JPH069420A - Prolactin or growth hormone secretion promoter - Google Patents

Abstract

PURPOSE:To obtain prolactin and/or growth hormone secretion promoter having low toxicity and side effects, capable of being applied in a large amount, comprising a squeezed component of green leaves of gramineous plant, especially wheat, barley, etc., as an active ingredient. CONSTITUTION:Green leaves of gramineous plant, especially wheat, barley, etc., are squeezed by a mechanically grinding means such as mixer or juicer under a condition not causing improper thermal denaturation and filtered to give a squeezed solution. The squeezed component is used as an active ingredient to give prolactin and/or a growth hormone secretion promoter. Vitamin E succinic ester of the formula occurs in the active fraction of the squeezed component. Blue juice, a supernatant liquid component or an active fraction thus obtained is directly blended as it is with milk, de-fatted milk, colloidal protein-containing material, sweetener and is orally administered. The medicine has both prolactin action and growth hormone secretion promoting action and is extremely unique.

Description

Detailed Description of the Invention

[0001] The present invention relates to a prolactin or growth hormone secretagogue, and more specifically, it is a substantially nontoxic and highly safe compound containing as an active ingredient the squeezed component of green leaves of gramineous plants, especially barley plants. The present invention relates to a prolactin or growth hormone secretagogue.

[0002] The present inventors have been focusing on the physiological action of juicing juice or a dry powder of young leaves of grasses, especially wheat plants before ripening, and have been conducting researches for many years. Preventive or curative action (Japanese Patent Publication No. 46-385)
48), anti-cancer effect (Japanese Patent Publication No. 59-36890).
Japanese Patent Publication), blood pressure lowering effect (Japanese Patent Publication No. 59-36888).
Japanese Patent Publication No. 59-39410 and Japanese Patent Publication No. 59-39410), blood glucose lowering action (see JP-A-62-226927), antiviral action (see JP-A-3-169823),
Antioxidant activity (see Japanese Patent Application No. 2-220398) has been found, and for some of them, isolation and confirmation of the active substance have been successful.

In the course of the above series of studies, the present inventors have
This time, we found that the squeezed components of green leaves of gramineous plants, especially malted plants, had a prolactin and / or growth hormone secretagogue action, and confirmed vitamin E succinate as one of its active substances. This has led to the completion of the present invention.

Thus, according to the present invention, there is provided a prolactin or growth hormone secretagogue, which comprises, as an active ingredient, a green leaf squeezed component of a grass family plant.

The prolactin or growth hormone secretagogue of the present invention will be described in more detail below.

As a grass family plant which is a raw material of the active ingredient of the secretagogue of the present invention, a barley plant is preferable, and examples of the barley plant include barley, wheat, naked barley, oats, and oats. Examples include corn, millet, and Italian diegrass, and barley, wheat, and barley are particularly suitable.

In the present invention, among these malted plants, fresh stem and / or leaf portions of young plants harvested before maturity (in the present specification, the stem and / or leaf portions are collectively referred to as "green leaf"). Is especially suitable.

First, the green leaves of grasses, especially barley plants,
Squeezing juice by mechanical crushing means such as a mixer or juicer under conditions that do not cause undue heat denaturation, and removing coarse solids by means such as sieving and filtering (hereinafter, this is referred to as "green juice"). )) Is prepared. The green juice thus obtained can be used as it is as the juice component of the present invention. In general, it is preferable to sterilize the green leaves with a sterilizing agent such as sodium hypochlorite before squeezing the juice.

The green juice obtained as described above is preferably further centrifuged to collect a supernatant, and if necessary, the supernatant may be subjected to sterilization filtration treatment with a sterile filter or the like to obtain a supernatant component. Can also be used as the juice component of the present invention.

Further, the green juice or supernatant component obtained as described above is neutralized to a pH of about 5 to 9 according to the method described in, for example, Japanese Patent Publication No. 38548/46, and then spray-dried or freeze-dried. It can be dried and powdered by an appropriate means that does not cause heat denaturation, to obtain green juice powder or supernatant powder. These green juice powder or supernatant powder can also be used as the juice component of the present invention.

Furthermore, a liquid obtained by re-dissolving the green juice powder or the supernatant powder in water, or a liquid obtained by removing insolubles from the redissolved aqueous solution can be used as the juice component of the present invention, or A liquid obtained by extracting green juice powder or supernatant powder with an aqueous alcohol, for example, 50% aqueous methanol, or a powdered product obtained by removing the aqueous alcohol therefrom can also be used as the juice component of the present invention.

A liquid obtained by removing the solid content from the green juice or a liquid obtained by redissolving the green juice powder in water to remove the solid content, for example, the above-mentioned supernatant component is a reverse phase chromatograph, an ion exchange chromatograph, A fraction having prolactin and growth hormone secretagogue activity can also be collected by subjecting to gel filtration or the like. Reversed phase chromatography
For example, Delta-Pak 300Å 15 μm C
_{Using 18} columns (manufactured by Waters), 0.01-0.0.
The active fraction can be eluted using acetonitrile containing 5% trifluoroacetic acid and 0.01-0.5% trifluoroacetic acid as the eluent. Further, as the ion exchange chromatography, for example, Protein-Pak DEAE is used.
-5 PW (manufactured by Waters) at pH 8.7 of 1-
100 mM Tris-HCl buffer and 0.1-1 M N
0-100 with 20 mM Tris-HCl buffer containing aCl
The active fraction can be obtained by performing chromatography with a linear concentration gradient of%. Further, gel filtration is performed by, for example, Protein-Pak 300SW (Wate
(manufactured by rs) can be used.

The present inventors further analyzed this active fraction, and in the active fraction, the following formula

[0014]

[Chemical 1]

It was confirmed that the vitamin E succinate represented by the formula (1) was present and that this vitamin E succinate had prolactin and growth hormone secretagogue activity.

The green juice, supernatant component or active fraction obtained as described above can be orally administered as it is, or can be mixed with, for example, milk, skim milk, other colloidal protein-containing substances, sweeteners and the like. However, in general, it is preferable to use the green juice powder or supernatant powder having a constant quality and better stability, or a redissolved product or an extract thereof.

The "squeezing component of green leaves of gramineous plants" used as an active ingredient in the present invention is not only green juice obtained as described above from green leaves of gramineous plants, especially barley plants, but As described above, it is intended to include all treatment components or fractions having a prolactin and / or growth hormone secretagogue action obtained by further treatment.

The prolactin or growth hormone secretagogue of the present invention may consist essentially of the above-mentioned squeezed components, but generally, various pharmaceutically acceptable additives are blended. Then, it is preferable to formulate into a dosage form suitable for administration.

Examples of the additives that can be used include additives for freeze-drying or spray-drying, as well as optional additives for preparation usually used for formulation into a desired dosage form. Specifically, for example, ascorbic acid, biotin, calcium pantothenate, carotene, choline chloride, magnesium oxide, niacin, pyridoxine chloride, lipofurapine, sodium pantothenate, thiamine hydrochloride, tocopherol, vitamin A,
Nutrients such as vitamin B ₁₂ and vitamin D ₂ ; sodium metaphosphate, sodium phosphate (first, second, third
Salt), sodium pyrophosphate, sodium tripolyphosphate and the like; preservatives such as calcium sorbate, benzoic acid, methyl paraoxybenzoate, sodium benzoate and the like; arabic gum, tragacanth, sodium alginate, methylcellulose, carboxymethylcellulose ,
Examples include carriers or diluents such as calcium alginate, aluminum silicate, calcium silicate, mannitol, sorbitol, lactose, fructose, soluble starch, amino acids, glucose, sugar, honey, sucrose, and fatty acid ester.

The secretagogue of the present invention can be administered orally or parenterally, and can be formulated into any dosage form suitable for each administration route, for example, powder, granules, pellets or tablets, Coated tablets, capsules,
The dosage form suitable for oral administration such as troche, solution, syrup; the dosage form suitable for parenteral administration such as injection, drip, suppository, eye drop, nasal drop, and spray can be prepared.

Further, it is also possible to use an external preparation type such as an ointment, a cream, a tincture and a poultice.

The dose of the secretagogue of the present invention depends on the degree of purification of the squeezed component, the lightness of the patient's symptoms, sex, age, body weight,
It can be varied within a wide range depending on the judgment of the doctor, etc., but as a rough guideline, it is generally about 0.0 as the active ingredient.
1 to about 20 mg / kg body weight / day, preferably about 0.05
A range of about 10 mg / kg body weight / day can be exemplified. The above dose can be administered once or divided into several times a day. However, in the case of oral administration, the secretagogue of the present invention is substantially nontoxic and causes no side effects as described below, and therefore, it can be administered in a large amount beyond the above range.

The acute toxicity LD ₅₀ of the juice component of green leaves of grasses, which is the active ingredient of the secretagogue of the present invention, such as green juice powder from young leaves of barley before ripening, is 12,000 mg /
Substantially nontoxic with kg (oral, mouse), 100
From the results of the subacute toxicity test of 0 mg / kg continuous administration (oral, mouse), virtually no toxicity or side effect was observed.

Therefore, in view of its great pharmacological effect, toxicity and no side effect, the juice component of green leaves of the Gramineae plant of the present invention has a practical prolactin and / or growth hormone secretagogue action. It was found to be an extremely unique prolactin or growth hormone secretagogue that is both nontoxic and capable of large-scale administration.

The present invention will be described in more detail below with reference to examples.

[0026]

【Example】

Example 1: Preparation of green juice powder of young barley leaf 1 kg of young barley leaf before maturity was washed with water, crushed with a crusher, and squeezed with a squeezing machine to obtain 1 liter of green juice of young barley leaf. . Add calcium hydroxide to this green juice and p
H was adjusted to 7.0 and freeze-dried by a conventional method to obtain 40 g of green juice powder of barley young leaves (hereinafter referred to as BLE powder).

Example 2: Measurement of secretagogue action of prolactin and growth hormone Preparation of sample solution 5 g of BLE powder prepared in Example 1 was added to RPMI medium 10
Dissolved in 0 ml, insoluble material was removed by centrifugation,
The supernatant was serially diluted with RPMI medium at a predetermined ratio, and then a 0.22 μm sterile filter (Millip) was used.
ore) to obtain a sample solution.

Assay method Anterior pituitary cells of Sprague-Dawley rats were treated with Cr.
Onin and Thorner's method [J. Cyclic
Nucleotide Res. , 8 , 267 (19
82)]].

On the other hand, fetal bovine serum 2.5%, horse serum 7.5
%, Streptomycin 7.5 μg / ml, gentamicin 15 μg / ml, penicillin 10 μg / ml and phandizone 0.6 μg / ml RPMI 16 supplemented
24-well tissue culture plate (F
Alcon) is inoculated with the prepared anterior pituitary cells at a rate of 0.4 × 10 ⁶ viable cells / well.

37 in an atmosphere of 5% CO ₂ -95% air
Serum-free RPM containing antibiotics after culturing at 4 ℃ for 4 days
After washing the cells with I 1640 medium, 1 ml of the sample solution prepared above is added to the wells and incubated at 37 ° C. for 30 minutes.

After culturing, the medium was taken out from the well and the NI
DDK Rat Pituitor Hormon
Radioimmunoassay is performed according to the Distribution Program to quantify prolactin and growth hormone in the medium. The results are shown in Table 1.
Shown in.

[0032]

[Table 1] Table 1: Secretory action of prolactin and growth hormone by BLE BLE * prolactin Growth hormone (μg / ml) (ng / well) (ng / well) 0 230 50 0.5 350 350 75 5 400 110 110 50 410 140 500 590 320 * Indicates the weight (μg) of solid content remaining after 1 ml of the sample solution was evaporated to dryness under reduced pressure.

Example 3 Model 990 Photo set to a wavelength of 214 nm
Model 600 HPLC System (Wat equipped with an instrument array (manufactured by Waters))
ERS) 300 × 19 mm Delta-Pak
A 300Å 15 μm C ₁₈ column (manufactured by Waters) was incorporated, and 250 mg of the BLE powder prepared in Example 1 in the HPLC _water solution (BLE
250 mg of the powder was dissolved in 5 ml of HPLC _water , the solution was centrifuged at 3000 rpm for 30 minutes to remove insolubles, and the supernatant was added to Millipore DEP.
HType Filter 0.45 μm Sterile Filter and 0.22 μm Sterile Filter (Millipore
(Manufactured by Mfg. Co., Ltd.)
Linear gradient between eluent A 0% to eluent B 80% between eluent A consisting of% trifluoroacetic acid and eluent B consisting of acetonitrile containing 0.1% trifluoroacetic acid Elution was carried out. The elution curve is shown in FIG.
In the graph of FIG. 1, the vertical axis represents the absorbance (%) at 214 nm, and the horizontal axis represents the elution time (minutes). With respect to each fraction collected by the fraction collector, the prolactin radioactivity was measured by the same method as described in Example 2. As a result, the fraction flown out during 30 to 40 minutes showed activity (FIG. 1). The shaded area). Particularly, the fraction eluted after 38 minutes was found to have a particularly strong activity, so this fraction was freeze-dried, and 50 mg of this freeze-dried powder was added to 2 ml of HPLC water.
200 μl of the resulting solution was subjected to HPLC in the same manner as above. As a result, three active fractions having prolactin-releasing activity were obtained.

Each active fraction was further added with 3.9 × 30.
0 mm Delta-Pak 300Å ₁₅ μm C ₁₈ column (Wate
(manufactured by rs) and chromatographed for 40 minutes at a flow rate of 0.5 ml / min with a linear concentration gradient within the range from 0% eluent A to 80% eluent B.

As a result, purified fractions F54 and F showing strong prolactin-releasing activity after 26 minutes, 26.5 minutes and 27 minutes from the above three active fractions, respectively.
55 and F56 were obtained. F56 was 0.02 mg.

Example 4 The purified preparation of the fraction F56 obtained in Example 3 was treated with V.
G ZAB-T Mass Spectrometer (VG Instruments,
VG Analytical LTD., Wythenshawe, Manchestter
England) using direct chemical ionization (DCI), electron impact (EI) and fast atom bombardment (FAB). FAB was analyzed by high resolution mass spectrometry (HRMS) to analyze the elemental composition of compounds.
Mode. FAB by high resolution mass spectrometry
-MS analysis results are shown in FIG. From this FAB-MS analysis result, [M + H ⁺ ] at m / z = 531, m / z =
Since the peak of [M + Na ⁺ ] is observed at 553,
The molecular weight of this compound is 530, and when considered together with the results of elemental analysis, the molecular formula is C ₃₃ H ₅₄ O ₅ , and the maximum absorption wavelength of the UV spectrum of this compound is 286 nm.
Therefore, this compound was determined to be a vitamin E succinate (Vitamin E acid succinate).

Example 5 The active fraction F56 obtained in Example 3 was used in Example 2
The activity was measured by the method described in 1. above, and as shown in Table 2, it was revealed that the active fraction F56 containing vitamin E succinate shows remarkable activity for prolactin or growth hormone production.

[0038]

[Table 2] * Similar results were obtained using a commercial product of vitamin E succinate.

Example A: Tablets BLE powder 1.0 mg Lactose 100.0 mg Crystalline cellulose 91.4 mg Talc 5.0 mg Binder (carboxymethyl cellulose) 2.0 mg Magnesium stearate 0.6 mg 200.0 mg BLE powder, lactose, crystals Cellulose, talc and a binder are uniformly mixed to form a granule, and magnesium stearate is added to the mixture to form a tablet of 200 mg.

Example B: Enteric coated tablets The tablets obtained in Example A were coated with the enteric coating having the following formulation to give 1 tablet of 430 mg.

Methacrylic acid / ethyl acrylate copolymer 10.8% Polyethylene glycol 6000 1.6% Surfactant (Tween 80) 1.1% Talc 7.2% Purified water 79.3% 100.0% Examples C: Granules BLE powder 1.0 mg Lactose 700.0 mg Starch 289.0 mg Gelatin 10.0 mg 100.0 mg BLE powder, lactose and starch were uniformly mixed, and a small amount of water was further mixed and kneaded before granulation. And dry (particle size 0.8 mm columnar granules).

Example D: Capsule A granule is prepared according to the following formulation, an enteric coating is applied, and the capsule is filled with it.

Granules (particle size 0.8 mm, columnar granules) BLE powder 1.4 mg Lactose 140.0 mg Starch 56.6 mg Gelatin 2.0 mg 20.0 mg Enteric coating (coating amount 430 mg /
g Granules) The formulation is as in Example B above.

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

Example E: Lozenge BLE powder 1.0 mg white sugar 920.0 mg arabic gum 79.0 mg purified water appropriate amount 100.0 mg BLE extraction dry powder and white sugar were uniformly mixed, and arabic gum was added in a small amount of purified water. The mixture was melted, added and kneaded to give granules, which were dried and compressed into lozenges.

Example F: Suppository BLE powder 1.0 g Polyoxyethylene lauryl ether (21 E.O.) 30.0 g Polyoxyethylene sorbitan monostearate (6 E.O.) 100.0 g Lipophilic monoglyceryl monostearate 16 0.0 g polyethylene glycol 400 6.0 g polyethylene glycol 4000 proper amount polyoxyethylene lauryl ether, polyoxyethylene sorbitan monostearate, lipophilic glyceryl monostearate, polyethylene glycol 400 and polyethylene glycol 4000 were dissolved by heating at 60 ° C. After that, the mixture was cooled to 45 ° C., BLE powder was added thereto and mixed uniformly, and then molded into 2 g of suppository with a suppository molding machine.

Example G: Microcapsules Lactose 740.0 g BLE powder 20.0 g Eudragit RS 50.0 g Magnesium stearate 10.0 g 100.0 g Lactose was put into a vacuum mixing and drying coater while being rotary mixed. After heating to about 50 ° C., the inside of the tank was evacuated by a vacuum pump, and coating was performed with an aqueous solution of BLE powder. Eudragit after coating
Coating with RS in a methylene chloride solution (containing 1% magnesium stearate based on the total weight of the product) was also performed under vacuum to obtain BLE powder microcapsules.

Example H: Tablets Fraction F56 Lyophilized powder 10 mg Lactose 90.0 mg Crystalline cellulose 92.4 mg Talc 5.0 mg Binder (carboxymethyl cellulose) 2.0 mg Magnesium stearate 0.6 mg 200.0 mg Fraction F56 Lyophilized powder , Lactose, crystalline cellulose, talc and binder are mixed uniformly and made into granules, then magnesium stearate is added and 1 tablet 200m
Mold into tablets of g.

Example I: Enteric coated tablet The tablet obtained in Example H was coated with the enteric coating having the following formulation to give an enteric coated tablet of 430 mg.

Methacrylic acid / ethyl acrylate copolymer 10.8% Polyethylene glycol 6000 1.6% Surfactant (Tween 80) 1.1% Talc 7.2% Purified water 79.3% 100.0% Examples J: Granule Fluxion F56 freeze-dried powder 10 mg Lactose 700.0 mg Starch 280.0 mg Gelatin 10.0 mg 100.0 mg Fluxion F56 freeze-dried powder, lactose and starch were uniformly mixed, and a small amount of water was further mixed and kneaded. After combining, it is made into granules and dried (particle diameter 0.8 mm columnar granules).

Example K: Capsule A granule is prepared according to the following formulation, an enteric coating is applied, and the capsule is filled with the granule.

Granules (particle diameter 0.8 mm, columnar granules) Fluxion F56 freeze-dried powder 10 mg lactose 110.0 mg starch 78.0 mg gelatin 2.0 mg 200.0 mg enteric coating (coating amount 430 mg /
g Granules) The formulation is as in Example B above.

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

[Brief description of drawings]

FIG. 1 is a RP-HPLC column chromatogram of an aqueous solution of BLE powder.

FIG. 2 is a fraction F56 obtained in Example 3.
Is a FAB-MS of the purified standard of 1. by high resolution mass spectrometry.

Claims (3)

[Claims] 1. A prolactin or growth hormone secretagogue, which comprises, as an active ingredient, a juice component of green leaves of a grass family plant. 2. The secretagogue according to claim 1, wherein the gramineous plant is a barley plant. 3. The secretagogue according to claim 1, which contains vitamin E succinate.