JPH08245408A - Substance having analgesic activity - Google Patents

Abstract

PURPOSE: To obtain the subject substance composed of a water-soluble component of green leaf of wheat plants, free from adverse effect, being safe and mild, usable not only as medicines, but also as healthy food or food additive. CONSTITUTION: This substance comprises squeezed solution of wheats plant, preferably barley or its dried powder or their extract with water or an acidic aqueous solution, etc., or its purified material. Furthermore, the substance is preferably obtained by squeezing green leaf of a wheats plant by a mechanical means, preparing squeezed solution, extracting the squeezed solution with water or an acidic aqueous solution of hydrochloric acid having pH <=6 or further extracting the solution with a solvent such as dimethyl sulfoxide at ambient temperature to 95 deg.C and removing the solvent.

Description

Detailed Description of the Invention

The present invention relates to an analgesic active substance derived from the green leaf of a barley plant.

Analgesics are drugs that act on the central nervous system to eliminate pain without causing loss of consciousness or sleep state, and are roughly classified into narcotic analgesics and non-narcotic analgesics. In the case of sexual analgesics such as morphine, acute coma causes deep coma, respiratory depression, pupil dilation, etc., and chronic toxicity causes drug resistance, which also causes psychological and physical dependence. Exhibit symptoms. On the other hand, non-narcotic analgesics such as aspirin, indomethacin, ibuprofen, ethensamide, phenacetin, aminopyrine and the like often cause side effects such as allergic reaction and serious gastrointestinal disorder.

Therefore, although the analgesics that have been clinically put into practical use are effective for short-term use, there is a problem in safety when they are used for a long time.

The present inventors have been researching physiologically active substances in young leaves of barley plants, especially barley for many years, and this time, they have an analgesic effect on the water-soluble components of green leaves of barley plants. It was found that there is a substance having an action of enhancing and / or enhancing, and the present invention has been completed.

The present invention thus provides an analgesic active substance comprising a water-soluble component of the green leaf of a barley plant.

The analgesic active substance of the present invention is of natural origin, is safe without the above-mentioned side effects, and is used as a mild analgesic substance not only as a medicine but also as a health food or a food additive. can do.

As is clear from the results of the analgesic activity test shown in Example 4 which will be described later, the juice (green juice) of green leaves (young leaves) of barley plants also exerts an analgesic effect. The active substance shown can be extracted and purified from a barley plant such as barley green leaf (young leaf) as follows.

Therefore, the "analgesic active substance" of the present invention comprises:
The extract of green leaves of barley plants, its dry powder, its extract at any stage of extraction and purification with water or aqueous acid solution, or its purified product is included.

Barley is the most suitable barley plant as a raw material for the production of the analgesic active substance of the present invention, but wheat, barley, oats, doves, corn,
Millet, Italian diegrass, etc. can also be used. Among these barley plants, fresh stem and / or leaf parts of young plants harvested before maturity (these are collectively referred to herein as "green leaves") are particularly suitable.

The green leaves of the barley plant are first squeezed by a mechanical crushing means such as a mixer, juicer, etc., and if necessary, a crude solid content is removed by means such as sieving and filtration to obtain a squeezed juice ( Hereinafter, this is referred to as "green juice". Then, this green juice can be made into a solid powder by adjusting the pH, if necessary, and then dried by an appropriate drying means such as freeze-drying or spray-drying (hereinafter, this solid powder is referred to as “green juice powder”). ). As is clear from the results of the analgesic activity test described in Example 4 below, since the green juice powder already exhibits an analgesic effect, the green juice and the green juice powder are used as a health food having an analgesic effect or as a food additive. Can be used as

First, the green juice or green juice powder is first mixed with water or pH 6
The following acidic aqueous solution (for example, hydrochloric acid aqueous solution) or alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or an aqueous solution thereof, dimethylsulfoxide and a mixed solvent thereof are used at a temperature of about room temperature to about 95 ° C. Can be extracted with. The solvent can be removed from this extract at this stage, and it can be used as an analgesic active substance of the present invention as an extract of green leaves of barley plants. In addition, the extract is then ODS-
It can also be purified by subjecting it to column chromatography using an adsorption carrier such as A (YMC Co.), Amberlite XAD-4 (manufactured by Organo Corp.) and the like. The active substance adsorbed on the carrier can be eluted with an aqueous solution of alcohols such as methanol, ethanol, n-propanol, isopropanol, and n-butanol (alcohol concentration is usually preferably in the range of 20 to 90%). If necessary, the collected active fractions may be dried by, for example, a vacuum concentration method or the like and used as the analgesic active substance of the present invention.

The active fraction thus obtained or its dry powder is subjected to ion exchange chromatography,
For example, DEAE-Sephadex, QAE-Sephadex A-25, DEAE-Sepharose FF, etc. (Phar
It can be further purified by column chromatography (manufactured by macia). At that time, as the eluent, p
An acid-alcohol aqueous solution of H6 or less (preferably hydrochloric acid-methanol aqueous solution) can be used. The eluted active fraction can be dried at this stage to obtain the analgesic active substance of the present invention.

The above active fraction is (1) reverse phase chromatography (HPLC) using, for example, Capsule Bag C ₁₈ SG120 [manufactured by Shiseido Co., Ltd.] and a 0.1% aqueous trifluoroacetic acid solution as a developing solvent; 2) For example, RP-18W
Thin layer chromatography using F _254s (Merck) and 0.1% trifluoroacetic acid aqueous solution as a developing solvent; and (3) For example, Sephadex LH-20 (Pharmacia).
Column chromatography using an acidic 80% aqueous methanol solution (pH 2) as an eluent or a combination of 2 or 3 thereof, and any of those purified products of the present invention. Can be used as an analgesic active substance. Although the details of the mechanism by which the analgesic active substance of the present invention exerts an analgesic effect are unknown, the following Example 3 will be given.
As is clear from the above, since the substance of the present invention has an enkephalin-degrading enzyme inhibitory activity and an aminopeptidase B inhibitory activity, it is an endogenous pain regulator that is widely distributed in the brain of animals. X = Met or Leu via the enkephalin [enkephalin degrading enzyme at position (3) and aminopeptidase B
Cleave position (1)] is presumed to exert analgesic activity.

When the analgesic active substance of the present invention is used as an analgesic agent for the treatment of pain, the substance of the present invention can be administered orally or parenterally, and any dosage form suitable for each administration route can be used. Can be formulated into For example,
Suitable dosage forms for oral administration such as powders, granules, pellets or tablets, coated tablets, capsules, troches, solutions, syrups; parenteral injections, drops, suppositories, nose drops, sprays, etc. It can be in a dosage form suitable for administration. Further, it is possible to use external dosage forms such as ointments, creams, tinctures and poultices.

The dose of the substance of the present invention can be varied within a wide range depending on the administration route, dosage form, symptom of patient, sex, age, weight, judgment of doctor, etc., but generally about 0 as an active ingredient. An example is 0.001 to 100 mg / kg / day, preferably about 0.002 to 50 mg / kg / day. The above dose can be administered once to several times a day.

The analgesic active substance of the present invention is, for example,
Addition to green juice or green juice powder of green leaves of wheat plants or other food materials to enhance its activity, new green juice powder, tablet type foods, capsule type foods, wide range of foods such as drinks Can be used.

In that case, the green juice or green juice powder or the analgesic active substance extracted from the green juice as described above is extracted powder or dry powder of various edible plants, sweeteners such as fructose, glucose, starch syrup and the like, quenching agent. Acid, malic acid, tartaric acid,
It can be mixed with an organic acid such as succinic acid, various vitamins, colorants, flavors, various edible thickeners, and the like and mixed with food materials.

The amount of each component added to the food can be changed depending on the type and use of the food, but the daily intake is preferably 1 to 20 g in the substance of Example 2 regardless of the form of the food. 2 to 10 g is suitable.

The substance 2 of Example 3 had a daily intake of 0.
1-1000 mg / kg, preferably 0.2-500 mg
Is appropriate.

Next, the effects of the substance of the present invention and its use in medicines and foods will be specifically described with reference to Examples.

[0021]

Examples Example 1 : Preparation of green juice powder of young barley leaves 1 kg of young barley leaves before ripening was washed with water, crushed with a crusher, and squeezed with a squeezing machine to obtain green juice of young barley leaves. 1 l was obtained. Add sodium carbonate to this green juice and add
Was adjusted to 7.0 and freeze-dried by a conventional method to obtain 40 g of green barley green juice powder (hereinafter referred to as BLE powder).

Example 2 : Preparation of sample solution 100 g of BLE powder prepared by the method of Example 1 was dissolved in 2000 ml of physiological saline, insoluble matters were removed by centrifugation, and the supernatant was washed with physiological saline. After serially diluting with a predetermined ratio, 0.22 μm sterile filter (M
A sample solution was obtained by filtering with (manufactured by illipore). Further, this sample solution was spray-dried to obtain 48 g of a dried product.

Example 3 : Purification of an analgesic active substance 200 g of barley young leaf juice was treated with 10 liters of 2N hydrochloric acid to give 50 parts.
After extraction at ℃ for 3 hours, it was filtered. 5 l of the filtrate was adsorbed on 1 l of an ODS-A column (YMC), washed with 5 times the column volume of distilled water, and then eluted with 2.5 l of 80% methanol solution. The eluate was concentrated under reduced pressure to obtain 5 g of a dry powder (Substance-1).

Further, 500 ml of the above dry powder was added to 500 ml of a DEAE-Sephadex column activated by a conventional method.
g was dissolved in a 30% aqueous methanol solution adjusted to pH 2.0 with hydrochloric acid to be adsorbed, and adjusted to pH 2.0 with hydrochloric acid 3.
It was eluted with a 0% aqueous methanol solution. The eluate was dried under reduced pressure to obtain 15 mg of dry powder (substance-2).

Next, 15 mg of the sample was dissolved in 0.1% trifluoroacetic acid solution and adsorbed on a high performance liquid chromatography equipped with Capsule Pack C ₁₈ , 10 mmφ, and eluted with a 0.1% trifluoroacetic acid aqueous solution. , The eluate was dried under reduced pressure 2.1
mg dry powder was obtained (Substance-3).

Furthermore, the powder was dissolved in 0.1% trifluoride acetic acid aqueous solution, using a 0.1% trifluoride acetic acid aqueous solution as a developing solvent was performed by thin layer chromatography by _RP-18WF 254s. Enkephalin-degrading enzyme inhibitory activity was found at Rf = 0.43. This active fraction was extracted with methanol and dried under reduced pressure to obtain 1.3 mg of powder (substance-4).

This powder was adjusted to pH 2.0 with hydrochloric acid 8
Dissolve in 0% methanol aqueous solution to Sephadex LH-
It was adsorbed on 20 columns and eluted by adjusting to 80% aqueous methanol solution (pH 2.0 with hydrochloric acid) to obtain 0.23 mg of a purified product (Substance-5). The elution curve of this substance is shown in FIG. 1, and the enzyme inhibitory activity of the purified preparation obtained in this purification process is shown in Table 1.

The enzyme inhibitory activity of the substance of the present invention was measured by the following method.

(1) Measurement of Enkephalin Degrading Enzyme Inhibitory Activity Enkephalin degrading enzyme inhibiting activity was measured by Shimamura,
Method of M., Hazato, T. and Katayama, T. [Biochim. Bio
phys. Acta., 1756, 223-229 (198
3)].

(2) Measurement of Aminopeptidase B Inhibitory Activity Aminopeptidase B was prepared from monkey brain by the method of Hopsu, V.
K., Makinen, KK and Glenner, GG [Arch. Bi
ochem. Biophys., 114 , 557 (1966)]. In addition, the measurement of aminopeptidase B is Sud
a, H., Aoyagi, T., Takeuchi, T. and Umezawa, H. [Arch. Biochem. Biophys., 177 , 196 (198).
3)].

[0031]

[Table 1]

Example 4 Analgesic Activity by Peripheral Administration In order to measure the analgesic activity of the sample solutions and the preparations prepared in Examples 2 and 3, 4-week-old male ddy mice (Japan S
LC company: Hamamatsu city) was used. For the pain threshold of mice, the method of Moolfe, G. and MacDonald, AA [M. J. Pharm, Muromachi Kikai Hot Analgesia Meter (MK-350) was used.
acol. 80 , 300 (1944)].
That is, after the mouse was placed on a hot plate at 55 ° C., the time until the mouse jumped, licked, or the like was measured in accordance with a conventional method, and the hot plate latency was obtained. The maximum measurement time was 25 seconds to prevent burns on the limbs of the mouse.

100 mg / kg of the dried product obtained in Example 2 was dissolved in physiological saline and intraperitoneally administered to a mouse. Immediately after that, 15, 30, 45, 60, 120 and 180 minutes later, the pain threshold value was measured. . Aminopyrine (30 as a control group
(mg / kg) physiological saline solution was used.

In the peripheral analgesic effect, the sample liquid of Example 2 independently showed a significant pain threshold from 15 minutes to 120 minutes after intraperitoneal administration (FIG. 2).

Example 5 : Analgesic activity by central administration 30 μg / monse of substance-4 obtained in Example 3 was placed in the cerebellar medulla oblongata of mouse, Ueda, H., Amano, H., Shiomi, H. and Tak.
agi, H method [Eur, J. Pharmacol. 56 , 265 (19
79)]. As a control group, a single administration of an endogenous analgesic substance, Met-Enkephalin 70 μg / mouse, and a combined administration of the above substance-4 and Met-Enkephalin were also examined.
For analgesic activity, 15 minutes before administration of the sample in the cisterna magna 4
The pain threshold of the mouse was measured after 10 minutes.

The above substance-4 showed analgesic activity when administered in a large tank. Further, the combined administration of the above substance-4 and Met-Enkephalin significantly enhanced the analgesic effect as compared with the administration of Met-Enkephalin alone (FIG. 3).

Example A: Tablets Substance-4 0.10 mg Lactose 71.90 mg Crystalline cellulose 25.25 mg Talc 2.50 mg Binder (carboxymethylcellulose) 1.00 mg Magnesium stearate 0.25 mg 100.00 mg Substance-4, lactose , Crystalline cellulose, talc and binder are uniformly mixed to form granules, and magnesium stearate is added to the mixture to form 100 mg tablets.

Example B: Granules Substance-2 2.0 mg Lactose 750.0 mg Starch 238.0 mg Gelatin 10.0 mg 100.0 mg Substance-2, lactose and starch were mixed homogeneously and a small amount of water was added. After further mixing, granulate and dry. (Particle size 0.8 mm columnar granules) Example C: Enteric coated tablet The tablets obtained in Example A were subjected to enteric coating according to the following formulation to produce 400 mg of enteric coating.

Methacrylic acid / ethyl acrylate copolymer 10.8% Polyethylene glycol 6000 1.6% Sea surface active agent 1.1% Talc 7.2% Purified water 79.3% 100.0% Example D: Capsule Granules are made according to the following formulation, enteric coated, and filled into capsules.

Granules (particle size 0.8 mm columnar granules) Substance-4 0.50 mg Lactose 140.00 mg Starch 57.50 mg Gelatin 2.00 mg 200.00 mg Enteric coating (coating amount 400 mg / g)
Granules) The formulation is as in Example C above.

Capsule filling Gelatin capsule No. 2 is filled with 200 mg.

Example E: Lozenge Substance-3 1.00 mg White sugar 370.000 mg Dextrin 540.000 mg Arabic gum 78.00 mg Purified water suitable amount 1000.00 mg Substance-3, sucrose and dextrin are uniformly mixed, and gum arabic is added. Was dissolved in a small amount of purified water, added and kneaded to give granules, which were then dried and tabletted to give a troche. Example F: Aqueous Injection An aqueous injection was prepared by dissolving 0.1 mg of the substance-5 in 0.5 ml of physiological saline.

Example 6 After washing the green leaves of young barley leaves, the green juice obtained by squeezing the juice was spray-dried to powderize 1 kg of green juice powder, and 25 g of the substance obtained in Example 2 was mixed. To produce a processed barley green leaf food containing an analgesic substance.

Example 7 Spinach was blanched and dried, and 50% by weight of the barley young vegetable extract obtained in Example 2 was added to the dried powdered vegetable to prepare a spinach powder having an enhanced analgesic effect.

Example 8 To 100 g of green juice powder of young barley leaves, 1.0 g of the substance-1 of Example 3 was added to obtain a processed barley young leaf food product having an enhanced analgesic effect.

Example 9 Using the substance-1 obtained in Example 3, a beverage having the following composition was produced.

Granulated sugar 7.0% Liquid sugar 3.0% Citric acid 0.2% Vitamin C 0.1% Substance-1 0.2% Example 10 Substance-2 obtained in Example 3 was used as follows. A beverage having the composition of was produced.

Granulated sugar 7.0% Liquid sugar 3.0% Citric acid 0.2% Vitamin C 0.1% Substance-2 2.0 mg%

[Brief description of drawings]

FIG. 1 is an elution curve during purification of an enzyme inhibitor by Sephadex LH-20 column chromatography in the process of extracting and purifying the analgesic active substance of the present invention from juicy barley juice according to Example 3.

FIG. 2 is a graph showing the effect of the sample solution obtained in Example 2 on mouse latency by the hot plate method.

FIG. 3 is a graph showing the effect of substance-4 obtained in Example 3 on the latency of mice by the hot plate method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinji Ona 2-24-9, Migamioka Nishi, Midori-ku, Yokohama-shi, Kanagawa (72) Inventor Yuka Ichijo 571-5 Kaneda, Atsugi-shi, Kanagawa (72) Inventor, Tadahiro Kanazawa 5-11-37 Tokiwadaira, Matsudo City, Chiba Prefecture

Claims (8)

[Claims] 1. An analgesic active substance comprising a water-soluble component of green leaf of a barley plant. 2. The analgesic active substance according to claim 1, which is a juice of green leaves of a barley plant, a dry powder thereof, an extract thereof with water or an aqueous acid solution, or a purified product thereof. 3. The analgesic active substance according to claim 1, wherein the barley plant is barley. 4. An enkephalin-degrading enzyme inhibitor comprising a water-soluble component of green leaves of a barley plant. 5. An aminopeptidase B inhibitor comprising a water-soluble component of green leaf of a barley plant. 6. The enzyme inhibitor according to any one of claims 4 to 5, which is a juice of green leaves of a barley plant, a dry powder thereof, an extract thereof with water or an aqueous acid solution, or a purified product thereof. 7. The enzyme inhibitor according to claim 4, wherein the barley plant is barley. 8. A food containing the analgesic active substance according to any one of claims 1 to 3 or the enzyme inhibitor according to any one of claims 4 to 7.