JPS63198627A - Improver for functional disorder caused by stress comprising cistanocides as active ingredient - Google Patents

Abstract

PURPOSE:To obtain the titled safe functional disorder improver effective for preventing and treating psychosomatic disease, alleviating or remedying disorder of various functions caused by stress, especially sexual function hypogonadism and amnesia, having low toxicity, containing a cistanocide as an active ingredient. CONSTITUTION:The titled improver containing a cistanocide shown by the formula [R1 is H, hydroxyl or methoxyl; R2 is alpha-L-rhamnopyranosyl-(1-3)-beta-D- glucopyranosyl or alpha-L-rhamnopyranosyl-( 1-3 )-beta-D-(2-0-acetyl)-glucopyranosyl] as an active ingredient. The compound shown by the formula is a drug, an extract especially from Cistanche salsa (parasitic plant of the family Orobanchaceae). The extracted compound shown by the formula or a fraction containing the said compound can be directly administered as it is or pharmaceu tically manufactured and administered.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to pharmaceuticals, particularly to an agent for improving functional disorders caused by stress, which contains cystunosides extracted from Physcomitrella sinensis as an active ingredient.

(Prior art) ``Niku Zhu Rong'' is the oldest herbal book in the People's Republic of China.
It is one of the herbal medicines that has been used since ancient times, as listed in the ``Shin-nong Bencao-jing'', and is used in Chinese herbal medicine prescriptions such as ``Jiyu-Ryo-gan'', ``Kanshaodan'', and ``Hush-Rong Junchogan'' as a supplement and tonic herbal medicine. used,
It has also been used alone or together with other herbal medicines to make medicinal sake, and even today it is considered a valuable straw material.

C15tanchesalsa'' (C15tanchesalsa) is a parasitic plant of the family Morayaceae, which grows in the People's Republic of China, the People's Republic of Mongolia, and the Siberian region.
C15tanche deserticola, a plant of the same genus, is originally derived from the dried whole plant of C.
,C.

Ma, “C15tanche a+++bigaa’
(Bge,) G, Beak and I″C15tanc
he 5inensis''G, Beak is also considered to be the source plant of the Physcomitrium ["Zhongwei Daeng Dictionary" and Ma Zunquan, "Inner Mongolia University Bulletin (Natural Science)", p. 69, 1977. Another parasitic plant of Bo
schniak1a rosgica”Fedtsch
, et Flerov) is called Wasan Niku Hose Ryo or Kusaburi Ryo, and this is sometimes used for the same purpose as Nik Hose Ryo.

Regarding the pharmacological and physiological effects of ``Niku Yakuryo'', there is a blood pressure lowering effect (Chinese Academy of Medical Sciences r 1956 Rinbun Report Conference Paper Abstracts 11J)
70, 1956, 1 and saliva secretion stimulating effect ("Nakahyo Research Bungoku Summaries", p. 259, 1975), and the presence of alkaloids and crystalline substances in the constituent components of Nikuhoseyo. have been reported, but the details are still unknown and have not been fully elucidated.

On the other hand, compounds chemically structurally close to the cistunosides according to the present invention include phenylpropanoid glycosides represented by the general formula. Known compounds belonging to the phenylpropanoid glycosides include actiosides (Rs, R, = hydroxy Examples include the following compounds, including R5, R6 and hydrogen atoms).

Ball, fJ Nishikusu- (Rs, R, = hydroxyl group, R5/hydrogen atom, R6 = β-D-glucopyranosyl group) 2'-acetic acid
Luid (R3, R4 = hydroxyl group, R5, acetyl group, R, = hydrogen atom) Sumanth 1B (Rs, R4, R5, R6 = hydrogen atom), Flucy'-'B (Rs, R4, hydroxyl Regarding the pharmacological and physiological effects of these phenylpropanoid glycosides, it has been reported that both ethinacoside and forcisoside B have antibacterial effects. It has been done (^, 5tall
etc. “He1v.

Chin, Acta, p. 238, 1950 and Endo et al., Proceedings of the 28th Annual Meeting of the Japanese Society of Herbal Pharmaceutical Sciences, p. 20, p. (West German Patent Application Publication No. 2609533), and it has also been reported that it has antitumor effects (Numata et al. ``No. 3'').
Collection of abstracts from the 3rd Annual General Meeting of the Kinki Branch of the Japanese Society of Pharmacological Pharmacology,” p. 76,
(1983).

These pharmacological and physiological effects possessed by the above-mentioned known phenylpropanoid glycosides, as well as the previously described pharmacological and physiological effects that have been reported regarding meat stimulation, such as blood pressure lowering effect and saliva secretion promoting effect, are゛The traditional efficacy of ``promotion'' is ``
It is thought that the nuance is different from "five labors, seven wounds." lIL, ``Go-ro-shichi-in'' is understood to refer to the alleviation and healing of various symptoms caused by functional decline of vital organs and mental fatigue caused by various types of stress. It is from.

In modern times, social institutions are becoming more complex and technological innovation is progressing extremely rapidly, and the reality is that people living under such environmental conditions are subject to and accumulate a great deal of stress without realizing it. be. In these modern times, anti-stress substances are extremely useful for leading a healthy social life.

The present inventors have focused on the above-mentioned traditional effects of Nikkutsuyou and have conducted research on Nikkutsuyou. In addition to various phenylpropanoid glycosides such as B, actioside, and ethinacoside, we have discovered that the following phenylpropanoid glycosides (cystanesides) are contained, and we report this and describe their isolation method. Kobayashi et al. Chew, Pharm, without patent application.
B1!11. J Vol. 32, pp. 3009 and 3880, 1984 and Japanese Patent Application No. 6O-29335), and further discovered that these cystanesides were effective as ameliorating agents for functional disorders caused by stress, and filed a related patent application. (Patent application 183953/1983).

cis ^ (In the above general formula II, RS: hydroxyl group,
R4: methoxyl group, R5: hydrogen atom, R6: β-D-glucopyranosyl group) cis B (In the above general formula II, R,, R,: methoxyl group, R: hydrogen atom, R6: β-D-glucopyranosyl (R3 in the above general formula II: hydroxyl group,
R4: methoxyl group; As a result of further research on the constituent components, it was found that nikuhōrō has the general formula (in the formula, R1 means a hydrogen atom, a hydroxyl group, or a methoxyl group, and R2 represents α-L-rhamnopyranosyl-(1
) 3) -β-toglucobyranosyl group or α-L-rhamnopyranosyl-(143)-β-D-(2-0-acetyl)-glucopyranosyl group) It turned out that it was.

Among these cystanesides, 2'G [R1: hydrogen atom, R2: α-L-rhamnopyranosyl-
(1>3)-β-D-glucopyranosyl group 1 and cis-noid H [R1: hydroxyl group, R2: α-L-rhamnopyranosyl-(l÷3)-β-D-(2-0-acetyl) A patent application was filed for the glucopyranosyl group (Japanese Patent Application No. 71320/1983).
).

As a result of preliminary tests, it was assumed that these cystansides G and H have an anti-stress effect, but this has not been fully elucidated.

(Objective of the Invention) The object of the present invention is to provide an anti-stress agent containing as an active ingredient a substance obtained from herbal medicines, particularly Physcomitrella sinensis, and thereby improve functional disorders caused by stress.

(Means and Effects for Achieving the Object) According to the present invention, the above object is achieved by containing as an active ingredient at least one of the cistunosides represented by the general formula (wherein R1 and R2 have the above-mentioned meanings). This is achieved by an agent for improving stress-induced dysfunction.

In addition to the above-mentioned cystanesides G and H, the active ingredients of the stress-induced dysfunction improving agent according to the present invention include, for example, cystaneside E (R1: methoxyl group, R2: α-rhamnopyranosyl(1+3)-β -D-glucopyranosyl group).

Although these active ingredients are preferably isolated and purified, they can also be used in both forms containing at least one of them.

(Dosage form and dosage) There is no particular restriction on the dosage form of the agent according to the present invention, that is, the agent for improving functional impairment caused by stress. Both components can be administered separately, or they can be formulated and administered. When formulating powders, fine granules,
It can be administered orally, such as granules, tablets, capsules, liquids, syrups, etc., or parenterally administered, such as injections, enteric preparations, etc.

The dosage depends on the type of active ingredient, dosage form, patient's age, weight, symptoms, etc., but in general, when administered orally to adults, the active ingredient compound is O.Q5-5 g/day. It is preferable to take the drug in 1-3 divided doses.

(Effects of the Invention) By administering the agent according to the present invention, various functional disorders caused by stress, especially sexual dysfunction and amnesia, can be alleviated or cured, and psychosomatic disorders can be prevented and treated. be able to.

Incidentally, the cistunosides used as the active ingredients of the agent according to the present invention have extremely low toxicity, and therefore the agent according to the present invention has excellent safety in use.

(Formulation examples, etc.) Next, production examples of active ingredients used in the agent according to the present invention,
The present invention will be explained in more detail with reference to pharmacological test examples and formulation examples.

111 YU (manufacturing example) 10 kg of meat soup root (market product from China) was cut into pieces, 36 liters of methanol was added, and the mixture was heated under reflux while stirring. This was repeated twice for 2 hours, and the resulting extracts were combined and concentrated under reduced pressure to obtain 4.5 kg of crude extract. This crude extract was suspended in 1.5 liters of water, washed twice with 3 liters of ethyl acetate, the aqueous layer was extracted twice with 3 liters of n-butanol, and the n-butanol extracts were combined. It was concentrated under reduced pressure to obtain 300 g of n-butanol soluble portion. Dissolve this in 5 liters of water and use Diaion HP-20.
The mixture was passed through a column filled with 2 liters of chlorine, washed with 20 liters of water, and eluted with 10 liters of methanol to obtain 75 g of diamond ion adsorption area. This diamond ion adsorption part was dissolved in water 500+Al, and polyamide C-200 was dissolved in 1
kg packed column, washed with 3 liters of water,
Elute with 5 liters of methanol to obtain crude cystane side (
40 g (hereinafter referred to as 1 fraction 1) was obtained. This fraction 1 was subjected to chromatography using a column packed with 5QOg of Wako Gel C-300, and the TLC gram as shown in the attached drawing [thin plate Nijirica Gel 60 F254
(manufactured by Merck & Co., Ltd.), developing solvent tanhol/water (6:4
: 1), color development: heating at 105°C after spraying with 20% sulfuric acid solution] using chloroform/methanol/water (6
:4:1) to obtain both fractions 1 and 2.

Fraction 1 was subjected to chromatography using a column packed with 200 g of Wakogel e-300, and using the above TLC gram as an indicator, chloroform/methanol/
The fraction was eluted with water (14:6:1), then subjected to chromatography using a column packed with 100 g of Sephadex 20, and fractionated with water/methanol (1:1) using the above TLC gram as an indicator. The product was purified by elution to obtain cisternside E (150 tg) and cisternside I (100 mg).

Further, the above fraction 2 was subjected to chromatography using a column packed with 300 g of Wako Gel C-300, and fractionated and eluted with chloroform/methanol/water (6:4:1) using the above TLC gram as an indicator. Next, it was subjected to chromatography using a column packed with 100 g of Sephadex LH-20, and using the above TLC gram as an indicator, it was purified by fractional elution with water/methanol (1':l) to obtain Cistanside G (12 hg). I got it.

The physicochemical properties of these cystafsides were as follows.

Cystane E Optical rotation (MeOH): IR spectrum (νS'a'X ''-') '345
0, 1620 and 1525 tOH uv spectra (λ, AX nm): 227 and 281 FD-MS spectra (1/Z): 476 (M”), 477 (M + 1) and 499 (M 10 Na) + Ito NMR Spectrum (methanol-44) δ pp+
s: 1.25 (3■, d, J = 6Hz, CH3 of rhamnose) 2,! 14 (2H, t, J ・
7) 1z, input r-CH2) 3.82 (
3H,s, 0CH3)4.50 (IN,
d, J = a) Iz, glucose 8-1) 5.14 (IH, br s, rhamnose ■
-1) 6.6 - 6.9 (3B, Ar-11)"C
-NMR chemical shift (methanol-d4) Near glycon 1; 131.6 27 113.9 3; 148.8 4; 145.9 5; 116.2 6, 122.4 α; 72.3 β, 36.7 Glucose 1; 104.2 2; 75.5 3; sa, a 4; 70.1 5; 77.7 6: 62.8 Rhamnose 1, 102.7 2, 71.9 3; 72.3 4; 74 .0 5: 70.3 6; 17.9 0CRs; 56.6 cis No. G Optical rotation (MeOH): 3420, 1616 and 1520 224, 279 and 285sh' H-NMR spectrum (methanol-44) δ P
P11 = 1.26 (3H, d, J = 6 Hz, C1(3) of rhamnose 2.84 (2H, t, J = IHz,
Ar-CHz) 4.31 (IH, d, J
= 8 Hz, H-1 of glucose) 5.18 (I) I, d, 6. of rhamnose.
72 (2H, d, J = 9 Hz, H-3,5 of aglycon) 7.09 (2H, d, J = 9 Hz, H-2,6 of aglycone) '3C-NMR chemical shift (methanol-d4) Ni Aglycone 1; 130.7 2, 130.8 3: 116.1 4, 156.7 5; 116.1 6; 130.8 α, 72.1 β, 36.3 Glucose 1; 104.1 2; 75 .5 3; 84.6 1 7 [1,1 5; 77.7 6; 62.7 Rhamnose 1: 102.7 2; 72j 3; 72j 4; 74.0 5; 70.2 6, 17.9 Elemental analysis (CzoH3oOtx・1/2 HzO):
Theory: C, 52, 74, H, 6, 86 Measurement: C
,52,67,H,6,72cis Noid H Optical rotation (Meoll): Samurai [α], -58.9° (c = 1.6) IR square)
k (1/l"'c+s-'): y+tLX 3430, 1740.1616 and 1532M@0H uv spectrum (λm□l): 221sh and 283' NMR spectrum (methanol-d4) δ pp ugly: 1.22 ( 3H,d, J = 6Hz, CH of rhamnose,) 1.96 <38.s, C0CH5)2.
66 (2H, t, J = 7Hz, Ar-
CH2) 4.41 (IH, d, J = 8
Hz, glucose 1) 5.15 (IO, br s, rhamnose H-1) 6.4 -6.8 (3H, Ar-H)'C-NMR
Chemical shift (methanol-d4) near glycon
1; 131.8 2: 116.2 3; 144.5 4; 145.9 5: 117.1 6: 121.2 α; 72.4 β, 36.3 Glucose t: iot, a 2; 74. 8 3; 83.1 4; 70.3 5: 77.9 6; 62.5 Rhamnose 1; 102.9 2; 71.6 3; 72.1 4: 73.7 5; 70.3 6: 17 .8 Co; 171.6 CH,, 20,9 Elemental analysis (C22H3201S・HzO): Theory:
C, 50, 57, H, 6, 56 Actual measurement: C, 50,
82 H
Soak TO-8 in v% ethanol aqueous solution twice for 2 hours.
0℃), the extracts were combined and concentrated under reduced pressure to obtain 4.2k crude extract.
I got g. This crude extract was suspended in 1.5 liters of water and washed twice with 3 liters of ethyl acetate. The aqueous layer was extracted twice with 3 liters of n-butanol and
The butanol layers were combined and concentrated under reduced pressure to obtain 285 g of lobetanol soluble portion. Dissolve this in 5 liters of water,
The column was passed through a column filled with Diaion HP-20, washed with 20 liters of water, and then eluted with 10 liters of ethanol to obtain 63 g of Diamond ion adsorption area. This diamond ion adsorption part was dissolved in 500 ml of water, passed through a column filled with 1 kg of polyamide C-200, washed with 3 liters of water, and then eluted with ethanol to obtain 35 g of crude cisternide. The following treatments were performed in the same manner as in Reference Example 1: cisternside E (180 μg), cisternside G (150 mg), and cisternside H (120 μg).
).

LULi (manufacturing example) 10 kg of meat soup root (market product from China) was cut into pieces, 36 liters of methanol was added thereto, and the mixture was heated under reflux while stirring. This was repeated twice for 2 hours, and the resulting extracts were combined and concentrated under reduced pressure to obtain 4.6 kg of crude extract. This crude extract was suspended in 1.5 liters of water and washed twice with 3 liters of ethyl acetate. The aqueous layer was separated, 4 liters of water was added thereto, it was passed through a column filled with 2 liters of Diaion IP-20, washed with 30 liters of water, eluted with 36 liters of methanol, and the Diaion adsorption section 82
I got g. Hereinafter, the same treatment as in Reference Example 1 was performed to obtain cisternide E <185 mgg), cisternide G (16
0 gag) and cystane side) (120 mg) were obtained.

Mth 141st Doctor”.

(1) Purpose In accordance with the method of Saito et al. ("The 16th Japanese and Chinese Medicine Symposium Lecture Abstracts", p. 66, 1983), the present invention is used to treat sexual behavior disorder and learning memory disorder in laboratory animals that occur after stress loading. Examine the effects of the active ingredients of the drug.

(2) Experimental Animals Select male mice of the IV-C8 strain (weight 2g - 32g) that are 9 weeks old or older and have normal sexual behavior, under conditions of temperature 23 ± 1' C and humidity 55 ± 5% for one week. Preliminary breeding was carried out.

In this case, "normal sexual behavior" refers to male mice kept in private cages, and female mice brought into estrus by daily subcutaneous administration of lOμg/kg of estradiol.
If you live with 0 animals for 10 minutes every day, aim to successfully complete intromissions at least 5 times a week.

3) Stress loading A male mouse, which is an experimental animal, was suspended in the air and fixed at a height such that the tip of its nose touched the water surface.This state was maintained for 30 minutes on the first day of the test, and from the second day onwards, the hanging time was increased. This was gradually extended, and on the final day of the test, the 15th, the subjects were kept suspended in the air for two hours to stress them.

Even when stress was applied every day over a 15-day test period, no weight loss was observed compared to the control non-stressed group, and there was also impaired motor coordination, decreased muscle tone, and decreased locomotor activity. Although no decrease in exploratory movement was observed, a decrease was observed in sexual behavior and learning and memory behavior.

(4) Experimental method and method for determining the effect of the test drug The stress load described above was carried out every day from the specified time (1:00 p.m.), and for the test group, the test drug was orally administered after the stress load, and the study group began at 9:00 a.m. the next morning. Memory) behavior is examined, and this is repeated for 15 days to examine the effects or effects of the test drug.

(Direct) Determination of sexual behavior A cage DO containing 10 female mice each to which estradiol was administered subcutaneously at a dose of IOμs/kg every day.
One male mouse, an experimental animal, was housed in a 40 x 20 cm cage for 10 minutes once a day, and the number of times each sexual behavior (licking, mounting, and introduction) was performed during that time and the time until the behavior occurred were recorded. It was measured. In this case, there were 10 animals per group, and analysis of variance was used to test for significant differences between groups.

(b) Judgment of learning (memory) behavior A rubber stopper (diameter 5c+s, height 5cm) was placed on the floor through which a current (DO36V, 0.1mA) was flowing, and a male mouse, which was an experimental animal, was allowed to use this for 5 minutes. A male mouse that had been trained not to get off the rubber stopper was placed on the rubber stopper, and the number of times it accidentally got down from the stopper within 5 minutes was measured. Analysis of variance was used to test for significant differences between groups.

(5) Results and Analysis (&) Regarding Sexual Behavior i) Results The results of investigating the decrease in sexual behavior due to stress load and the degree of recovery thereof by administration of each test drug were as shown in the table below.

ii) The sexual behavior of male mice follows the steps of licking, mounting, introduction, and ejaculation. Therefore,
The influence or effectiveness of the treatment can be determined by examining what changes occur in the sexual behavior of male mice treated with a female mouse in heat. That is, it can be determined that the more times each of the 10 animals in a group performs sexual behavior within a certain period of time, and the shorter the time until sexual behavior occurs, the more active the sexual behavior is.

As is clear from the table above, the sexual behavior of stressed male mice is generally reduced. When comparing the stressed test group and the non-stressed control group, a more pronounced difference is observed in the later stages of sexual behavior compared to licking, which is the early stage of sexual behavior. Regarding the number of times each sexual behavior occurs and the time required to initiate it, stress load reduced the number of times each individual mouse engaged in sexual behavior, and it was observed that there was a delay in the time required to initiate sexual behavior. .

Thus, by administering the test drug to male mice whose sexual behavior had decreased due to stress, a clear improvement in sexual behavior was observed. This indicates that the test drug has the effect of effectively improving the decline in sexual function caused by stress.

(b) Regarding learning (memory) behavior 1) Results The test results are shown in Figures 2 and 3. It was on the street.

li) Analysis The average number of times during the 15-day test period of accidentally falling off the top of the rubber stopper during the 5-minute test period was higher in the stressed test group than in the non-stressed control group. It can be seen that there is a significant decrease in learning behavior due to stress load.

By administering the test drug to male mice whose learning behavior had decreased in this way due to stress, there was a clear tendency to reduce the number of times they accidentally fell off the rubber stopper.

These facts indicate that the test drug has the effect of improving memory ability decline caused by stress.

Drug mbum (acute toxicity test) ddY mice (body weight 25-30g) were kept at a temperature of 23±1°C.
, preliminarily reared for one week under conditions of ad libitum feeding and water supply in a room with a humidity of 55 ± 5x, selecting individuals in good health.
Groups of 5 animals were given 500 mg/kg each of the test drugs (cystanside E and fraction I) after fasting for 18 hours.
Do not administer orally at a dose of

For all test drugs, a decrease in body temperature was observed 30 minutes after administration, but recovery occurred approximately 3 hours later, and during this period, signs of sedation were observed, albeit weakly. There were no cases of death. Thereafter, body temperature measurements and general symptoms were observed for one week, but no significant differences were found between the animals and the control group, and autopsy revealed no abnormalities.

This shows that the toxicity of the test drug, which is the active ingredient of the agent according to the present invention, is extremely low, and therefore suggests that the agent according to the present invention has excellent safety in use.

WLMJLi (granules) 20g of fraction 1 obtained in Reference Example 1 was collected, and 95% of starch was added to it.
g and 80 g of lactose were added thereto and mixed uniformly, and then wet granulated in a conventional manner using a hydroxypropyl (5 g) ethanol solution, dried, and sized to obtain granules.

1 Tashoyu (capsules) Cisternside E Ig with lactose tag and starch q, 5g
Add and mix uniformly, then add hydroxypropyl (
0.5 g) Wet granulation was carried out in a conventional manner using an ethanol solution, dried, sized, and 200 mg was filled per 1 capsule to obtain capsules.

1 milk 1 unit (granules) 9 g of starch, 9 g of lactose, and 0.5 g of hydroxypropyl cellulose are added to Cistanside G Ig, mixed uniformly, and then wet granulated by a conventional method, dried, and sized. Granules were obtained.

Reidu Salmon 1 (Liquid for internal use) Cistanside HIg, 10 g of glucose, and an appropriate amount of fragrance were dissolved in a 14% ethanol aqueous solution to make a total volume of 200 ml to obtain a liquid for internal use.

[Brief explanation of the drawing]

Figure 1 shows Cisternsides E and G extracted from Nikkusao.
Figures 2 and 3 are graphs showing the reducing effect of stress on the learning and memory behavior of mice and the improving effect of cystaneside E and fraction I, respectively, among the agents according to the present invention. be. Patent applicant: Yomeishu Seisaku Co., Ltd. Figure 3 A1t=h C1 Figure 1 1: 2゛3゜Figure 2 BC if≠ B-nistress negative

Claims (1)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R_1 means a hydrogen atom, hydroxyl group or methoxyl group, and R_2 is α-L-rhamnopyranosyl-
(1→3)-β-D-glucopyranosyl group or α-L-
Rhamnopyranosyl-(1→3)-β-D-(2-0-acetyl)-glucopyranosyl group) A functional impairment improving agent.