JP2000302784A - Apocynins and prophylactic / therapeutic agents containing liver diseases containing them - Google Patents

Abstract

(57) [Problem] To provide a novel plant-derived compound and a prophylactic / therapeutic agent for liver disease. SOLUTION: Novel compounds, apocynins A to D and cinchonaines, were isolated from Rafu hemp and extracts of Rafu hemp. Apocynins A to D and cinchonines exhibit hepatoprotective activity and are useful as agents for preventing and treating liver diseases such as chronic active hepatitis caused by viruses, drug poisoning, alcohol and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to apocynins, which are novel compounds isolated from Rafu-ma, and a prophylactic / therapeutic agent for liver diseases containing apocynins or cinchonines as active ingredients.

[0002]

[Prior art] Rafu hemp (scientific name: Apocynum venetum L.)
Is a perennial inhabiting herbaceous plant of the Oleander family widely distributed in China (the herb is what is commonly called "grass"). In China, Lufu hemp leaf has been used as a substitute for tea leaves since ancient times, and Rabo hemp tea has also been used as a folk medicine such as antipyretic. In addition, several research institutes in China have been conducting medical and pharmacological studies on Lufu Asaba, which is used as a folk medicine, and reported that it is effective for hypertension, heart failure, bronchitis, edema, etc. Have been. Rabo hemp leaves include tannins such as gallotannin; quercetin, isoclurcitrin,
It is known that flavonoids such as hyperin are included. On the other hand, the liver is also called "silent organ" because the liver has a strong natural healing power and does not show noticeable symptoms with a few disorders, it regulates substance metabolism, regulates blood sugar, detoxifies, regulates bile circulation,
It plays an essential role in maintaining human life, such as storing nutrients. The etiology and pathology of liver dysfunction vary widely,
The most demanding therapeutic drug development is chronic active hepatitis, which has high medical needs. Active research and development of drugs.

[0003]

The treatment of chronic active hepatitis and the like requires the administration of a drug for a long period of time due to its nature, and the side effect becomes a problem. Therefore, development of a better drug for preventing or treating liver disease, which has a high therapeutic effect regardless of the cause of liver disease such as virus, drug poisoning and alcohol, has been awaited.

[0004]

Means for Solving the Problems The present inventors have found that some crude drugs, such as assembly, have a hepatoprotective effect. They have found that they exhibit an effect, and have further advanced their research to find novel compounds, apocynins. In addition, the present inventors have found that cinchonines isolated from the water extract of Rafu-maha have a remarkable hepatoprotective effect, and completed the present invention.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. In order to isolate apocyanins and cinchonaines from Lufu hemp leaf, for example, the method shown below may be used.

[0006]

(Equation 1)

When apocyanines and cinchonaines are used as prophylactic / therapeutic agents for liver diseases, various pharmaceutical preparations such as solutions, suspensions and syrups can be prepared by combining them with excipients, auxiliaries and additives. , Elixirs, extracts, powders, granules, fine granules, tablets, capsules and the like.

EXAMPLES The hepatoprotective action of apocyanines and cinchonines will now be described.

[Inhibitory activity on TNF-α-induced primary mouse hepatocyte death] Method: Hepatocytes isolated from the liver of male ddY mice by a modified collagenase perfusion method were isolated from 10% bovine serum and penicillin G1OOIU / ml. The cells were suspended in William's E medium supplemented with 100 μg / ml of streptomycin, 100 μM of dexamethasone, and 50 ng / ml of insulin, and 1.5 × 10 ⁴ cells were plated per well in a 96-well plastic plate. After preculture for 2 hours, the medium is replaced with a fresh medium containing 0.5 mM D-galactosamine and a test compound. After 30 minutes,
Add 100 ng / ml of TNF-α. Eighteen hours later, MT hepatocyte survival
Assay by T color reaction. Table 1 shows the results.

[0009]

[Table 1]

Next, apocynins A to D from Rafu hemp leaf
And the physical properties of apocynins AD will be described below. Isolation and Extraction a) Twice roasted 7.8 kg of Rafu hemp leaf was extracted with boiling water and freeze-dried to obtain 1.4 kg of a water extract. 170 g of this water extract was refluxed with 1 L of methanol for 1 hour, followed by filtration.
0 g was obtained.

B) The methanol-soluble portion was suspended in 100 mL of water, and chloroform, ethyl acetate and butanol were sequentially extracted (4 times with 150 mL of each solvent) to give a chloroform-soluble portion of 0.7.
g, ethyl acetate-soluble part 10.9 g and butanol-soluble part 14.4
g was obtained.

C) Sephadex LH
The column was subjected to -20 column chromatography. Elution was performed with an eluate having a methanol content of 0% to 100% in water to obtain 8 fractions.

D) Fraction 7 was collected by reversed-phase preparative thin-layer chromatography (developing solution; methanol: water = 1: 1 or 11: 9) to obtain 13.7 mg of hyperin and 1.9 mg of (+)-catechin. , (+)
-9.9 mg gallocatechin, 2.3 mg kaemferol-6'-O-acetate, isoquercetin-
6'-O-acetate 7.8 mg, catechin- [8,7-e] -4α- (3,4-
(Dihydroxyphenyl) -dihydro-2 (3H) -pyranone 2.4m
g, apocynin B 2.4 mg, apocynin A 3.0 mg, cinchonine Ia 7.2 mg, apocynin C 4.4 mg, apocynin D 3.9 mg
I got The chemical structural formulas and physical properties of apocynins A to D are shown below.

[0014]

Embedded image

Apocynin A: brown solid [α]_D ^{twenty five}-46.9 ° (c 0.19, MeOH) UVλmax (MeOH) nm (logε): 210 (1.6), 230 (1.0), 270 (0.
3) FAB-MS m / z: 469 [M + H]⁺  CD (MeOH, c 2.91 × 10^-Five): Δε-4.0 (λ / nm 233) .PMR (CD_ThreeOD) ppm: 2.58 (1H, dd, J = 16.5, 8.2Hz, C4-H),
2.81 (1H, dd, J = 15.7, 1.6Hz, C8 "-H), 2.91 (1H, dd, J
= 16.5, 5.6Hz, C4-H), 3.02 (1H, dd, J = 15.7, 7.0Hz, C
8 ”-H), 3.92 (1H, ddd, J = 8.2, 7.5, 5.6Hz, C3-H), 4.3
8 (1H, dd, J = 7.0,1.6Hz, C7 ”-H), 4.52 (1H, d, J = 7.5H
z, C2-H), 6.20 (1H, s, C6-H), 6.25 (2H, s, C2'-H, C
6'-H), 6.44 (1H, dd, J = 8.0, 2.0Hz, C6 "-H), 6.53 (1
H, d, J = 2.0Hz, C2 "-H), 6.65 (1H, d, J = 8.0Hz). CMR (CD_ThreeOD) ppm: 28.8 (C4), 35.2 (C7 "), 38.6 (C8"),
68.5 (C3), 83.6 (C2), 96.4 (C6), 105.7 (C8), 106.1 (C
4), 107.3 (C2 ', C6'), 115.1 (C2 "), 116.6 (C5"), 1
19.5 (C6 "), 130.9 (C1 '), 134.1 (C4'), 135.2 (C1"),
 146.2 (C4 "), 146.5 (C3"), 146.7 (C3 ', C5'), 152.
1 (C7), 153.2 (C8), 156.8 (C5), 170.6 (C9 '').

[0016]

Embedded image

Apocynin B: brown solid [α] _D ²⁵ + 49.9 ° (c 0.46, MeOH). UVλmax (MeOH) nm (logε): 218 (2.1), 230 (1.8), 270
(0.5) FAB-MS m / z: 469 [M + H] ⁺ . CD (MeOH, c 5.2 × 10 ^-5 ): Δε + 2.3 (λ / nm 233) .PMR (CD ₃ OD) ppm: 2.59 ( 1H, dd, J = 16.5,7.0Hz, C4-H),
2.80 (1H, dd, J = 16.5, 5.0Hz, C4-H), 2.86 (1H, dd, J =
15.7, 1.6Hz, C8 "-H), 3.08 (1H, dd, J = 15.7, 6.7Hz,
C8 "-H), 4.02 (1H, ddd, J = 7.0, 6.3, 5.0Hz, C3-H),
4.45 (1H, dd, J = 6.7, 1.6Hz, C7 ”-H), 4.64 (1H, d, J =
6.3Hz, C2-H), 6.19 (1H, s, C6-H), 6.35 (2H, s, C2'-
H, C6'-H), 6.41 (1H, dd, J = 8.0, 2.0Hz, C6 "-H), 6.
51 (1H, d, J = 2.0Hz, C2 ”-H), 6.60 (1H, d, J = 8.0Hz, C
5 "-H). CMR (CD ₃ OD) ppm: 27.5 (C4), 35.2 (C7"), 38.5 (C8 "),
68.0 (C3), 82.5 (C2), 96.3 (C6), 105.7 (C8), 106.7 (C
4), 107.0 (C2 ', C6'), 115.1 (C2 "), 116.3 (C5"), 1
19.2 (C6 "), 131.4 (C1 '), 134.0 (C4'), 135.1 (C1"),
145.3 (C3 "), 145.9 (C4"), 146.9 (C3 ', C5'), 152.
2 (C7), 156.9 (C5), 170.7 (C9 ”).

[0018]

Embedded image

Apocynin C: brown solid [α] _D ²⁵ -57.6 ° (c 0.28, MeOH). UVλmax (MeOH) nm (logε): 210 (1.6), 230 (1.0), 270
(0.3). FAB-MS m / z: 469 [M + H] ⁺ . CD (MeOH, c 2.0 × 10 ^-5 ): Δε-3.0 (λ / nm 233) PMR (CD ₃ OD) ppm: 2.82 ( 1H, dd, J = 16.0, 3.0Hz, C4-H),
2.87 (1H, dd, J = 15.7,1.9Hz, C8 "-H), 2.89 (1H, dd,
J = 16.0, 4.5Hz, C4-H), 3.05 (1H, dd, J = 15.7,6.7Hz, C
8 ”-H), 4.22 (1H, dd, J = 4.5, 3.0Hz, C3-H), 4.45 (1H,
dd, J = 6.7, 1.9Hz, C7 "-H), 4.79 (1H, s, C2-H), 6.1
9 (1H, s, C6-H), 6.46 (1H, dd, J = 8.0,2.0Hz, C6 "-H),
6.50 (2H, s, C2'-H, C6'-H), 6.53 (1H, d, J = 2.0Hz,
C2 "-H), 6.60 (1H, d, J = 8.0Hz, C5" -H). CMR (CD ₃ OD) ppm: 29.2 (C4), 35.3 (C7 "), 38.5 (C8"),
66.7 (C3), 79.7 (C2), 96.2 (C6), 105.1 (C4), 106.0 (C
8), 106.7 (C2 ', C6'), 115.0 (C2 "), 116.4 (C5"), 1
19.2 (C6 "), 131.2 (C1 '), 133.7 (C4'), 135.3 (C1"),
145.1 (C4 "), 146.2 (C3"), 146.6 (C3 ', C5'), 152.
0 (C7), 153.3 (C8), 157.2 (C5), 170.7 (C9 '').

[0020]

Embedded image

Apocynin D: brown solid [α] _D ²⁵ + 45.5 ° (c 0.23, MeOH). UVλmax (MeOH) nm (logε): 210 (1.8), 230 (1.4), 270
(0.3). FAB-MS m / z 469 [M + H] ⁺ . CD (MeOH, c 5.3 × 10 ^-5 ): Δε + 2.1 (λ / nm 233) .PMR (CD ₃ OD) ppm: 2.68 ( 1H, dd, J = 16.5, 7.0Hz, C4-H),
2.84 (1H, dd, J = 16.5,5.6Hz, C4-H), 2.87 (1H, dd, J =
15.7, 1.6Hz, C8 "-H), 3.02 (1H, dd, J = 15.7,7.0Hz, C
8 ”-H), 4.06 (1H, ddd, J = 7.0, 6.5, 5.6Hz, C3-H), 4.
44 (1H, dd, J = 7.0, 1.6Hz, C7 "-H), 4.67 (1H, d, J = 6.
5Hz, C2-H), 6.22 (1H, s, C8-H), 6.37 (2H, s, C2'-H,
C6'-H), 6.46 (1H, dd, J = 8.0, 2.0Hz, C6 "-H), 6.54
(1H, d, J = 2.0Hz, C2 ”-H), 6.65 (1H, d, J = 8.0Hz, C5”
-H). CMR (CD ₃ OD) ppm: 27.3 (C4), 35.1 (C7 "), 38.4 (C8"),
68.0 (C3), 82.7 (C2), 99.7 (C8), 96.2 (C6), 101.3 (C4),
106.8 (C6), 106.0 (C8), 107.1 (C2 ', C6'), 115.0 (C
2 "), 116.4 (C5"), 119.2 (C6 "), 131.1 (C1 '), 134.0
(C4 '), 134.8 (C1 "), 146.3 (C3', C5 ', C4"), 146.
9 (C3 "), 154.8 (C5), 155.6 (C7), 170.3 (C9").

[0022]

The apocynins and cinchonines are
It has a hepatoprotective effect and is useful as a prophylactic / therapeutic agent for liver diseases.

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Claims (2)

[Claims] (1) The following general formula: Wherein R ¹ and R ² are represented by the following formula: R ³ is a hydrogen atom, R ⁴ is a hydroxyl group, R ^5a is a hydroxyl group, or R ³ and R ⁴ are represented by the following formula: R ¹ is a hydroxyl group, R ² represents a hydrogen atom, apocynin such that R ^5a is represented by showing a hydroxyl group ". 2. The following general formula: Wherein R ¹ and R ² are represented by the following formula: R ³ is a hydrogen atom, R ⁴ is a hydroxyl group, R ⁵ is a hydrogen atom or a hydroxyl group, or R ³ and R ⁴ are represented by the following formula: R ¹ represents a hydroxyl group, R ² represents a hydrogen atom, and R ⁵ represents a hydrogen atom or a hydroxyl group ”. A preventive / therapeutic agent for a liver disease containing apocynins or cinchonines.