JPS5914451B2 - Plant green leaf source anti-inflammatory agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an anti-inflammatory agent which is characterized by containing as an active ingredient an anti-inflammatory component in the green leaf juice of a natural grass plant, which has a strong anti-inflammatory effect and is substantially non-toxic. Regarding inflammatory agents.

More specifically, the present invention provides a compound that is soluble in an approximately 40% saturated ammonium sulfate aqueous solution system and separated from a water-soluble protein-containing component in the green leaf green juice of a grass family plant, and is soluble in an approximately 80% saturated ammonium sulfate aqueous solution system. An anti-inflammatory agent characterized by containing an anti-inflammatory component that can precipitate as an active ingredient.

The green juice obtained by squeezing the unripe green leaves (leaves and stems) of a natural rice-forming plant, Toku (this is a general term for the leaves and stems) of wheat, is rich in a wide variety of natural useful ingredients. Knowing this, some people including the present inventors discovered that it was possible to make this green juice component into a stable powder while maintaining the original state in the green juice, and patented Japanese Patent No. 645378 (corresponding). U.S. Patent No. 3,787,591; British Patent No. 1,358;
No. 052, etc.).

According to the proposal disclosed herein, green juice obtained by separating and removing coarse solids from mechanically crushed green leaves of barley before the ripening stage is neutralized to pH 6 to 9, and then spray-dried or By freeze-drying, a stable powder of green juice components of green barley leaves can be obtained.

Furthermore, this proposal states that the obtained green juice component powder is useful in a wide range of fields such as foods including luxury items, health drugs, and pharmaceuticals including cosmetics. An example was shown in which tablets were manufactured using green juice powder, Bofutsu bedding extract powder, starch, lactose, talc, magnesium stearate, and ethyl alcohol, and this agent was used as a health drug for the prevention and treatment of arteriosclerosis. It is disclosed that this will happen.

In addition, JP-A-54-129111 discloses that such green juice components can be used to prevent cancer and its prodromal symptoms, including ulcerative tumors.
It is disclosed that it has excellent therapeutic effects and is also expected to have preventive and therapeutic effects on liver disorders.

However, these conventional proposals do not mention at all that such green juice components contain anti-inflammatory components and their use.

The present inventors have been conducting research for many years on the useful ingredients in the green leaf juice of rice plants, and this time, we have discovered that one useful ingredient in the green juice is substantially non-toxic and exhibits excellent anti-inflammatory effects. We have discovered that this anti-inflammatory component exists and that this anti-inflammatory component can be easily isolated and obtained from green juice.

The details of its active ingredient are still unknown, but it is presumed to be a complex ingredient containing a mixture of complex protein substances found in the green leaf juice of grasses.

For example, about 5 m, as will be shown in detail experimentally later.
y/kg (intravenous injection) in some cases approximately 0.1 Tv/k
y (intravenous injection), approximately 20 μg/kg (subcutaneous injection) in some cases, and in some cases approximately 1 kg (subcutaneous injection), which is equivalent to the anti-inflammatory drug phenylbutacin 50~/ky (subcutaneous injection) 1. We discovered that it is an anti-inflammatory ingredient that exhibits an amazingly unique anti-inflammatory effect that surpasses that, and has significantly lower toxicity compared to the anti-inflammatory dose.

Therefore, an object of the present invention is to provide an anti-inflammatory agent having excellent anti-inflammatory effects derived from a naturally sourced green leaf juice from a grass family plant.

The above objects and many other objects and advantages of the present invention will become more apparent from the following description.

The anti-inflammatory component used in the present invention is separated from the green juice of the green leaves (in the present invention, the leaf and stem are collectively referred to) of the grass family.

As such grass family plants, wheat is preferably used, but other grass family plants can also be used, for example,
Other grasses such as adlay, rice corn, and milo can be exemplified.

A more detailed explanation will be given below using wheat as an example.

Japanese Patent No. 6
A green juice obtained by redissolving the green juice powder obtained as described in detail in Japanese Patent No. 45378 in an aqueous medium can be used.

In order to separate and massage the anti-inflammatory components in the green leaf juice, the green juice is preferably centrifuged to remove chlorophyll, crushed fat, grana, and water-insoluble surrounding proteins. After separating and removing water-insoluble polysaccharides and other solid components, for example, ammonium sulfate or an aqueous ammonium sulfate solution is added to the water-soluble protein-containing component system in the obtained green leaf juice to form an aqueous ammonium sulfate solution system with about 40% saturation. , Separate the liquid phase component from the precipitate component precipitated from the system, add ammonium sulfate or ammonium sulfate aqueous solution to this liquid phase to form an approximately 80% saturated ammonium sulfate aqueous solution system, and collect the precipitate component precipitate from the system. You can do it by doing it.

For example, the anti-inflammatory component separated from green leafy green juice that is soluble in an aqueous ammonium sulfate solution system of about 40% saturation and can be precipitated in an aqueous solution system of about 80% saturated ammonium sulfate, which can be collected as described above, is preferably It can be purified by dissolving it in water, using this solution as an internal solution, dialysis treatment using a dialysis membrane using water as an external solution, and collecting the dialysis internal solution.

Furthermore, the anti-inflammatory components can be re-deposited by salting out the obtained dialyzed fluid using ammonium sulfate.

The anti-inflammatory component isolated from the green leaf juice of the grass family plant used in the present invention is substituted for ammonium sulfate in the above-mentioned exemplary means.
For example, salting out agents such as ammonium chloride, common salt, ethyl alcohol,
Other precipitating agents such as polar organic solvents such as methyl alcohol and acetone can also be used for separation.

For example, the water-soluble protein-containing component system in the green leaf green juice can be treated with the salting-out agent in the same manner as the ammonium sulfate example.

Alternatively, for example, the water-soluble protein-containing component system may be treated to have an organic solvent concentration of about 20 to about 60%, the precipitate may be collected, and the anti-inflammatory component may be obtained by dialysis treatment in the same manner as above. I can do it.

Further, the anti-inflammatory component used in the present invention can be obtained by column chromatography using various columns such as carboxymethyl cellulose, DEAE-Sephadex, activated carbon, alumina, silica gel, etc., and gel filtration agents such as Sephadex-0. by other purification means such as gel filtration, electrophoresis, isoelectric focusing, ultrafiltration, suitable combinations thereof, or by dialysis and combination with these other purification means. , can be further purified.

The anti-inflammatory component isolated from the green leaf juice of the grass family plant, which can be obtained as described above, exhibits excellent anti-inflammatory effects and has extremely low toxicity, making it useful as an anti-inflammatory agent.

Its acute toxicity LD, o is 200 ~ /
kg or more, subcutaneous administration (mouse) of 2000 to 9 or more, and oral administration (mouse) of approximately 5g1kg or more.

The anti-inflammatory component can be administered in a wide variety of dosage forms, including oral administration, subcutaneous injection, intramuscular injection, intravenous injection, herbal administration, sublingual administration, intrarectal administration, and external administration.

The dosage may be changed as appropriate depending on the symptoms, administration form, etc., but for example, about 10 to about 507n? per kg, subcutaneous, intravenous or intramuscular doses of about 0.05 to about 10/ky, about 0.1 to about 40 Tn9/kg
Examples include sublingual or rectal administration.

The anti-inflammatory component can be prepared into various thin compositions by using various pharmaceutically acceptable carriers or diluents, as well as various adjuvants. Oral dosage forms such as granules, granules, tablets, solutions, syrups, etc.; subcutaneous, intravenous, intramuscular, intravenous, intraperitoneal,
Injectable dosage forms such as intrathecal; rectal, anal, urethral, etc.
Vagina, etc.; ointment, plaster, pasta, liniment,
lotion, bag, plaster, application, inhalation, poultice, spray,
It can be in the form of external preparations such as eye drops, rinses, mouthwashes, etc.; sublingual tablets; and various other small forms.

Examples of gaseous, liquid or solid carriers, diluents, and various auxiliary agents used to prepare such fine forms include:
Any pharmaceutically acceptable gaseous, liquid or solid additive can be utilized.

Examples of such additives include gas carriers or diluents such as Freon gas, compressed air, carbon dioxide, argon, etc.; purified water, purified water for injection, sterile water, sterile water for injection;
Physiological saline, ethanol, methanol, chrycerin, phlopylene gelicol, chlorophthanol, acetone, petroleum benzine, benzyl alcohol, polyethylene glycol, polyoxyethylene glycol and its alkyl ethers, polyhydric alcohols, olive oil, peanut oil, Camellia oil, soybean oil, tung oil, linseed oil, corn oil, germ oil, coconut oil, lard, lanolin, paraffin, liquid paraffin, wax, stearic acid, pine resin, rosin, cacao butter,
Liquid carriers or diluents such as alcohols or fats and oils such as beef tallow, hydrogenated oils, calcicum oleate, calcium stearate, sodium soap, potassium soap, benzalkonium chloride, benzethonium chloride, sorbitol, mannitol, starch syrup, and xylit. Agents; lactose, sucrose, starches, dextrin, pheasant, coupling sugar, CMC's alkali metal salts, methylcellulose, hydroxyalkylcellulose, agar, gelatin, egg yolk,
Egg white, casein, lecithin, gum arabic, gum tragacanth, pectin, alkali metal pectate, alginic acid and its salts and esters, magnesium stearate, asbestos, curcum, bentonite, kaolin,
zinc oxide, magnesium hydroxide, aluminum hydroxide,
Solid carriers such as citric acid or its salts, benzoic acid or its salts, paraoxybenzoic acid or its salts or esters, sorbic acid, dehydroacetic acid, boric acid, sodium sulfite, acetone acid sodium sulfite, ascorbic acid, tocopherol, yeast powder, or Diluent; Furthermore, other bases known to the above-mentioned various thin shapes can be used.

The anti-inflammatory agent of the present invention can be used for various types of inflammation, such as various dermatitis, myositis, neuritis, mucositis, periostitis, visceritis, keyring inflammation, arthritis, periarthritis, and various infections. Inflammations such as sexual or allergic inflammation, rheumatoid arthritis, gout, burns, contusions, contusions, incisions, post-surgical and post-traumatic inflammation, swelling, wet examination, hiccups, otitis media, and odontitis can be exemplified.

Hereinafter, several examples of the collection and pharmacological effects of the anti-inflammatory component of the present invention will be shown in Examples.

Example 1 Spray-dried powder of green juice obtained by separating coarse solids from juice of green barley leaves (generally referred to as stems and leaves) before maturity 1. 1M water was added to Okp to make a suspension, which was centrifuged to obtain furnace solution 91.

Ammonia sulfate was added to this to make it 40% saturated, the resulting precipitate was suction filtered, and ammonia sulfate was added to the p solution to make it 80% saturated.
The mixture was brought to saturation, left to stand in a cold place, and the precipitate layer was centrifuged once to collect the precipitate.

Add the precipitate to 50rrLM'J potassium acid buffer 500rfL
1, and dialysis was performed against 50 rfLM potassium phosphate buffer.

Add ammonia sulfate to the dialysis solution to achieve 100% saturation.
Same as above (collect the precipitate, dissolve it in 10rrLM potassium phosphate buffer, perform dialysis,
I got rnl.

This was vacuum freeze-dried to obtain 5 g of physiologically active substance.

Example 2 1 kg of spray-dried powder of green juice obtained by separating the coarse solid content from the juice of green barley leaves (collectively referred to as stems and leaves) before the ripening stage was mixed with 50 mM') acid buffer (50 mM'). pH7,8
) 107 was added, dispersed, and suction filtered to obtain R liquid 850IIL.
I got l.

Ammonium sulfate is added to the P solution to make it 40% saturated, and the resulting precipitate is vacuumed and separated.

Add ammonia sulfate to the obtained P solution to make it 80% saturated.

Leave this in a cold place and centrifuge the sediment layer at 12,000G.
) A precipitate was obtained.

This was diluted with 500 rIL of 50 mM potassium phosphate buffer, placed in a dialysis tube, and dialyzed against 50 rm potassium phosphate buffer for 2 days.

The dialysis fluid was passed through a DEAM-Sephadex-A40 column (
4,5CrIlX 80cm bed volume 112 pieces) 50
The solution was sequentially dissociated with rrLM, 150rrLM, and 300rrLM potassium phosphate buffers to obtain about 51 ml of eluate.

Add ammonium sulfate to saturate the mixture, collect the precipitate by centrifugation, and add 50 rfLM potassium phosphate buffer to saturation.
It was dissolved in rILl and dialyzed again.

The dialyzed solution was passed through the DF and AE Sephadex columns again and treated in the same manner as described above to obtain purified Compound 54.

Ammonium sulfate was added again to achieve 100% saturation, and the resulting precipitate was dissolved in 10rrLM potassium phosphate buffer and dialyzed against 10rrLM potassium phosphate buffer.

The dialyzed fluid was vacuum freeze-dried to obtain 4.5 g of physiologically active substance.

Example 2' 3 g of the physiologically active substance obtained in Example 2 above was added to 3 g of water (distilled water).
Column packed with Sephadex G-50 gel dissolved in 0ml (bed volume 1200rIL11 inner diameter 5C1rL)
) for adsorption, and using water as an eluent, the sequentially flowing effluent was fractionated into three fractions.

After concentrating each physiologically active substance fraction by ultrafiltration, lyophilization was performed to obtain fraction 11 fraction (■) and fraction (2), each having a concentration of 1.25
g, 0.95 g, and 0.1 g.

Example 3 (anti-inflammatory effect test) experimental method All experiments below were performed at room temperature of 25±1°C.

The experimental animals used were male Wistar rats weighing around 100 g, each group consisting of 6 rats.

The physiologically active substance D-01 of Example 1 or the physiologically active substance DA-02 of Example 2 was dissolved in physiological saline.

Phenylbutacin was ground by adding one drop of Tween 60 in an agate mortar, and then suspended by adding 15 WLl of physiological saline.

All of these test solutions were injected subcutaneously or intravenously to rats at a dose of 10 ml/kg.

30 minutes after administration of the test solution, 0.1 ml of a 1% saline suspension was subcutaneously injected into the right hind paw of the rat.
At the same time, 0.1 rnl of physiological saline was subcutaneously injected into the left leg.

In the control group, the same amount of physiological saline as the test solution was injected subcutaneously or intravenously.

The paw volume was determined by immersing the rat's paw in a mercury bath to the depth of the carp.
Go Ba5ile Volume Medi ff
It was recorded on a recorder via an erential meter.

Paw volume was measured over 8 hours after carrageenan injection;
The edema intensity was expressed as the difference between the left and right foot volumes (I721), and the edema suppression rate of the test drug was calculated according to the following formula.

Foot volume was measured in a blinded manner to prevent the subjectivity of the measurer from contaminating the measurements.

Experimental results: D-0.1 was subcutaneously injected into rats at 38.6 μ/day or 154.2 days/day (Fig. 1 shows the edema intensity of the group injected with this and that of the control group).

FIG. 2 shows the edema intensity of the group in which DA-02 was subcutaneously injected at 20 Tng/kg, the phenylbutacin 50 Tng/kg subcutaneous injection group, and the control group.

Figure 3 shows DA-02, 5 da/kg intravenous injection, DA-0220
2 shows the edema intensity of the subcutaneous, DA-02, 10 ■/ky intravenously injected group, and the control group.

Figure 4 shows DA-0210/kg subcutaneous injection, DA-025
The edema intensity of the group treated with 9 intravenous injections at 0.05 m/day and the control group is shown.

Table 1 shows the maximum edema suppression rate by D-01, DA-02 and phenylbutacin.

As shown in Figures 1, 2, 3, and 4, DA-02 was administered intravenously at 5 μ/kg, D-01 was administered subcutaneously at 38.6 da/kg, and DA-01 was administered subcutaneously at 38.6 da/kg.
Subcutaneous administration of 02101n9/kg showed a significant carrageenan edema suppressing effect (risk rate of 1% or less).

In addition, as shown in Table 1, D-0138, 6■/ky subcutaneous injection, DA-0210■/ky subcutaneous injection, DA-025m9/
Intravenous injection of 50 ~/kg of phenylbutacin shows a stronger anti-edema effect than subcutaneous injection of phenylbutacin.

When DA-02 and D-01 were administered at the same dose, they did not cause any abnormality in the behavior or other behavior of the rats after administration.

Example 4 (Anti-inflammatory effect test) The maximum inhibition rate of rat carrageenan edema was measured and calculated in the same manner as in Example 3 for each of fractions 1 and 2 obtained in Example 2'. , 0.2■/! 9・
Edema suppression rate: 31.3% with intravenous injection, 50.8% with intravenous injection of 1~/kg, edema suppression rate of 3 with 1~/kg/subcutaneous injection
It was 5.1%.

[Brief explanation of drawings]

In the accompanying drawings, Figure 1 shows the physiologically active substance D- obtained in Example 1.
FIG. 2 is a graph showing the influence of the physiologically active substance DA-02 obtained in Example 2 and phenylbutacin (control) on the edema intensity of rat paws, and FIG. and FIG. 4 are graphs showing the influence of DA-02 on edema intensity.

Claims (1)

[Scope of Claims] 1. Soluble in an approximately 40% saturated ammonium sulfate aqueous solution system separated from a water-soluble protein-containing component in the green leaf green juice of a grass family plant, and in an approximately 80% saturated ammonium sulfate aqueous solution system. An anti-inflammatory agent characterized by containing a precipitable anti-inflammatory component as an active ingredient. 2. The anti-inflammatory agent according to claim 1, wherein the green leaf juice is a green leaf juice of barley before the ripening stage.