JPH0623110B2 - Agent for improving dysfunction caused by stress containing cystanoside as an active ingredient - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of use) The present invention relates to a medicine, particularly to a dysfunction-improving agent due to stress, which contains cystanosides extracted from bean sprouts as an active ingredient.

(Prior art) As mentioned in "Shinno Honzo Sutra," which is the oldest book in the People's Republic of China, Nikufu Ryo is one of the herbal medicines that have been used since ancient times. It is used as a herbal medicine in Kampo prescriptions such as Niku-Yo-gan, Kaotan-tan, Kayo-Jun-en-maru, etc., and has been used alone or together with other herbal medicines in medicinal liquor. It is said that.

Meat 蓉 is a parasitic plant of the family Moray eel and originally refers to dried whole grass of "Cistanche salsa" (CAMey.) G.Beck, which is produced in the People's Republic of China, Mongolia and Siberia. However, its cognate plant "Cistanche deserticola" YCMa, "Cistanc
he ambigua "(Bge.) G. Beck and" Cistanche sinensis "GB
eck is also considered to be the source plant of the meat roe
And Izumi Magi, Inner Mongolia University Academic Journal (Natural Science), page 69, 1977.
Year]. On the other hand, other parasitic plants of the family Nematode, "Boschniakia rossica" Fedtsch.et Flerov, are called Japanese meat roe or grass roe, which serve the same purpose as meat roe. Sometimes.

Regarding the pharmacological and physiological actions of 蜫 艉, blood pressure lowering action (Chinese Medical Science, "1956 Thesis Briefing Paper Summary II", page 70,
1956] and salivary secretion-promoting action ("Summary of anther research literature")
(P. 259, 1975) is known, and the existence of alkaloids and crystalline substances has been reported for the constituents of flesh carp roe, but the details are still unknown, and they have been fully elucidated. It has not been done.

On the other hand, as the compound structurally closely related to the cystanosides according to the present invention, a compound represented by the general formula: There are phenylpropanoid glycosides shown in. Known compounds belonging to the phenylpropanoid glycosides include acteoside (R ₃ , R ₄ = For example, the following compounds include a hydroxyl group, R ₅ and R ₆ = hydrogen atom).

Ethinacoside (R ₃ , R ₄ = hydroxyl group, R ₅ = hydrogen atom group, R ₆ = β-D-glucopyranosyl group) 2′-acetylacteoside (R ₃ , R ₄ = hydroxyl group, R ₅ = Acetyl group, R ₆ = hydrogen atom), Osmans side B (R ₃ , R ₄ , R ₅ , R ₆ = hydrogen atom), Forcisoside B (R ₃ , R ₄ = hydroxyl group, R ₅ = hydrogen) Atom, R ₆ = β-D-apiofuranosyl group) Regarding the pharmacological or physiological action of these phenylpropanoid glycosides, it has been reported that both etinacoside and forcisoside B have antibacterial action ( A.Stoll
"Helv. Chim. Acta." P. 238, 1950 and Endo et al. "Abstracts of the 28th Annual Meeting of the Biomedical Society of Japan" p. 20, 1981), and acteoside has anti-Parkinsonism and β-blocking action. Has been reported (West German Patent Application Publication No. 2609533), and further has an antitumor effect (Numata et al., "33rd Annual Meeting of the Kinki Branch of the Japanese Society of Biopharmacy" Pp. 76, 1983).

The above-mentioned known phenylpropanoid glycosides have these pharmacological or physiological actions and the above-mentioned pharmacological or physiological actions that have been previously reported with respect to meat syrup, which are hypotensive action and salivary secretion promoting action. It is thought that the traditional effect of Kaoru, that is, "five to seven wounds", has a different nuance. It is understood that "five to seven wounds" means to alleviate and eventually cure various symptoms caused by functional depression of the living organs and mental fatigue caused by various stresses. Because.

In the present age, the social mechanism is complicated and technological innovation is progressing extremely rapidly. Therefore, it is the actual situation that a great deal of stress is accumulated and accumulated unknowingly to people living under such environmental conditions. is there. In such modern times, anti-stress substances are extremely useful for healthy social life.

The inventors of the present invention have focused on the above-mentioned traditional effects of flesh rooster, and have conducted repeated research on flesh rooster from the past. As a result, 2'-acetylacteoside and osmansside were found in flesh roe. In addition to various phenylpropanoid glycosides such as B, acteoside, etinacoside, etc., it was found that the following phenylpropanoid glycosides (cystanosides) were contained, and it was reported and the isolation method thereof was patented. No application (Kobayashi et al. "Chem.Pharm.Bull." Volume 32,
Nos. 3009 and 3880, 1984, and Japanese Patent Application No. 60-29335), and further found that these cystanosides are effective as agents for improving functional dysfunction due to stress, and filed a related patent application (Japanese Patent Application No. 60- 1839533).

Shisutanosaido A (R in the general formula II of the _3: hydroxyls groups, R _4:
Methoxyl group, R ₅ : hydrogen atom, R ₆ : β-D-glucopyranosyl group cystanoside B (in the general formula II, R ₃ , R ₄ : methoxyl group, R ₅ :
Hydrogen atom, R ₆ : β-D-glucopyranosyl group) cystanoside C (in the above general formula II, R ₃ : hydroxyl group, R ₄ : methoxyl group, R ₅ , R ₆ : hydrogen atom) cystanoside D (the above general formula in II R _3, R _4: methoxyl groups, R _5, R _6: a hydrogen atom) the present inventors have made the continued further studies on the components of the meat慫蓉, the meat慫蓉formula (In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂
Is α-L-rhamnopyranosyl- (1 → 3) -D-glycopyranose-
It also proved that cystanosides represented by (meaning 4-yl group) are also contained.

As a result of earnest research, it was found that these cystanosides, ie, cystanoside F (R ₁ : hydroxyl group) and cystanoside I (R ₁ : hydrogen atom), have anti-stress action, and the present invention was completed. Came to

(Object of the invention) Therefore, an object of the present invention is to provide an anti-stress agent containing a crude drug, in particular, a substance obtained from flesh roe as an active ingredient, thereby improving functional impairment due to stress.

(Means and Actions for Achieving the Object) According to the present invention, the above-mentioned object is represented by the general formula It is achieved by a stress dysfunction-improving agent characterized by containing at least one cystanoside represented by the formula (wherein R ₁ and R ₂ have the above meanings) as an active ingredient.

Although these active ingredients are preferably isolated and purified, they can be used even in the state of a fraction containing at least one of them.

(Dosage Form and Dosage) There is no particular limitation on the dosage form of the stress dysfunction-improving agent according to the present invention, and therefore it contains the compound represented by the above general formula I or the compound extracted from the Fusarium cypress. The fraction can be administered as a single dose, or can be formulated and administered. Upon formulation, it may be administered orally such as powder, fine granules, granules, tablets, capsules, liquids, syrups, or parenterally administered agents such as injections and enteral injections. You can also

Although the dose depends on the type of active ingredient, dosage form, age, weight, symptoms of patient, etc., generally, when orally administered to an adult, 0.05-5 g / day as an active ingredient compound
It is preferable to take 3 divided doses.

(Effects of the Invention) Administration of the agent according to the present invention can reduce or cure various functional disorders caused by stress, especially sexual dysfunction and amnesia, and prevent or treat psychosomatic disorders. be able to.

The cystanosides used as an active ingredient of the agent of the present invention have extremely low toxicity, and therefore the agent of the present invention is excellent in safety in use.

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

Reference Example (Production Example) 10 kg of meat roe (marketed in China) was shredded, 36 liters of methanol was added, and the mixture was heated under reflux with stirring. This was repeated twice for 2 hours, and the obtained extracts were combined and concentrated under reduced pressure to obtain 4.5 kg of a crude extract. This crude extract was suspended in 1.5 liters of water, washed twice with 3 liters of ethyl acetate, and the aqueous layer was extracted twice with 3 liters of n-butanol. The n-butanol extract was combined and concentrated under reduced pressure. Then n
-300 g of butanol-soluble part was obtained. This was dissolved in 5 liters of water, passed through a column packed with 2 liters of Diaion HP-20, washed with 20 liters of water, and added with methanol 10
It was eluted with liter to obtain 75 g of a diaion adsorption part.
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 eluted with 5 liters of methanol to obtain 40 g of crude cystanoside (hereinafter referred to as "fraction 1"). Called). This Fraction 1 was subjected to chromatography on a column packed with 500 g of Wakogel C-300, and TLC grams [thin layer plate: silica gel 60 F ₂₅₄ (manufactured by Merck & Co.), developed tanol / Water (6: 4: 1), color development: 20%
After spraying the sulfuric acid solution and heating at 105 ° C] as an index, the product was repeatedly purified with chloroform / methanol / water (6: 4: 1) and then subjected to chromatography on a column packed with 100 g of Sephadex LH-20. Fractional elution with water / methanol (1: 1) using gram as an index gave cystanoside F (150 mg) and cystanoside I (120 mg).

The physicochemical properties of these cystanosides were as follows:

Cystanoside F Optical rotation (MeOH): [α] ²⁵ _D -83.5 ° (c = 0.9) IR spectrum : 3430,1698,1634,1606,1520 FD-MS spectrum (m / z): 511 ( M + Na) + 1 H-NMR spectrum (methanol _{-d 4) δppm: 1.11 (3H} , d, J = 6Hz, rhamnose CH ₃ ) 4.57 (1H, d, J = 8Hz, H-1 of glucose) 5.15 (1H, brs, = H-1 of rhamnose) 6.28 (1H, d, J = 16Hz, Ar-CH = C H-) 6,7-7.2 (3H, Ar-H) 7.60 (1H, d, J = 16Hz, Ar-C H = CH-) 13 C-NMR chemical shifts (methanol -d _4): caffeic acid 1; 127.8 2; 114.9 3; 149.6 4; 146.7 5; 116.6 6; 123.1 α; 168.3 β; 115.4 γ; 147.8 glucose 1α; 94.0 β; 98.1 2α; 71.2 β; 74.7 3α; 79.2 β; 81.6 4α; 70.3 β; 70.3 5α; 76.1 β; 77.4 6α; 62.5 β; 62.5 Rhamnose 1; 102.9 2; 72.2 3; 72.3 4; 73.9 5; 70.9 6; 18.4 Cystannoside I optical rotation (MeOH): ▲ [α] ²⁰ _D ▼ -82.2 ° (c = 1.5) ) IR spectrum : 3430,1698,1634,1606,1518 ¹ H-NMR spectrum (methanol-d ₄ ) δppm: 1.10 (3H, d, J = 6Hz, CH _{3 of} rhamnose) 4.60 (1H, d, J = 8Hz, of glucose H-1) 5.18 (1H, brs, H-1 of rhamnose) 6.36 (1H, d, J = 16Hz, Ar-CH = C H- ) 6.84 (2H, d, J = 9Hz, H of p-coumaric acid -3,5) 7.51 (2H, d, J = 9Hz, p- coumaric acid H-2,6) 7.70 (1H, d, J = 16Hz, Ar-C H = CH-) 13 C-NMR chemical shift (Methanol-d 4 ): p-coumaric acid 1; 127.2 2; 116.9 3; 131.3 4; 161.3 5; 131.3 6; 116.9 α; 168.3 β; 115.0 γ; 147.6 glucose 1α; 94.1 β; 98.2 2α; 71.2 β; 74.7 3α; 79.2 β; 81.7 4α; 70.4 β; 70.4 5α; 76.1 β; 77.4 6α; 62.4 β; 62.5 Rhamnose 1; 103.0 2; 72.1 3; 72.3 4; 73.8 5; 70.9 6; 18.4 Elemental analysis (C ₂₁ H _{28 O 12 · 1 / 2H 2} O): theory: C, 52.54; H, 6.02 Found: C, 52.39; H, 6.07 pharmacological test example 1 (1) of equal interest Saito method ( 16th oriental medicinal Shinpojiumu Abstracts "66, pp accordance 1983), with respect to sexual behavior disorders and learning and memory disorders experimental animals that occurs after stress, examined the effect of the active ingredient of the composition according to the invention have on.

(2) Experimental animals IV-CS male mice (body weight 28-32 g) of 9 weeks or older with normal sexual behavior were selected, and the temperature was kept at 2 for 1 week.
Preliminary breeding was carried out under the conditions of 3 ± 1 ° C. and humidity of 55 ± 5%.

In this case, “normal sexual behavior” means that when male mice bred in a single-chamber cage are allowed to live together with 10 female mice in an estrus state by daily subcutaneous administration of 10 μg / Kg of estradiol for 10 minutes every day, 1 It refers to successful intromissions at least five times a week.

(3) Stress load A male mouse, which is an experimental animal, is suspended in air and fixed at a height level position where the tip of the nose comes into contact with the water surface, and this state is maintained for 30 minutes on the first day of the test and suspended for the second and subsequent days. Was gradually extended and on the 15th day of the last day of the test, stress was applied by keeping it suspended for 2 hours.
Thus, even if daily stress was applied for the 15-day test period, no weight loss was observed as compared with the control stress-unloaded group, and there was impaired motor coordination, decreased muscle tone, and decreased locomotor activity. Also, there was no decrease in exploratory movement, but there was a decrease in sexual behavior and learning / memory behavior.

(4) Experimental method and method for determining effect of test drug The above stress is applied every day from the specified time (1:00 pm), and the test drug is orally administered after the stress for the test group, and learning is started from 9:00 the next morning ( (Memory) behavior is examined, and this is repeated for 15 days to examine the effect or effect of the test drug.

(a) Judgment of sexual behavior A cage (30 x 40 x) containing 10 female mice to which estradiol was subcutaneously administered at a dose of 10 µg / Kg every day.
One male mouse as an experimental animal within 20 cm) once a day
They were allowed to live together for 0 minutes, and during that period, the number of sexual actions such as licking, mounting and intromission, and the time until they took action were measured. In this case, each group consisted of 10 animals, and the analysis of variance was adopted for the significance test between groups.

(b) Judgment of learning (memory) behavior Rubber floor (diameter 5) on the floor where current (DC 36V, 0.1mA) is flowing.
cm, height 5 cm) is placed, and male mice, which are experimental animals, are trained for 5 minutes so that they will not descend from the rubber stopper. The male mouse that underwent this learning treatment was placed on the above rubber stopper, and the number of times of accidentally descending from the rubber stopper within 5 minutes was measured. In this case, the analysis of variance was adopted for the significant difference test between groups.

(5) Results and analysis (a) Sexual behavior i) Results The results of examining the reduction of sexual behavior due to stress loading and the degree of recovery by administration of each test drug were as shown in the table below.

ii) Analysis The sexual behavior of male mice follows licking, mounting, intromission, and ejaculation. Therefore, the influence or effect of the treatment can be determined by investigating what kind of change occurs in the sexual behavior of the male mouse which has been treated with the certain treatment when it cohabits with the female mouse in the estrus state. That is, it can be determined that sexual behavior is more active as the number of times each sexual behavior among 10 animals in a group is performed within a certain period of time and as the time until sexual behavior is shortened.

As is evident from the above table, the sexual behavior of stressed male mice is generally reduced. When comparing the stress-loaded test group with the non-stress-loaded control group, a significant difference is observed in the later sexual behavior than in the initial sexual behavior, licking. Regarding the number of sexual behaviors and the time required for the initiation of each sexual behavior, the stress load was found to decrease the number of sexual behaviors of individual mice and delay the time required to initiate sexual behavior.

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

(b) Learning (memory) behavior i) Results The test results were as shown in Figs.

ii) Analysis The average number of times in the test period of 15 days that accidentally got off the rubber stopper within the test time of 5 minutes was higher than that in the stress-unloaded control group in the stress-loaded test group. There is a significant increase, which indicates that stress load causes a significant decrease in learning behavior.

When the test drug was administered to male mice whose learning behavior was thus reduced by stress, the number of accidentally getting off the rubber plug tended to decrease obviously.

These facts indicate that the test drug has an effect of improving the decline in memory ability due to stress.

Pharmacological and pharmacological test example 2 (acute toxicity test) A ddY mouse (body weight 25-30 g) was heated at a temperature of 23 ± 1 ° C and a humidity of 5
Individuals in good condition were selected by preliminarily breeding in a room of 5 ± 5% under conditions of free feeding and free water for 1 week, and 5 animals per group were selected. Then, after fasting for 18 hours, the test drugs (cystanoside F and I) were orally administered at a dose of 500 mg / Kg each.

A decrease in body temperature was observed 30 minutes after the administration of any of the test drugs, but the temperature recovered within about 3 hours, during which weak sedation was observed. There were no deaths. After that, the body temperature was measured and the general symptoms were observed for one week, but no significant difference was observed between the control group and the autopsy, and no abnormality was observed by autopsy.

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

Formulation Example 1 (Granule) To 20 g of Fraction 1 (not yet fractionated into cystanoside F and I) obtained in the course of Reference Example, 95 g of starch and 80 g of lactose were added and mixed uniformly, then Hydroxypropyl cellulose (5 g) in ethanol was used for wet granulation by a conventional method, dried and sized to obtain granules.
This granule is packaged in 1 g of laminated paper. The dosage of this granule is 3 times a day, one for each packet.

Formulation Example 2 (Capsule) 10 g of lactose and 8.5 g of starch were added to 1 g of cystanoside F and uniformly mixed, and then wet-granulated with hydroxypropylcellulose (0.5 g) ethanol solution by a conventional method, dried and conditioned. Granules were filled in 200 mg per capsule to obtain capsules. A capsule was obtained. The dose of this capsule is 3 times a day for 2 capsules each.

Formulation Example 3 (liquid drug for internal use) 1 g of cystanoside F, 10 g of glucose, and an appropriate amount of fragrance were dissolved in a 14% aqueous ethanol solution to make a total amount of 200 ml to obtain an internal drug solution. The daily dose of this liquid is 2
Times, 10 ml each.

[Brief description of drawings]

Fig. 1 shows cystanosides F and I extracted from bean curd roe.
2 and 3 are graphs showing the lowering effect of stress on learning and memory behavior of mice and the improving effects of cystanoside F and fraction 1 among the agents according to the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kumiko Kobayashi 2132-37 Nakaminowa, Minowa-cho, Kamiina-gun, Nagano Inside the Central Research Laboratory, Yomeishu Brewing Co., Ltd. (56) References ・ Chemical & Pharmaceutical Bulletin, Vol. 7 (1985) ・ Pharmaceutical magazine, Vol. 106, No. 7, pp. 562-6 (1986)

Claims (1)

[Claims] 1. A general formula (In the formula, R ₁ represents a hydrogen atom or a hydroxyl group, and R ₂
Is α-L-rhamnopyranosyl- (1 → 3) -D-glucopyranose-
(Meaning 4-yl group), containing at least one cystanoside represented by the formula (1) as an active ingredient.