JPS6072821A - Production of antigonadotropic hormone substance - Google Patents

Abstract

PURPOSE:To obtain an antigonadotropic hormone substance useful for treating menopausal disorder, and tumors promoting the multiplication by hormones, etc., by extracting a bulbil of a plant of the genus Humulus of the family Moraceae with an alkaline solution. CONSTITUTION:A bulbil of a plant of the genus Humulus of the family Moraceae, e.g. Humulus lupulus (hop) or Humulus scandens Merrill, is extracted with an alkaline solution to give an antigonadotropic hormone substance. 1-5% aqueous ammonia, aqueous solution of sodium hydrogencarbonate, 0.001-0.01 N aqueous solution of NaOH, boric acid buffer solution of 8-11pH and tris buffer solution are preferred for the alkaline solution. The extraction temperature is preferably 3-20 deg.C. The above-mentioned substance is capable of antagonizing the biological action of gonadotropic hormone and reducing the sensitivity of the ovary to the gonadotropic hormone. Menopausal disorder, etc. can be treated by the antagonism of this substance to the gonadotropic hormone.

Description

[Detailed description of the invention] The present invention relates to a method for producing an anti-gonadotropin substance.

Gonadotropin is produced in the anterior pituitary gland, enters the bloodstream, reaches target organs such as the ovaries and testes, and has the function of promoting the production and secretion of steroid hormones. In recent years, gonadotropin secretion promoting factors or inhibitors have been discovered in the hypothalamus, and gonadotrophin inhibitors have been discovered in the ovaries and testis as factors that regulate the secretion of gonadotropins.

However, regarding anti-hormone-like substances derived from plants, for example, there are reports that hops have estrogen-like activity and reports denying their existence, but the existence of anti-gonadotropin substances such as those mentioned above is still unknown. unknown.

The present inventors conducted repeated research on plants of the genus Humulus of the Mulberry family, represented by hops, and discovered that a substance with anti-gonadotropin action was present in the flower cones of the plant. It was completed.

The present invention is a method for producing an anti-gonadotropin substance, which is characterized by extracting the cone of a plant of the genus Humulus in the family Mulberry family using an alkaline solution.

Hop (Humulus 1) is a plant of the genus Humulus of the Mulberry family.
upulu8 L, ) is typical, and others include Canum/La (H, japonicus 5ieb,
et Zucc, ), Karahanasou (H, 1, L,
var, cordifolius Maxim,
)and so on.

Prior to extracting the cones of the above plants with an alkaline solution,
It is desirable to crush the cones in order to improve extraction efficiency, and further crush the cones in advance with acetone.

It is desirable to perform pretreatment to remove fats and oils and pigments using an organic solvent such as alcohol. This pretreatment is usually 0 to
10-120 at a temperature of 30°C, preferably 0-10'C
The heating time is preferably 20 to 60 minutes.

Next, examples of the alkaline solution used in the present invention include ammonia aqueous solution, hydroxide aqueous solution, hydrogen carbonate aqueous solution, and boric acid buffer.

Examples include Tris buffer. In this case, ammonia aqueous solution and sodium bicarbonate aqueous solution are 1 to 5% hydroxide)
The IJium aqueous solution is preferably used at a concentration of 0.001 to 0.01N, and the borate buffer and Tris buffer preferably have a pH in the range of 8 to 11.

However, the concentration, pn, amount used, etc. of the alkaline solution depend on the specific operating conditions during extraction, such as the extraction temperature.

It should be determined appropriately, taking into account the pulverized particle size of the raw material, stirring conditions, etc.

The extraction of cone in the present invention is carried out at 0 to 30°C, preferably 3 to 20°C, with stirring for at least 1 hour, usually 3 to 5 hours. The desired antihormonal substance can be separated and purified from the extract by conventional methods. An example is shown below. The pH of the p solution obtained by filtering the extract is adjusted to 3 to 5 using hydrochloric acid or the like, and the resulting precipitate is collected by centrifugation. This precipitate was dissolved in distilled water, and an alkaline solution was added to adjust the pH to 8-9.
Dialyze and equilibrate with /buffer (pus~9). This solution was then adsorbed onto a DEAE-cellulose column,
0.05M containing 1M or 2M salt
Elution with C7 buffer yielded two halves, F1 and F2;
Concentrate both parts separately. Next, fraction F1 is 0.05
M)') Pass through a Sephadex G-75 or Sephadex G-1000 column equilibrated with a SuHc1 buffer and collect the first eluted peak (Fla). This was adsorbed onto DEAR-Sephadex A-25 or DEAE-Cellulofine AH equilibrated with 0.1 M) soot (pH 7 to 8), and 0.5 M or IM salt was added. Fraction F1. obtained by elution with 0.01 M Tris-HC1 buffer (pH 6-7) containing F1. Each of a-i and F1a-1 is desalted by dialysis and then freeze-dried to obtain a powder of the desired antihormonal substance. Note that the antihormone substance can be obtained in the same manner by using fraction F2 instead of fraction P1.

There are two types of anti-gonadotropin substances obtained by the present invention: one in which the salt concentration in the buffer used for elution is 0.5M (Fla-I substance) and one in which it is IM (F1a-[substance). The molecular weight of these substances is approximately 20,000 for Fla-I substance by gel p-filtration method using Sephadex G-100, and 7,000 to 10 for F1a-[substance.
It is 000. Further, elemental analysis values of these substances are shown in Table 1.

4- Carbon (%) Hydrogen (sub)) Nitrogen (%) Ashbo%) pl
a-I substance 45.5 7.0 5.4 3.2Fl
a-]1 substance 40.4 6.1 4.8 5.6 Furthermore, the ultraviolet absorption spectra of these substances are shown in FIG. 1, and the infrared absorption spectra of these substances are shown in FIGS. 2 and 3. Table 2 shows the results of chemical analysis of each substance.

Neutral sugar D-glucose orcinol-H, 80, method
22.4 10.4 Pentose D-Xylose Orcinol #-Fe3+-12,73,6110j Method The anti-gonadotropin substance obtained by the present invention is pretreated with PMS serum gonadotropin (PMS). It has the biological effect of suppressing the increase in ovarian weight in young rats. Normally, ovaries increase in weight due to gonadotropic hormones such as PMS. From this, the mechanism of biological action of this substance is +1.
) Antagonizing the biological effects of gonadotropin (2) Decreasing the sensitivity of the ovaries to gonadotropin.

Clinically, there are many diseases and symptoms that are thought to be caused by hormonal imbalance, but appropriate treatments for these have not been established. Conventionally, when anti-gonadotropin therapy is performed, large doses of steroid hormones are administered to weaken the pituitary gland. There are only methods that indirectly utilize negative feedback, such as suppressing gonadotropin secretion from the anterior lobe.

Menopausal disorders, which many women suffer from, can be treated by this substance's antagonism with gonadotropin, and it is also thought to be effective in treating tumors whose growth is promoted by hormones.

Next, the present invention will be explained in detail with reference to examples.

Example 1 10 crushed hop cones degreased with acetone in a container of 21
Pour 1,500 ml of 0.2 M* uric acid buffer (pH 10) containing 1,000 ml of ascorbic acid and 1,500 ml of ascorbic acid (1,000 ml of polychlor-ATJ (manufactured in the United States, sold by Gokyo Sangyo), seal it tightly, and incubate at 4°C The extract was filtered with suction and the resulting solution was adjusted to pH 3 to 5. The resulting precipitate was collected by centrifugation and dissolved in 0.05M Tris binding buffer H9). M ) !jsu buffer (pH 8-9
) and eluted with the same buffer containing 1M sodium chloride (fraction Fl).
. Sephadex G-
The first peak eluted through the 75 column (Fla)
were collected (see Figure 4). This was adsorbed onto D:EAE-Sephadex A-25 equilibrated with 0.1 M) Lys buffer (pH 7-8), and the same buffer containing 0.5 M or IM sodium chloride (pH 6-7) was adsorbed. ), the F1a-1 fraction and Fla-
An f1 fraction was obtained (see Figure 5). After dialysis and desalination of both obtained fractions, freeze-drying was performed to obtain each powdered 501R.
I got a 9.

Fraction F1 and purified powder substance F1a-■, F1. For each of a4, 22 treated with PM825IU in advance
Five day-old female rats were administered VC by subcutaneous injection of a prescribed amount twice a day for 3 days, and changes in ovarian weight were examined. As a result, when F1 was administered at 20 μ/mouse, the weight of the ovaries decreased by 25.7% compared to the control.

Further, FIG. 6 shows the results when F1a-1 and F1a, -fl were administered at a rate of 1 Da/mouse or 41 V/mouse. As is clear from the figure, the reduction rate of ovarian weight when 49/mouse was administered was 42.0% for F1a-1, and 42.0% for F1a-1;
It was found that the weight increase was 33.1% at 1 a-1, and the weight increase was suppressed statistically significantly.

Example 2 In a 2 Il container, 100 g of crushed hop cones defatted with ethanol, 10 g of ascorbic acid, [Polyclar-AT-
150 Qm of an aqueous solution containing 12 og of o, o o I N caustic soda 8 was added, the mixture was tightly stoppered and stirred at room temperature for 1 hour, and the extract was prepared in the same manner as in Example 1. After purification by column, 0.1 M) Lys buffer (pH 7~
It was adsorbed onto DEAE-Cellulofine AH equilibrated in step 8). Next, the eluted fraction was desalted with the same buffer containing 1M sodium chloride and lyophilized to obtain about 50 m9 of powder. When this substance was tested by the rat ovary weight method in the same manner as in Example 1, injections of 4 to 5 In9 per animal showed a significant weight increase suppressive effect compared to the control value.

[Brief explanation of the drawing]

Figure 1 shows Fla-) substances obtained according to the present invention and F
Ultraviolet absorption spectrum of la-fJ material, Figure 2 is Fl
a-) Infrared absorption spectrum of the substance, Figure 3 is F1a-
[This is the infrared absorption spectrum of the substance. Figure 4 shows Sephadex G-
The elution pattern when gel filtration was performed using a 750 column, and FIG. 5 is a chromatogram when a DEAE-Sephadex A-25 column was used. In addition, Figure 6 shows the F1a-4 substance or FIA patent applicant Sapporo Breweries Ltd. 11-2r Figure 1 Figure 6 Procedural amendment (voluntary) January 1980 It Japan Patent Office Commissioner Kazuo Wakasugi 1. Indication of the case Patent application No. 58-179285 2 Title of the invention Process for producing anti-gonadotropin substances 5 Person making the amendment Relationship to the case Patent applicant (219) Sapporo Breweries Ltd. 4, Agent Address: 104 Chuo-ku, Tokyo Kyobashi 1-1-10 & Detailed explanation of the invention of the specification subject to amendment & Contents of amendment Add [(Pharmacia Co., Ltd. W) J after G-100J. (2) [7ooo~i oo on page 5, second line from the bottom]
o. It is. ] should be corrected as follows. “7ooo~1 anon, Sephacryl S-2
In the gel filtration method using 00 Super Fine (manufactured by Pharmacia), the F1a-1 substance is approximately aooooo, IFl
The m-■ substance is 70,000 to 75,000. ” (or more) 2-

Claims (1)

[Scope of Claims] 1. A method for producing an anti-gonadotropin substance, which comprises extracting the cone of a medicinal plant of the genus Humulus using an alkaline solution. 2. The production method according to claim 1, wherein the medicinal Humulus plant is a hop. 3. The alkaline solution is an ammonia aqueous solution, a sodium hydroxide aqueous solution, or a sodium hydrogen carbonate aqueous solution. The method according to claim 1, wherein the method is one selected from borate buffer and Tris buffer.