JP2003113107A - Antiarrhythmic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antiarrhythmic agent comprising an extract from Leonurus heterophyllus Sweet or β-sitostenone having antiarrhythmic actions, in which the β-sitostenone is readily obtained by separation from the Leonurus heterophyllus Sweet or can be produced from a readily available raw material by organic synthesis at a low cost in a relatively high yield. SOLUTION: This antiarrhythmic agent comprises the extract from the Leonurus heterophyllus Sweet, preferably an acetone extract or the β-sitostenone or its pharmaceutically acceptable salt as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an extract of Yakusou, or β-sitostenone (stigmast-4).
-En-3-one) as an active ingredient, and a method for producing an antiarrhythmic agent.

[0002]

2. Description of the Prior Art As antiarrhythmic agents, quinidine, procainamide, β-blocker and the like have been used for a long time, and further development of new drugs is desired. By the way, Yakusou is a Liliaceae plant, Leonurus.
Heterophyllus Sweet. (Labiatae) is the above-flower part of the flower stage. It grows naturally in the wilderness of the mountains, rice fields, grasslands, and around the Tanigawa River, and is widely distributed from Japan, the Korean Peninsula, and China to Southeast Asia. Since ancient times, it has been said that maternal herbs are highly effective in treating irregular menstruation, difficult birth, and menstrual cramps. Also,
It is already known that several tens of kinds of compounds such as alkaloids such as leonurine and stachydrin, steroids, vitamin A, oleic acid, and diterpenes are contained as its components. Recently, in 1997, the discovery of six new diterpenes was reported by Ihsan Calis of Turkey.

[0003]

An object of the present invention is to search plants for new active substances having antiarrhythmic activity.

[0004]

[Means for Solving the Problems] The present inventor has conducted a screening test on various solvent-extracted extracts of maternal herbs for antiarrhythmic action against digoxin-induced arrhythmia using antiarrhythmic drug disopyramide as a control drug. , It was found that this maternal herb has an antiarrhythmic effect. Further research on the extract found that the active form was β-sitostenone (stigmast-4-en-3-one). Therefore, the present invention provides an antiarrhythmic agent containing a botanical extract as an active ingredient. The present invention also provides an antiarrhythmic agent comprising β-sitostenone (stigmast-4-en-3-one) represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0005]

[Chemical 1]

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION β-Sitostenone can be isolated from benign herbs by the following steps: (a) Extract benign herbs with an aqueous acetone solution, concentrate and freeze-dry to obtain an acetone extract; ) The above-mentioned acetone-extracted extract is subjected to a liquid separation operation using ether and water to obtain an ether fraction extract; (c) The ether fraction extract is subjected to chromatography to isolate β-sitostenone. Alternatively, β-sitostenone can be synthesized by the following steps: (A) A raw material containing stigmast-5-en-3-ol is oxidized with pyridinium chlorochromate to be β-sitostenone, stigmast-5. Ene-3-one and stigmast-4-en-3,6-dione are obtained; (B) stigmast-5-en-3-one by adding p-toluenesulfonic acid in methylene chloride The double bond is isomerized and converted to β-sitostenone.

The maternal herb extract is preferably an maternal herb acetone extract (acetone extract), and particularly preferably an ether fraction extract of the acetone extract. The acetone solution used in the step (a) obtained by isolating β-sitostenone from the maternal herb and used to obtain the above-mentioned medicinal herb acetone extract is preferably 50 to 90% by weight, preferably 60 to 80% by weight. %, Especially about 70%
Acetone in water is used. The ether extract can be prepared by using a separating funnel and using ether and water. The ether fraction is concentrated and freeze-dried to obtain an ether fraction extract. Chromatography of the ether fraction extract is performed by silica gel column chromatography, high performance liquid chromatography (HPLC) or the like.

Β-sitostenone is added to the above-mentioned step (A) to
In the method of synthesis by (B), β-sitosterol (stigmast-5), which is relatively inexpensive and easily available, is used.
A mixture of -en-3-ol) and campesterol (ergost-5-en-3-ol) can be used as starting material. Β-sitosterol, which may include campesterol, is preferably methylene chloride (CH ₂ C
Oxidation with pyridinium chlorochromate in l ₂ ) to give β
-Sitostenone, stigmast-5-en-3-one and stigmast-4-en-3,6-dione are obtained. Oxidation can usually be carried out at room temperature. The reaction time is usually about 1 to 6 hours. Next obtained Stigmast-5
En-3-one is converted to β-sitostenone by isomerizing its double bond by adding p-toluenesulfonic acid in methylene chloride. Isomerization can usually be carried out at room temperature. The reaction time is usually about 10 minutes to 1 hour.

Examples of pharmaceutically acceptable salts of β-sitostenone include inorganic acid salts such as hydrochlorides and sulfates, and organic acid salts such as formic acid, citric acid, acetic acid, fumaric acid and maleic acid.

[0010] The botanical herb extract, β-sitostenone or a pharmaceutically acceptable salt thereof, which is an active ingredient of the antiarrhythmic agent of the present invention, is combined with a pharmaceutically acceptable carrier according to a conventional method to prepare pharmaceutical compositions of various dosage forms. It can be a thing. Further, the administration form is not particularly limited and may be appropriately selected according to the purpose of treatment, and may be, for example, an oral preparation, an injection, a suppository or the like. Can be manufactured by When preparing a solid preparation for oral use, an excipient, if necessary, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, a flavoring agent, etc. are added to the active ingredient, and then a tablet is prepared by a conventional method. Coated tablets, granules, dispersions, capsules and the like can be manufactured. When preparing a liquid preparation for oral use, a flavoring agent, a buffering agent, a stabilizer, a flavoring agent, etc. are added to the active ingredient as necessary, and an internal solution, syrup, elixir, etc. are produced by a conventional method. You can When preparing injectables, pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc. are added to the active ingredients, and subcutaneous, intramuscular and intravenous injections are manufactured by conventional methods. can do. When a suppository is prepared, the active ingredient is a carrier for formulation known in the art, such as polyethylene glycol,
Lanolin, cacao butter, fatty acid triglyceride and the like can be produced by a conventional method after optionally adding a surfactant and the like.

The amount of β-sitostenone or a pharmaceutically acceptable salt thereof to be blended in each of the above dosage unit forms is not constant depending on the symptoms of the patient to which it is applied or the dosage form thereof, but it is generally administered. Oral formulation per unit form is about 0.1-10 mg at a time, and injection formulation is about 0.00
It is desirable to administer 1 to 1 mg once to three times a day. The daily dose of the drug having the above dosage form varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally, but is usually about 0.
005-2 mg / kg, preferably about 0.01-0.1
mg / kg may be used once a day, or 2 to 4
It is preferable to administer in divided doses. When the acetone extract of benign mother is contained as an active ingredient, it is generally administered in a dosage unit form of about 0.5 to 50 mg in an oral preparation and about 0.05 to 5 mg in an injection preparation, which is administered 1 to 3 times a day. desirable. In addition, one of the drugs having the above administration form
The daily dose is usually about 0.01 to 1 per adult per day.
10 mg / kg, preferably about 0.05-1 mg / kg
It is preferable to administer this once a day or in 2 to 4 divided doses.

[0012]

INDUSTRIAL APPLICABILITY The extract of the beneficial herb and β-sitostenone isolated from the beneficial mother plant have an antiarrhythmic action. β-Sitostenone can be obtained either easily from the maternal plant or can be produced in relatively high yield by organic synthesis from inexpensively available raw materials.

[0013]

EXAMPLES The present invention will be specifically described with reference to examples. Test Example 1: Screening test In order to examine the presence or absence of the antiarrhythmic effect of maternal herbs, a screening test was performed for the antiarrhythmic effect against digoxin-induced arrhythmia. The antiarrhythmic drug Disopyramide (disopyramide) was examined as a control drug using papillary muscle preparations of Hartley guinea pigs. With the water extract of Japanese medicinal herbs, 1 mg / mL was effective in 1 of 3 cases, the remaining 2 cases were transiently effective, and 3 mg / mL was effective in all 4 cases. . The same effect was observed in all 3 cases with 1 mg / mL of the ethanol extract of Japanese medicinal herbs. With the water extract of Chinese medicinal herbs, 1 mg / mL was found to be effective in 1 of 3 cases, and the remaining 2 cases were transiently effective.
At 3 mg / mL, an effect was observed in all 3 cases (Table 1).

[0014]

[Table 1]

From these results, it was suggested that the maternal herb had an antiarrhythmic action.

Example 1 (Preparation of 70% Acetone Extract of Benthic Herb) Grinded Chinese Producer [Leonurus heterophyllus Swee
t. (Labiatae)] was placed in a 3000 mL eggplant-shaped flask, immersed in a 15-fold amount (1.5 L) of 70% acetone aqueous solution, stirred with a stirrer for 10 minutes, and filtered with a cotton plug three times.
The filtrate was concentrated under reduced pressure, and a small amount of water was added to the residue to disperse and freeze-dry it to obtain 70% acetone extract. The above operation was repeated 10 times to obtain 47.8 g of a 70% acetone-extracted extract from 1 kg of the maternal herb.

Reference Example 2 (Preparation of 70% Methyl Alcohol Extraction Extract of Bengal Herb) In the same manner as in Example 1, using 70% MeOH (methyl alcohol) aqueous solution instead of 70% acetone solution,
78.4 g of 70% MeOH extract extract was obtained from 1 kg of Chinese medicinal herbs.

Reference Example 3 (Preparation of 70% Ethyl Alcohol Extraction Extract of Maternal Mother) The same operation was carried out by using 70% EtOH (ethyl alcohol) aqueous solution instead of the 70% acetone solution in Example 1.
28.8 g of EtOH extract was obtained from 1 kg of Chinese medicinal herbs.

Test Example 2 The antiarrhythmic action of each extract of Example 1 and Reference Examples 2 to 3 against digoxin-induced arrhythmia was examined. Digoxin-induced arrhythmia model specimen: Hartley guinea pig (male) isolated right ventricular papillary muscle (diameter: 1 mm, length: 2 mm) Buffer: Krebs-Henseleit buffer (Kreb
s-Henseleit buffer) (in 1 L: NaCl 6.95g, KCl 0.36g,
NaHCO ₃ 2.09g, glucose 1.8g, 2M CaCl ₂ solution 1.25mL,
1M MgSO ₄ solution 1.2mL, 1M KH ₂ PO ₄ 1.2mL) Method: 2 with guinea pig sample perfused with buffer
A field stimulus of Hz, 10 V was applied. After stabilization of muscle contraction, if 1 μM of digoxin is applied to the buffer and left to stand,
Arrhythmia occurred in 2 hours. When the arrhythmia did not occur, the arrhythmia was induced by further stimulating at 10 Hz for 10 minutes. Then, the test sample was injected and the antiarrhythmic effect was observed. In addition, digoxin 1 μM is a continuous adaptation. Observation: The specimen was put in a sample in which arrhythmia was induced, and the effect was observed and evaluated on a monitor. At that time, an arrhythmic action was expressed during the adaptation of the sample, and the effect was considered to be effective even after washing. Those that did not develop an antiarrhythmic effect during sample adaptation or those that lost their effect after washing were considered invalid.

When the above-mentioned activity test was conducted, 70% Me
The OH extract (Reference Example 1) had no effect at a concentration of 0.1 mg / mL, and an effect was observed in 1 of 3 cases at a concentration of 0.3 mg / mL, and at 3 mg at a concentration of 1 mg / mL. 3mg in 2 of the examples
An effect was observed in all cases at a concentration of / mL. Its ED ₅₀ is 0.
It was 55 mg / mL. EtOH extract (Reference Example 2)
No effect was observed at concentrations of 0.03 mg / mL and 0.1 mg / mL, and effects were observed in 2 out of 3 cases at concentrations of 0.3 mg / mL and 1 mg / mL, and at all concentrations of 3 mg / mL Was confirmed to be effective. Its ED ₅₀ was 0.4 mg / mL. For the 70% acetone extract (Example 1), 0.01 mg / mL, 0.0
No effect appeared at a concentration of 3 mg / mL, an effect was observed in 1 of 3 cases at a concentration of 0.1 mg / mL, and a concentration of 1 mg / mL in 2 of 3 cases at a concentration of 0.3 mg / mL The effect was confirmed in all cases. Its ED ₅₀ was 0.17 mg / mL. The above results are shown in Table 2.

[0021]

[Table 2]

Example 2: (Preparation of ether extract) 47.8% of 70% acetone-extracted extract obtained in Example 1
g ether (1500mL × 3), ethyl acetate (1500mL ×
3), chloroform (1500mL × 3), n-butanol (15
Water (1 mL) using a 5000 mL separating funnel in the order of 00 mL x 3)
(500 mL) and liquid separation operation was performed. After that, each fraction was concentrated under reduced pressure, and a small amount of water was added to the residue to disperse and freeze-dry, thereby obtaining 10.3 g of ether fraction extract (Fr-01).
(Yield 21.5%), ethyl acetate fraction extract 1.8 g (yield
3.8%), chloroform 0.3 g (yield 0.6%), n-butanol fraction extract 5.1 g (yield 10.7%), and aqueous layer fraction 29.0 g (yield 60.7%).

Test Example 3 For each of the fraction extracts obtained in Example 2, the same activity test as in Test Example 2 was conducted. The ether fraction extract is
No effect appeared at concentrations of 0.01 mg / mL and 0.03 mg / mL, an effect was observed in 1 of 3 cases at a concentration of 0.1 mg / mL, and an effect was observed in all cases at a concentration of 0.3 mg / mL. Was recognized. Its ED ₅₀ was 0.13 mg / mL. The ethyl acetate fraction extract and the n-butanol fraction extract were 0.01 mg / mL and 0.1 mg / mL, respectively.
No effect appeared at the concentrations of 03 mg / mL, 0.1 mg / mL, and 0.3 mg / mL, and the effect was observed at 1 mg / mL in 1 of 3 cases. Chloroform fraction extract, water layer fraction 0.01
mg / mL, 0.03 mg / mL, 0.1 mg / mL, 0.3 mg / m
No effect was observed at all concentrations of L, 1 mg / mL (Table 3).

[0024]

[Table 3]

Example 3 (Isolation of β-Sitostenone from Bengal Herb Extract) The ether fraction extract (Fr-1) 10.32 obtained in Example 2
g was chromatographed as shown in FIG. That is, the ether fraction extract (Fr-1) was first subjected to silica gel column chromatography and eluted in the order of ethyl acetate: n-hexane = 1: 5, 1: 3 and 1: 1, methanol: chloroform = 1: 5. , Fr-2a 4.59g (44.5
%), Fr-2b 5.03 g (48.7%), total 9.62 g
(93.2%) obtained. 4.59 g of Fr-2a was subjected to reverse phase DM1020T column chromatography, and methanol: water = 1: 9,
Chloroform (100%) was eluted in order of 1:15 and 1:19 to obtain 4 fractions (Fr-3a to 3d). The yield of each obtained fraction was 33 for Fr-3a.
3.8 mg (7.3%), 2611.1 mg of Fr-3b (56.9
%), Fr-3c is 598.6 mg (13.0%), Fr-3d is 96
The total amount was 2.7 mg (21.0%), 4506.2 mg (98.2%). Furthermore, 962.7 mg of Fr-3d was subjected to silica gel column chromatography, and ethyl acetate: n-hexane =
Elution was performed in the order of 1:10, 1: 8, 1: 6, 1: 4 and 1: 1, and methanol: chloroform = 1: 9 to obtain four fractions (Fr-4a to 4d). The yield of each obtained fraction was 176.7 mg (18.4%) of Fr-4a and Fr-4b.
116.6 mg (12.1%), Fr-4c 224.5 mg (23.
3%), Fr-4d 443.1 mg (46.0%), total 960.
It was 9 g (99.8%). An activity test was conducted for each fraction in a series of operations. ED ₅₀ is 0.1 for Fr-1
It was 3 mg / mL, whereas ED ₅₀ was 50 μg / Fr-3d.
The action became stronger up to mL. Next, 116.6 mg of Fr-4b
When fractionated using HPLC, compound (1) was 12.7.
mg (0.0014%), 10.7 mg (0.0010) of compound (2)
%) And 54.1 mg (0.0054%) of compound (3) were obtained. In addition, the percentage in the parentheses is the percentage based on the weight of the raw material of the mother-to-be herb. The HPLC conditions are as follows. Column: Mightysil RP-18 (5mm, 20 × 250mm) Solution: MeOH: H ₂ O = 98: 2 Flow rate: 6mL / min. Measurement UV: 254nm Temperature: 38 ℃

The compound (3) obtained in Example 3 was subjected to electron impact mass spectrometry,
EI-MS), High resolution mass spectrometry
The structure was determined by ectrometry, HR-MS), ¹ H-NMR, and ¹³ C-NMR. The results were as follows: ¹ H-NMR (300MHz, CDCl ₃ ): δ 5.71 (s, 1H, H-4) ¹³ C-NMR (75MHz, CDCl ₃ ): δ 35.66 (C-1) , 33.96 (C-2),
199.69 (C-3), 123.71 (C-4), 171.75 (C-5), 32.94 (C-6),
32.03 (C-7), 35.60 (C-8), 53.79 (C-9), 38.58 (C-10),
21.01 (C-11), 39.60 (C-12), 42.37 (C-13), 55.85 (C-1
4), 24.16 (C-15), 28.18 (C-16), 55.98 (C-17), 11.93 (C-
18), 17.36 (C-19), 36.10 (C-20), 18.68 (C-21), 33.85
(C-22), 26.07 (C-23), 45.80 (C-24), 29.12 (C-25), 19.
80 (C-26), 19.01 (C-27), 23.04 (C-28), 11.96 (C-29) EI-MS m / z (%): 412 (M ⁺ , 100%), 397 (M- 15, 11.1%),
370 (M-42, 26.5%), 355 (M-57, 6.9%), 289 (M-123, 3
4.3%), 229 (M-183, 47.4%), 124 (M-288, 78.9%) HR-MS (EI) C ₂₉ H ₄₈ O Measured value: 412.3699, Calculated value: 412.370
5 [α] _D ²⁵ : + 68.6 ° (c 0.218% CHCl ₃ )

Compound (3) was analyzed by EI-MS to give m / z: 412.
(M ⁺ ) molecular ion peak was observed, which was 4 in HR-MS (EI)
It was determined to be 12.3699 and the molecular formula was C ₂₉ H ₄₈ O. ^{In 1} H-NMR, 4 of δ5.71 (s, 1H)
A signal based on the proton at the position was observed, and from these facts, β-sitostenone (stigmast-4-en-3-
On), and the ¹³ C-NMR spectral data are also based on literature values (JW Blunt, JB Stothers, ORGANIC MAGNETI
C RESONANCE, 9, 439-464, (1977) ； EJ Corey, Jaemoo
n Lee, J. Am. Chem. Soc., 115, 8871-8873, (1993)), and was identified as β-sitostenone. The remaining compound (1, 2, 4, 5,
When the structure of (6, 7) was determined, the structure was determined as follows.

[0028]

[Chemical 2]

[0029]

[Chemical 3]

[0030]

[Chemical 4]

[0031]

[Chemical 5]

[0032]

[Chemical 6]

[0033]

[Chemical 7]

Test Example 4 An activity test was conducted on the six kinds of compounds obtained in Example 3. The method of the activity test was the same as that of the screening test, and the antiarrhythmic effect of each fraction on digoxin-induced arrhythmia was examined. as a result,
Only compound (3) had a strong antiarrhythmic action, and other compounds (1,2,4,5,6,7) had no action. The ED ₅₀ of Compound (3) in which antiarrhythmia was observed was 35.
3 μg / mL, which is about 3 times that of ether extract,
The value was about 30 times stronger than that of the extract of Ascophyte extract at the time of the screening test (Table 4).

[0035]

[Table 4]

The ED _{50 of} this compound (3): 35.3
The value of μg / mL is the ED _{50 of the} antiarrhythmic drug Disopyramide:
Even if compared with 17.0 μg / mL, its effect can be expected sufficiently although it is slightly weak.

Example 4 (Synthesis of β-sitostenone) MERCK β-sitosterone [Stigmast-5-E
N-3-ol (7): Ergost-5-en-3-o
(5) = 40:55 mixture] 500 mg, CH ₂Cl₂ (salt
Pyridinium chlorochlor dissolved in 10 mL of methylene chloride
Add 500 mg of mate (MW = 215.56) and stir at room temperature for 4 hours.
I stirred. After reaction, apply to silica gel column chromatography.
, Ethyl acetate: n-hexane: 1:10, 1:18
By eluting in this order, a mixture of compounds (2) and (3)
Mixture 2 of 3.5 mg (0.7%), compounds (8) and (9)
A mixture of 50.8 mg (53%), compounds (10) and (11)
This gave 108.6 mg (22%) of the product. Next, the compound (8),
To 151.8 mg of the mixture of (9), CH₂Cl₂(Methyl chloride
P-Toluenesulfonic acid 1H dissolved in 5 mL₂O (MW
= 190.21) 140 mg was added, and the mixture was stirred at room temperature for 20 minutes.
After the reaction, it was subjected to HPLC to give 87.4 mg (56.5 mg) of compound (3).
%) And 58.1 mg (38%) of the compound (2) were obtained. In addition,
The HPLC conditions are as follows. Column: Mightysil RP-18 (5mm, 20 × 250mm) Solution: MeOH: H₂O = 98: 2 Flow rate: 6 mL / min. Measurement UV: 255nm Temperature: 38 ℃ Compound (3) is β-sitostenone,¹H-NMR
(300MHz, CDCl₃), EI-MS and HR-MS (EI)
It was

The mixture of (8) and (9) was analyzed by ¹ H-NMR (300 MHz,
The peaks at 3.25, 3.31, 5.33 ppm were confirmed by CDCl ₃ ). By ¹³ C-NMR (75 MHz, CDCl ₃ ), 122.88 (C-6), 138.53 (C-5), 21
A peak of 0.30 ppm (C-3) could be confirmed. From this, it was estimated that the mixture of (8) and (9) was a mixture of stigmast-5-en-3-one and ergost-5-en-3-one.

Test Example 5 The obtained compound (3) was subjected to the same activity test as in Test Example 2, and the compound (3) extracted from the maternal herb
The same antiarrhythmic effect was observed. From this, too
It was reconfirmed that the compound (3) synthesized in Example 4 is an active substance (β-sitostenone) from the maternal herb.

[Brief description of drawings]

FIG. 1 is a diagram showing a separation operation of an ether fraction extract from a 70% acetone extract of a maternal mother plant.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masakazu Matsunaga             1757 Kanazawa, Tobetsu-cho, Ishikari-gun, Hokkaido             Inside the university pharmacy (72) Inventor Sanbu             1757 Kanazawa, Tobetsu-cho, Ishikari-gun, Hokkaido             Inside the university pharmacy (72) Inventor Katsuyuki Uchida             540 Meiji Dairy Co., Ltd.Narita, Odawara, Kanagawa             Company Food Function Research Center (72) Inventor Kimiko Shimizu             540 Meiji Dairy Co., Ltd.Narita, Odawara, Kanagawa             Company Food Function Research Center (72) Inventor Tetsuya Kono             540 Meiji Dairy Co., Ltd.Narita, Odawara, Kanagawa             Company Pharmaceutical Division (72) Inventor Akinobu Kakuo             3-9-9 Shibuya, Shibuya-ku, Tokyo             In Nick F-term (reference) 4C086 AA01 AA02 DA11 MA01 MA04                       NA14 ZA38                 4C088 AB38 AC02 BA08 BA32 CA07                       NA14 ZA38

Claims (3)

[Claims] 1. An antiarrhythmic agent comprising an extract of a botanical herb as an active ingredient. 2. The maternal herb extract is an acetone extract.
The antiarrhythmic agent according to claim 1. 3. An antiarrhythmic agent containing β-sitostenone or a pharmaceutically acceptable salt thereof as an active ingredient.