JPS61204126A - Ovulatory agent and production thereof - Google Patents

Abstract

PURPOSE:To obtain an ovulatory agent, by adding ethyl acetate to bran of adlay, extracting a fat or oil fraction, recovering ferulic acid derivative of stanol, and incorporating the resultant derivative as an active constituent in the agent. CONSTITUTION:An ovulatory agent obtained by adding 3-5pts.wt. ethyl acetate to 1 pts.wt. bran of adlay, extracting a fat or oil fraction, removing impurities, adding 3-5pts.wt. ethanol to 1pts.wt. residue, extracting the residue, removing insoluble matter, distilling away the ethanol from the ethanol-soluble fraction to give an ethanol extract fraction, adding 3-5pts.wt. ethyl acetate to 1pts.wt. resultant fraction, extracting the fraction to give ethyl acetate-soluble fat or oil fraction, removing ethyl acetate from a mixture of the above-mentioned ethyl acetate fat or oil fraction with the ethanol extract-ethyl acetate fat oil fraction with the ethanol extract-ethyl acetate- soluble fraction, feeding the resultant fraction to a column packed with silica gel, carrying out the gradient elution with n-hexane-ethyl acetate, collecting fractions eluting with a solvent of the n-hexane and ethyl acetate at 30:1-10:1 mixing ratio, mixing the resultant fractions, and distilling away the solvent to give the aimed com pound, expressed by the formula (R is methyl or ethyl), and having the ovulatory action. The dose thereof is 40-80mg/time for oral administration.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides stigmastanol (in the above general formula, R is an ethyl group) and/or campestanol (in the above general formula, R is a methyl group) extracted from adlay. ) An ovulation-inducing agent containing a ferulic acid derivative as an active ingredient.

[Technical background and prior art]

Representative ovulation-inducing agents currently administered to humans include clomitene and cyclohexyl, and it is known that their pharmacological effects are clinically insufficient to some extent. However, these medicines are not necessarily sensitive and can cause abnormalities in the sexual cycle and the various disorders caused by them (
For example, multiple births, failure to conceive) and other side effects are known.

Although these medicines have been used for over 20 years, no medicines to replace them are known.

Research into new ovulation-inducing agents is also being carried out, and it is known that there are substances in the leaves of corn, rye, wheat, etc. that induce ovulation in domestic rabbits (Masasu Suzuki Niigata Medical Society Journal, Vol. 78, 305 Sada, (Showa 39).

On the other hand, some pharmacological effects of extracts of Coix barley or extracts of Coix barley (coix husk, husk and pericarp removed) are already known, and Inagaki et al. (Herbal Pharmacology, p. 162, Nankodo)
According to (1975):

l) Due to its diuretic effect, it reduces edema, nausea, kidney and bladder stones,
Used to treat neuralgia and cough.

2) It is used for muscle cramps because it has 411K pain and @I spasmodic effects.

3) Used for warts, rough skin, etc.

Furthermore, in Japan, it has been used as an emulsifying agent in folk medicine for a long time, but it has been revealed that protein extracted from powdered pearl barley that is not threshed promotes milk secretion (Masahiko Shigemitsu:
Japan Gynecological Society Kumamoto Regional Section Bulletin, Volume 3, Page 191, 1
944), a substance with cancer-causing effects has also been isolated from Yokuinin.

[Chemical and Pharmaceutical Company Lupretin Japan (
Chemical and Phar+aaceutl
calBulletin+ Japan) Volume 9, 4
3 pages, (1961)].

However, nothing is known about the ovulation-inducing effect of adlay or adlay or adlay extract.

As a result of repeated research on substances that have an ovulation-inducing effect, the present inventors have found that ferulic acid derivatives of stigmastanol and campestanol contained in coix seed have an ovulation-inducing effect, and that these derivatives are derived from coix seed bran. The present invention was completed based on the discovery that extraction can be achieved with high yield.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a novel ovulation-inducing agent that induces physiologically natural ovulation without causing abnormalities in the estrous cycle.

Another object of the present invention is to provide a method for producing a ferulic acid derivative of stanol having excretion fI8-inducing activity from coix seed in high yield.

The present invention provides an ovulation-inducing agent characterized by containing a ferulic acid derivative of stanol represented by the following general formula (I) as an active ingredient, and an ovulation inducer characterized by adding ethyl acetate to coix seed bran, extracting an oil and fat fraction, and The oil and fat fraction is also a method for producing an inducing agent.

General formula: (However, in the above general formula, R represents a methyl group or an ethyl group) [Detailed description of the invention] The ovulation-inducing agent of the present invention is produced as follows. The pearl barley is threshed and milled using a conventional method, and separated into yokuinin, bran, and outer shell. Add 3 to 5 parts CMMk (the same applies hereinafter) of ethyl acetate to 1 part of bran, and heat at 15 to 20°C to 5 to 10
Extract the fat and oil fraction while stirring for a period of time. Then, it is filtered to remove insoluble matter.

Add 3 to 5 parts of ethanol to 1 part of the residue, and remove insoluble matter at 15 minutes. Ethanol is removed from the ethanol-soluble fraction to obtain an ethanol-extracted fraction.

Add 3 to 5 parts of ethyl acetate to 1 part of this ethanol-extracted fraction, and extract with stirring at 15 to 20''C for 5 to 10 hours to obtain an ethyl acetate soluble fraction.

The ethyl acetate fat fraction was extracted with ethanol, and ethyl acetate was removed from the mixture with the ethyl acetate soluble fraction, and the mixture was fed to a column packed with silica gel, and gradient elution of n-hexane and ethyl acetate was performed. Mixing of hexane and ethyl acetate is 30:1! Collect fractions eluting with 0:1 solvent.

These fractions are mixed and the solvent is removed to obtain a substance having an ovulation-inducing effect. These substances are formulated into tablets, powders, capsules, external preparations or injections by conventional methods,
The ovulation-inducing agent of the present invention is obtained.

The ovulation-inducing agent of the present invention is administered orally or parenterally. The dosage varies depending on the symptoms to be cured and the administration method, but usually 40 rnli to 100 rnli per dose when administered orally to adults.
Valid at 80 mal.

Next, the ovulation-inducing agent of the present invention will be explained in detail. In addition, the physical and chemical @-shapes described below are shown for substances obtained by the same method as in Practical Example 1.

As a result of analyzing this substance by conventional thin layer chromatography, two spots (denoted as substances A and B, respectively) were observed that developed a blue-green color with sulfuric acid and were distinguishable under ultraviolet light.

Substance A was further purified by chromatography using an alumina column and isolated as colorless needle crystals with a melting point of 156°C. Substance A has a positive Gibbs reaction, and mass spectrometry shows m/
It has an M peak at e592, and in the infrared spectrum it has a peak at 3120-3500 ort- as shown in Figure 1.
'2k acid group, 1710 cat-'2k &-h)L
r F * yl group, 1640 m-' double bond of α, β-unsaturated carbonyl group, 1600 and U 1510 cm-'
Nihe:/ It has absorptions based on the C-C stretching vibration of the Sen ring and the out-of-plane bending vibration of the benzene ring at 840 m-', and the nuclear magnetic resonance spectrum (CDCI) shows 0.0 m as shown in Figure 2. In addition to having a signal pattern unique to butystyrol at 62 to 2.0 ppm, it also has a signal pattern of 3.88 ppm.
Methoxy group in pm, 6.22 ppm and 7.55 p
In pm, the hydrogen of the double bond part of the α,β-unsaturated carboxyl group of the cinnamic acid derivative is AB type (J == 16 Hz)
, the hydrogen on the trisubstituted benzene ring splits into the ABM type, and 5
．． Since the IH of 951)011 disappeared upon addition of singlet/XDO, it was recognized that it had a phenolic hydroxyl group.

Substance A was subjected to alkaline hydrolysis, separated into acidic and neutral fractions, and analyzed. As a result, only ferulic acid was isolated from the acidic fraction (identified by comparison with the standard); The neutral fraction was silylated by a conventional method and quantitatively analyzed by gas chromatography, and the neutral fraction was found to be a mixture of stigmastanol and campestanol in a ratio of 9:■.

Furthermore, when Substance A is acetylated with pyridine-acetic acid, a monoacetal of colorless plate-like crystals with a melting point of 155-156°C is produced, and the magnetic resonance spectrum (CDCI) shows that phytosterol is present at 0.6-2.0 ppm. Methyl group and methylene group, signal based on acetyl group at 2.32 ppm, methyl of methoxy group appears in 3H, singlet at 3.84 ppm, 6.32 ppm and 7
．． 60 pp+a, α, β-unsaturated carboxyl group olefinic hydrogen is AB type, 7. At 07 ppm, hydrogen on the trisubstituted benzene ring is observed to be split into ABM type.

From the above results, substance A was identified as a 9:1 mixture of trans-ferryl stigmastanol and trans-ferryl campestanol.

Based on the melting point and other analysis results, trans-feruloyl stigmastanol is the same substance as dihydro-β-sitosterin ferulic acid ester isolated from corn germ oil by Yamamura et al. (Nippon Kagaku M Magazine, 7).
9 volumes, 1000 pages, 1958).

Next, the present invention will be explained in further detail by showing test examples.

(Test 1) This test was conducted to compare the yields of active substances from whole grains, coix seeds, bran, and outer shells of adlay.

Whole grains of adlay were crushed in a conventional manner, threshed, polished, and divided into husk, bran, and kernel. If the whole grain is 100, the hull is 33, and the bran! 5 and 52 (weight ratio).

These 4m were used as starting materials, and 3 times each of n-hexane and ethyl acetate were added to each, and the mixture was heated at 15 to 20°C for 50 minutes.
The mixture was extracted by stirring for a period of time, and the solvent other than the extraction solvent was removed to obtain an oil portion. The weight ratio of each oil portion to the starting material was calculated and the results are shown in Table 1.

From the results shown in Table 1, it was found that the effective substance was contained in bran.

(Test 2) This test was conducted to recover the active ingredient by silica gel chromatography, that is, to determine the fraction containing the active ingredient.

1) Preparation of sample By the same method as in Example 1, an oil and fat fraction of 1,250.9 g was obtained from 5 kg of bran using ethyl acetate and ethanol.

Of this, 300 g was subjected to column chromatography using 5K9 silica gel, eluting first with n-hexane, then with a mixture of n-hexane and ethyl acetate, and gradually increasing the amount of ethyl acetate. ethyl 1
00: I elution fraction (F-1 fraction) 4.352
77, similarly 20:l elution fraction (F-1111 min)
283g and ethyl acetate elution fraction (F-Seal fraction) 8.
756 g was obtained. The F-1 fraction was further subjected to column chromatography using 25011 silica gel, and n-
It was purified by elution with a hexane-ethyl acetate (too:1) mixture, and the F-1 fraction was subjected to 500 g alumina column chromatography, and purified with an n-hexane-ethyl acetate (20:1) mixture. By elution, F
-1-111min 1281rLII, F-1-2mmin 1
．． 2849, F-1-3m minute, F-! -4 [min 210 fractions and F-N-5 fraction 55 fractions were obtained, F-1 and F-1
The fractions were combined to obtain seven fractions.

2) Physiological activity test method Regarding the above 7 heel fractions, golden hamster (5
~8 weeks old), and each side was treated with 0.2 tn/l and 0.
．． Dissolve 5 m in 0.2 M soybean oil and send to 10 animals in each group.
They were orally administered forcibly for 3 weeks, during which time their sexual cycles and natural ovulation numbers were observed, and compared with a control group in which only 0.21 soybean oil was administered.

3) Results The number of natural ovulations in each group is shown in Table 2.

(Left below) Table 2: Oestrous cycle (for human-related observations, including control and treatment groups)
In all cases, no disturbance in the estrous cycle was observed, lasting an average of 4 days. F
It has become clear that the -1 fraction and the F-1-4 fraction have an ovulation-inducing effect. The preparation according to the invention is P, -1-4
This fraction was completely different from the FI fraction.

(Test 3) This test was conducted to determine the effective dosage of the stanolferulic acid derivative, which is the active ingredient of the ovulation-inducing agent of the present invention.

1) Preparation of Sample A stanol ferulic acid derivative (a 9:1 mixture of ferulic acid derivatives of trans-stigmastanol and trans-cambestanol al!J) was prepared by the same method as in Example 1.

2) The same method as the test method (Test 2) was used. However, the dosage is 1 per day.
The times were 0.1ffLg, 0.2glue, and 1.0m17.

3) Test results 3-1) Sexual cycle Both groups showed a regular 4-day cycle.

3-2) Natural ovulation number Note: * indicates a significant difference at 1%.

A significant difference was observed in the 0.1 d, 0.2 mg administration group compared to the control group at P(0.01).

The average weight of the hamsters used in this test was 15
011, the effective dose for an adult is 40 to 80 ml once a day if the weight of the revenue is converted to 60 to 9.

Example 1 50 kg of adlays were milled and milled using a conventional method, and about 6.
I got 9 stars for 5. Of these, ethyl acetate of 5 to 9 was added to 5 to 9 and extracted while stirring at 20° C. for 5 hours. This operation was repeated three times and each fraction was collected to obtain an ethyl acetate extracted fraction. 10 kg of ethanol was added to the extraction residues 4 and 9, and the mixture was extracted with stirring at 20° C. for 5 hours. This operation was repeated twice, and each fraction was collected and filtered to remove insoluble materials. Ethanol was distilled off from the ethanol-soluble fraction by a conventional method, and 120 g of the obtained ethanol fraction was added with 36
0.9 of ethyl acetate was added and extracted by stirring at 20° C. for 5 hours to obtain an ethyl acetate soluble fraction.

Ethyl acetate was distilled off from the mixture of the ethyl acetate fraction and the ethyl acetate soluble fraction by a conventional method to obtain a fat and oil fraction of 1.310 ji.

Of these, 50ON was subjected to column chromatography using 8 kg of silica gel and eluted with a mixture of n-hexane and ethyl acetate (20:1) to obtain 450 g of silica gel. This fraction was subjected to alumina column chromatography (
800g) and n-hexane-ethyl acetate (20:
1) The mixture was eluted and about 540 fractions were obtained which were subjected to ultraviolet spotting using a fraction collector. Both fractions were subjected to high-speed jet chromatography using a reversed-phase column using Rp-18, eluted with a mixture of ethyl acetate and methanol (5:3), and a field spot with an ultraviolet spot was detected. Obtained 440#. This fraction was subjected to column chromatography using 50 g of silica gel, and n-
Elution was performed with a hexane-ethyl acetate (20:1) mixture to obtain about 330 fractions with ultraviolet spots. This fraction was tested using the same method as (Test 2) and found that no
8-induced effect was observed.

Example 2 3 to 9 parts of bran prepared from adlay, 3 to 9 parts of ethyl acetate
and extracted with stirring at 17'C for 8 hours.
This operation was repeated four times, and each fraction was collected to obtain an ethyl acetate extraction fraction. 8 ethanol for 2.2 kg of extraction residue
9 was added to the solution, extracted with stirring at 17° C. for 8 hours, and filtered to remove insoluble matter. Ethanol was distilled off from the ethanol-soluble fraction by a conventional method to obtain ethanol fraction 6.
5 g was obtained, 260 g of ethyl acetate was added thereto, and the mixture was heated at 17°C.
The mixture was stirred and extracted for 8 hours to obtain an ethyl acetate soluble fraction.

Ethyl acetate was distilled off from the mixture of the ethyl acetate extracted fraction and the ethyl acetate soluble fraction to obtain 790 g of fat and oil fraction.

Of this, 300 g was subjected to column chromatography using 5:9 silica gel to obtain 269 g of a fraction eluted with a mixture of n-hexane and ethyl acetate (30:l). This fraction was subjected to alumina column chromatography (50
n-hexane-ethyl acetate (2 (Ml
) Fraction 330 eluted with the mixture and having an ultraviolet spot
ml was obtained. This fraction was subjected to high performance liquid chromatography using a reverse phase column using Rp-18,
Elution was performed with a mixture of ethyl acetate and methanol (5:3) to obtain about 248 fractions having ultraviolet spots. This fraction was subjected to column chromatography using 3017 silica gel, and n-hexane-ethyl acetate (30:
1) Fractional 190d with an ultraviolet spot was obtained by elution with a mixed solution. As a result of testing this fraction using the same method as (Test 2), it was found to have an ovulation-inducing effect.

Example 3 Using 40 g of the fraction having an ovulation-inducing effect obtained by repeating Example 1 and commercially available medium chain fatty acid triglyceride 15011, 1,000 soft capsules were prepared in a conventional manner. One capsule contained 40mp of active ingredient.

〔Effect of the invention〕

The effects achieved by the present invention are as follows.

l) Ovulation can be induced without causing abnormalities in the sexual cycle.

2) Ovulation can be induced without the various disorders and side effects caused by abnormalities in the sexual cycle.

3) A substance having an ovulation-inducing effect can be produced with high yield.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 show the infrared spectrum and nuclear magnetic resonance spectrum of the ovulation-inducing shave of the present invention, respectively. Procedural amendment (formality) July 11, 1985 Michibu Uga, Commissioner of the Patent Office l Case description 1985 Patent Application No. 41932 2 Title of the invention Ovulation-inducing agent and its manufacturing method 3 Case made by the person making the amendment Relationship with Patent applicant Morinaga Milk Industry Co., Ltd. 5-33-1 Shiba, Minato-ku, Tokyo (612) Representative Mitsugu Monmae 4 Agent 5 Date of amendment order June 10, 1985 (Showa 60)
(Shipped on June 25, 2017) 6. Subject of amendment Description 7. Contents of amendment

Claims (6)

[Claims] (1) General formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the above general formula, R represents a methyl group or an ethyl group.) It is characterized by having a stanolferulic acid derivative represented by the following as an active ingredient. An ovulation-inducing agent. (2) The ovulation-inducing agent according to claim 1, wherein the active ingredient is a mixture of trans-ferulyl stigmastanol and trans-ferulyl campestanol. (3) 9 whose active ingredients are trans-ferulyl stigmastanol and trans-ferulyl campestanol
The ovulation-inducing agent according to claim 1 or 2, wherein the ovulation-inducing agent is a mixture of: :1 (by weight). (4) A method for producing an ovulation-inducing agent, which comprises adding ethyl acetate to coix seed bran, extracting an oil and fat fraction, and recovering an active ingredient from the oil and fat fraction. (5) Add ethanol to the ethyl acetate extract residue of Coix bran to extract, add ethyl acetate to the ethanol extracted fraction, and extract the resulting ethyl acetate extracted fraction with the first ethyl acetate extracted fraction. 5. A method for producing an ovulation-inducing agent according to claim 4, which comprises mixing. (6) Claim 4, characterized in that the active ingredient is recovered by silica gel column chromatography with gradient elution of n-hexane-ethyl acetate.
5. A method for producing an ovulation-inducing agent according to any one of Items 1 and 5.