is collected, and the liquid component obtained by sterilizing and filtering this can be used as it is as the juice component of the present invention.

Moreover, it is also possible to use the above-mentioned green juice as a juice component of the present invention. In addition, prior to the above-mentioned juice extraction, it is preferable to sterilize the green leaves with a disinfectant such as sodium hypochlorite and then perform the juice extraction treatment. Furthermore, green juice powder obtained by neutralizing the green juice obtained as described above to a pH of about 5 to 9 and pulverizing it by means such as spray drying or freeze drying that does not cause substantial thermal denaturation can also be preferably used.

The green juice powder obtained in this way can be used as it is, but it is also possible to redissolve the powder in water and use a solution after removing the insoluble matter from the redissolved product. A solution obtained by extracting the powder with an aqueous alcohol solution, a 50% aqueous methanol solution, or another powdered product can also be used.

However, in any case, it is best to use fresh green juice from green barley leaves without any undue heat history or chemical denaturation. If desired, the squeezed green juice component-containing liquid can be administered orally as it is or by adding, for example, milk, skim milk or other colloidal protein-containing substances, sweeteners, etc.
It is preferable to use the spray-dried or freeze-dried product, which has constant quality and good stability, as well as its redissolved product or extract.

The "juice component of pre-ripe green leaves of barley" used as an active ingredient in the present invention refers to the green juice obtained by squeezing pre-ripe green leaves of barley as described above, and the green juice obtained from this green leaf. This term is used to include all processing components having an immune-enhancing effect obtained by further processing the juice as described above.

The immune enhancer of the present invention may contain various additives, if desired, and may be in various dosage forms.

Such additives include additives for freeze-drying or spray-drying, as well as pharmaceutical additives for forming the desired dosage form. Examples of these additives include ascorbic acid, biotin, calcium pantothenate, carotene, choline chloride, magnesium chloride, niacin, pyridoxine chloride, riboflavin, sodium pantothenate, thiamine hydrochloride, tocopherol, vitamin A1, vitamin B□ 2,
Nutrients such as vitamin D2; sodium metaphosphate;
Hiding agents such as sodium phosphate (11th secondary and tertiary salts), sodium pyrophosphate, sodium tripophosphate, etc.;
Preservatives such as calcium sorbate, benzoic acid, paraoxic methyl benzoate, sodium benzoate, etc.: gum arabic, tragacanth, sodium alginate, methyl cellulose, carboxymethyl cellulose, calcium alginate, aluminum silicate, silicic acid, mannitol, Other additives or diluents such as sorbitol, lactose, fructose, soluble starch, amino acids, glucose, sugar, honey, sucrose, and fatty acid esters may be mentioned.

The immune enhancer of the present invention can be administered orally or parenterally, and can be formulated into any dosage form suitable for each administration route, such as powders, granules, pellets or tablets, coatings, It can be made into dosage forms suitable for oral administration such as capsules, liquids, and tablets; dosage forms suitable for parenteral administration such as injections, drips, herbal medicines, eye drops, nasal drops, and sprays.

Furthermore, it is also possible to formulate external preparations such as ointments, creams, tinctures, and poultices.

Can the dosage of the immune enhancer of the present invention be varied within a wide range depending on the type of target antigen, the severity of the patient's symptoms, gender, age, weight, doctor's judgment, etc.? As a guideline, the active ingredient is generally about 0.1 to about 5 mg/
kg body weight per day preferably about 0.5 to about 2 mg/kg
An example is within the range of body weight/day. The above dosage can be administered once a day or in divided doses. However, when administered orally, the immunopotentiator of the present invention is substantially non-toxic and causes no side effects, as described below, and therefore can be administered in large amounts exceeding the above range.

Acutely toxic LDs of green juice powder from green leaves of barley, such as green juice powder from young barley leaves, which is an active ingredient of the immune enhancer of the present invention. The value is 12.000 mg/kg (oral,
(mouse) and is virtually non-toxic at 1000 mg/mice).
Based on the results of subacute toxicity tests of continuous administration (oral, mouse) of kg, virtually no toxicity or side effects were observed, and the extract of the barley green leaf juice component of the present invention was found to have pharmacological effects, low toxicity, and no side effects. was found to be an extremely unique immunostimulant that has both a practical immunostimulatory effect and is substantially nontoxic and can be administered in large amounts.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Preparation of active ingredient) Freeze-dried powder of green juice (hereinafter referred to as BLE) obtained by separating coarse solids from the juice of green barley leaves (collectively referred to as stems and leaves) before the ripening stage. )1. og to RDF+l
After adding 100ml of O% FBS medium (2) and stirring well for about 10 minutes at room temperature, insoluble components were centrifuged (8000 rpm).
, 10mln) to obtain an amber colored supernatant liquid.

This supernatant was mixed with RDF + 10% FBS medium for 10
It was prepared in 7-fold dilutions (10° to 106) (hereinafter referred to as BLE dilution solution). Each BLE dilution solution is 0.2μm
The mixture was filtered through a sterile filter (manufactured by Gelman) to remove bacteria and suspended particles.

Example 2 (Preparation of antigen) 20 mg of ready-made albumin (OVA vehicle) was added to RDF 110%.
After dissolving in FBS medium, the volume was exactly 50ml. This solution was diluted sequentially with the same medium, and ready-made albumin 100.
pg/rnQ, 10 pg/m (1, lμg/ma
A solution was prepared. Each prepared solution was filtered through a 0.2 μm sterile filter to remove bacteria.

This ○vA: ○valbumin Example 3 (Preparation of lung cells) The lungs were removed from a BALB/cAcl mouse (41 weeks old male) and placed in a cage-shaped steel plate in a cold RDF medium prepared in a 50 mm diameter plastic dish. Metshu (#2
00; 20+nm") and crushed the lungs while pressing lightly with a spaticle until only white connective tissue remained on the mesh.The obtained cell suspension was transferred to a centrifuge tube, suspended well, and then centrifuged (1500 rpm, I 0m
1n) was performed. After centrifugation, the upper groove was removed by suction, 10 m+2 of new RDF + 10% FBS medium was added, the mixture was suspended again, and the same operation was repeated. The number of cells in the lung cell fluid was counted using a hemocytometer and 1-OXl
RDF+10 to become O'cells/mff
% FBS medium was added.

Example 4 (Immunization of lung cells with ready-made albumin) 24
Example 3: Each person in a microtiter plate with 3 holes.
500μQ (5X
106 cells).

Next, 100 μα of each prepared solution of the ready-made albumin prepared in Example 2 was dropped into each row (4 rows×6 holes) of a microtiter plate.

Rows to which no preparation was added were RDF+I as controls.
100 μα of O% FBS medium was added dropwise.

Furthermore, for the BLE dilution solution prepared in Example 1, each dilution solution was similarly dropped into the vertical columns (6 columns x 4 holes) of the microtiter plate by 100 μσ for each column, and the columns to which no BLE dilution solution was added were the same as before. As a control, TRDF + 10%F
100 tt (1 drop) of ES medium was added.

Subsequently, RDF + l O% FBS medium was added at 3 times per well.
After dispensing in 00μQ portions to make the total liquid volume in one large medium 1.0mQ, the liquid was heated to 37°0. Culture was performed for 5 days in an incubator with 5% C○2 conditions.

Example 5 (Confirmation of anti-valve albumin antibodies by enzyme-linked immunosorbent assay) Anti-valve albumin antibodies produced from immunized lung cells were measured using an enzyme-linked immunosorbent assay (ELISA). The enzyme antibody method is as follows. Pipette 200 μΩ of 1% OVA solution containing IOmMPBS into each person in a microtiter plate with 96 holes, let it adsorb to the plate for 30 minutes at 37°C, and add gelatin buffer (0.3% gelatin). The cells were washed three times with IOmMPBS).

Next, the 24-well microtiter plate cultured for 5 days in Example 4 was centrifuged (1500 rpm).

200 μρ per well of a 96-well plate was dropped onto 4 wells of a 96-well plate, reacted for 1 hour at 37° C., and washed three times with gelatin buffer.

Subsequently, 50% of peroxidase-conjugated anti-mouse Igs antibody was added.
μα was added dropwise, reacted at 37°C for 30 minutes, and washed three times with gelatin buffer. Furthermore, hydrogen peroxide and 0
Add a substrate solution containing -7 22 diamine and store in the dark for 1 hour.
The reaction was allowed to react for 0 minutes, and the reaction was stopped by adding 50 μα of 5N-H2SO4.

If the peroxidase-conjugated anti-mouse Ig antibody remains on the microplate, a substrate reactant with absorption at 492 nm will be produced.

As a result, an immune-enhancing effect was observed in a system in which lung cells were immunized with 0.1.1.0 μg/mΩ of ready-made albumin.
Particularly, high activity was shown at BLE 10,000 times diluted concentration. (See Figure 1) Example 6 (Preparation of active ingredient) 1.0 g of freeze-dried powder of green barley juice before ripening: R
Add DF+l O% FBS medium 100-100-1 at room temperature.
After stirring well for 0 minutes, insoluble components were centrifuged (8000
rpm, 10mln) to obtain an amber colored supernatant liquid. Ammonium sulfate was added to this supernatant liquid, and 30
After making w/w% saturated ammonium sulfate solution, centrifugation (6000r
pm, l0m1n) and the supernatant was collected. Kamizo further added ammonium sulfate to form a precipitate as a 70 W/w% saturated ammonium sulfate solution, and then centrifuged (600
0rpm, l0m1n).

The obtained precipitate was dissolved in a small amount of 10mMPBS and then dialyzed against IOmMPBS. After dialysis, centrifugation was performed and the same procedure as in Example I was performed using RDF+l Q% FBS to prepare a BLE diluted solution.

Each BLE dilution was filtered through a 0.2 μm sterile filter to remove bacteria and airborne particles.

Example 7 (Immunization of lung cells with sheep red blood cells) JCI:I
The lungs were removed from a CR mouse (21 weeks old male) and treated in the same manner as in Example 3, and a lung cell suspension (8X 10
'cells/m4) was obtained. The sheep erythrocytes used as antigens were washed three times with physiological saline and then suspended in RDF containing 10% FBS to give 8X 10' cells/m.
The concentration was adjusted to Q. Immunization in vitro was performed as follows. 1 mQ of lung cell fluid (8X IO'cells), 0.1 mff of sheep red blood cell fluid (8X IO' cells), 1
10μQ1 of 0mM 2-mercaptoethanol and 0°5rnQ of each BLE dilution prepared in Example 6 were added, and finally the final volume was adjusted to 2ml with RDF medium containing 10% FBS. Then 37°C 15% CO2
The cells were cultured under these conditions for 4 days, and the number of cells producing anti-sheep erythrocyte antibodies was measured using a PFC method*. The PFC method is as follows.

Cultured lung cells (100μa), sheep red blood cells (25%v
/V25μQ), guinea pig complement (25, uC) and 0.
6% agarose (0, 3n + 12) was spread on a 60 mm diameter plastic dish and left at 37°C overnight.
The number of plaques was counted. Guinea pig complement is obtained from Hemo-Lo guinea pigcom, which is absorbed by sheep red blood cells.
plement) was used.

As a result, the number of plaque cells increased twice by adding BLE at a 4000-fold diluted concentration. (See Figure 2) *5BRC: 5heep red blood
cells**PFC method: plaque form
ing cell assay Example A (tablet) BLE extracted dry powder 1.0mg lactose
100.0mg crystalline cellulose 91.4mg talc
5. Omg binder CMC2,
0mg Magnesium stearate 0.6tng2
00, Omg BLE Extraction After uniformly mixing the dry powder, lactose, crystalline cellulose, talc, and binder to form granules, magnesium stearate is added to form tablets of 200 mg each.

Example B (Enteric Coated Tablets) The tablets obtained in Example A were coated with enteric coating according to the following formulation to produce enteric coated tablets each weighing 430 mg.

Methacrylic acid Acrylic acid 1000% Ethyl copolymer PEG 6000 Tween 80 Talc Purified water 1.6% 1.1% 7.2% 79.3% 100.0% Example C (granules) BLE extracted dry powder 1.0 mg Lactose 700.0mg Starch 289.0mg Gelatin 10.0mg 1000.0
mg BLE extracted dry powder, lactose, and starch are mixed uniformly, dissolved in a small amount of water, mixed, kneaded, and then made into granules and dried (particle size 0.8 columnar granules).

Example D (Capsule) 1) Granules are made according to the following formulation, enteric coated, and filled into capsules.

■ Granules (particle size 0.8mm columnar granules) BLE extracted dry powder 1.4mg lactose
140.0mg starch
56.6mg gelatin
2.0mg200.0mg ■Enteric coating (coating amount 430mg/g
Granules) The formulation is as in Example B above.

■Capsule filling Fill gelatin capsule No. 2 with 200 mg.

Example E (lozenge) BLE extracted dry powder 1.0mg white sugar 920. (1 mg gum arabic 79.0 mg purified water appropriate amount 1000.0 mg BLE extracted dry powder and white sugar are mixed uniformly, gum arabic is dissolved in a small amount of purified water and added, kneaded, made into granules, dried, and pounded. It was made into a tablet and used as a lozenge.

Example FC Suppository BLE Extracted Dry Powder 1.0g Polyoxyethylene 30.0g Lauryl Ether (21E, O,) Polyoxyethylene Sorbitan 100.0g Monostearate (6E, O,) Lipophilic Glycerin Monostearate 16. 0g polyethylene glycol 400 6.0g polyethylene glycol 4000 Appropriate amount Lauryl ether, sorbitan monostearate, lipophilic glycerin monostearate, polyethylene glycol 400, and polyethylene glycol 4000 were heated to 60° and dissolved, then cooled to 45°C. The BLE extracted dry powder was added to this, mixed uniformly, and then molded into 2 g of crude drug using a crude drug molding machine.

Example G (Microcapsule) Lactose 740. OgBLE
Extracted dry powder 200.0gE udra
git R350,0g magyonium stearate
After putting 1O, oglooo, 0g lactose into a vacuum mixing dry coating machine, it was heated to about 50° while rotating and mixing, and then the inside of the tank was evacuated using a vacuum pump, and an aqueous solution of BLE extracted dry powder was coated. Ta. After coating, E u
A methylene chloride solution of dragit R3 (containing 1% magnesium stearate based on the total weight of the product) was similarly coated under vacuum to obtain microcapsules of BLE extracted dry powder.

[Brief explanation of drawings]

FIGS. 1 and 2 are graphs showing the effect of BLE treatment on immunized lung cells; FIG.
Barley green leaf extract (B L
E) Graph showing the effect of barley green leaf extract (BLE) on primary immune reaction in vitro.
This is a graph showing the effect of

Claims (1)

[Claims] An immune enhancer characterized by containing as an active ingredient a juice component of green leaves of barley before the ripening stage.