JPS60255727A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To provide a carcinostatic agent containing agrimoniin. CONSTITUTION:The root of KIN-MIZUHIKI (Agrimonia pilosa) is extracted with a highly polar solvent such as water, methanol, ethanol, acetone, etc., and the extract solution is subjected as it is or after the distillation of the solvent, to the adsorption chromatography to remove the amino acids, saccharides and low-molecular compounds from the extract. The obtained fraction is subjected as it is or after the removal of the solvent by distillation, to the fractionation and adsorption chromatography to obtain agrimoniin of formula which is a hydrolyzable tannin having a molecular weight of 1,870. A carcinostatic agent can be prpared by using the compound as an active component. Dose: 100-500mg/ dose, 300-1,500mg/day by oral administration, or <=500mg/day by intravenous drip injection or intramuscular injection.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anticancer agent characterized by containing agrimoniin having the chemical structure shown in the accompanying drawings.

The present inventors are currently researching the various components contained in Aphragmium japonica, a plant belonging to the Parafamily, and have discovered that the methanol extract of Aphthalmia japonica root (Chinese medicine name: Senkakuso m) has an antitumor effect. By repeating anti-tumor screening for each fraction obtained by fractionation using various chromatography methods, we separated and purified the active ingredient. As a result, we found that agrimoniin, a hydrolyzable tannin with a molecular weight of 1870, is highly potent. It was found that it has antitumor activity. The present invention has been made based on this knowledge.

Agrimoniin is a known compound that was isolated from the goldenrod tree by Aida et al. [T.

0kuda et al., J., Chem., S.
oc, , Chem, Commun11982, 35
1), its pharmacological action has hardly been clearly determined, so the present inventors investigated its antitumor action and safety and found that the previously known nucleic acid analogues and anticancer We have succeeded in providing the anticancer agent of the present invention, which contains as an active ingredient a hydrolyzable tannin with a large molecular weight that is structurally significantly different from anticancer agents such as alkaloids, antibiotics, alkylating agents, or polysaccharides.

Agrimonia pil
The above-ground part of osaLedeb is known as Senkakuso, a Chinese medicine, and is said to be used in prescriptions for cancer and to treat bleeding caused by plowing. It is not clear that Senkakuso Suikokisu shows weak antitumor activity against Sarcoma 180 [Sahara et al., Chinese medicine research 19]
79/2511979), and that the low polarity fraction of the Senzuru root, which contains almost no agrimoniin, exhibits in vitro antitumor activity in HeLa cells [Koshiura et al. Japanese Pharmacological Journal Vol. 77 No. 3 19P 1981)・
[Koshiura et al. Certain magazine 100 Qll 1167-1170
(1980) E et al. are only known.

The present inventors discovered for the first time that agrimoniin has strong antitumor activity and that this substance can be used as an anticancer agent.

Agrimoniin, which is an active ingredient of the anticancer agent of the present invention, can be used, for example, in Agrimoniin (Agrim Oniap Llosa).
Ledeb, ) roots are extracted with a highly polar solvent such as water, methanol, ethanol, or acetone, and the extract is strained or the solvent is distilled off, and then subjected to adsorption chromatography to form an extract. The fraction containing amino acids, saccharides, and low-molecular-weight compounds can be obtained as is, or if necessary, after distilling off the solvent, the fraction can be further subjected to distribution and adsorption chromatography.

The above-mentioned extraction using a highly polar solvent from the root of the Red Cornflower can be carried out at room temperature or by heating, but in order to improve the extraction rate, it is necessary to It is desirable to carry out the extraction by heating within a temperature range.

A specific example of the production of agrimoniin, which is the active ingredient of the drug of the present invention, is as follows.

Specific example: Agrimonia pilosa L
edeb, ) root powder 8007 was mixed with methanol 4.
Extract for 4 hours under reflux using 5t, filter after standing to cool, extract the extraction residue for 4 hours under reflux using methanol 4.54, filtrate after standing to cool, and filter the extraction residue. Furthermore, it was extracted with 4.5 t of methanol under reflux for 4 hours, left to cool, and then filtered. The above three methanol extracts were combined and the solvent was distilled off under reduced pressure (25, methanol extract 139°722 was obtained. This methanol extract 87.28 S' was subjected to column chromatography (diameter 7 Crn, length 35 CTn) using 1 t of Diaion HP-20 (manufactured by Mitsubishi Kasei), eluted with 9 t of water, and then eluted with 3 t of ethanol. The obtained ethanol eluate was dried under reduced pressure to obtain an ethanol eluate of 49.919.
12 is Celite Nn545 (domestic chemical system) 250 r
Column chromatography (diameter 7 cm, length 20 cm) was performed using 3 tons of methylene chloride and 31 tons of diethyl ether. h41 was sequentially eluted with 3 t of ethyl and 2 t of ethanol, and the ethyl acetate eluate was dried under reduced pressure to obtain an ethyl acetate eluate of 36.8 Or. Next, this acid-resistant ethyl elution part 35.20 r was subjected to column chromatography (diameter 5c) using Diaion HP-2 (1 (manufactured by Mitsubishi Chemical)).
m, length 35cn), and eluted with 1t each of 10% ethanol, 25% ethanol, 50% ethanol, and 99.5% ethanol, and the obtained 25% ethanol eluate was dried under reduced pressure. %ethanol elution area 19.7
I got 8 r. This 2!5 tanol elution part 15. O
Divide f into 5 times, 37 times each, Toyo Ni-ru HW-4O8
Column chromatography (manufactured by Toyo Soda Kogyo) (diameter 2
．． 5 crn, length 30 cm) and methanol 6
50 mg was then eluted with 320 ml of water-acetone (1:1), and the resulting 194 portions were collected in 5 d portions.While checking on a Merck TLC plate, the resulting 194 portions were combined into 11 portions and dried under reduced pressure. The mixture was solidified to obtain the 11th fraction, 7.76r, which was a water-acetone (1:l) eluted fraction. This amount], 09
2r 7 iodenox polymer no ξtsuku D 2014
By preparative high performance liquid chromatography using a high performance liquid chromatography column, 0.44 g of agrimoniin, the active ingredient of the drug of the present invention, was determined.
I got it.

The properties of agrimoniin obtained in the specific example were as follows.

Color/Properties “Brown powder Specific optical rotation, [α] +14σ (C = 1.94. F
, toH) Elemental analysis 9 Molecular formula C82H5405°3H2
0 Theoretical value (C251゜i6; H,3,14 Actual value 1 @ C,51,29; H,3,17 Fast atomic bombardment mass spectrum (FAB Mass Spectrum)
) m/Z 1893 ['(M+Na)+) Infrared absorption spectrum (IRSpectrum) 4B (:1n
-1:3392 (OH), 1738 (""0),
1620, 1516°1446 (aromatic
) Ultraviolet absorption spectrum (UV Spectrum) λ 7 nm (log ' ) '229 (5,20
), 270 (5holder, 4.g□)
Proton nuclear magnetic resonance (1H-NMR8pθc)
trum) δppm (200MHz, acetone-da)
: 7.39 (l H, d.

J=2Hz), 7.31 (I H,S), 6.
94 (IH, d.

J=2Hz), 6.663 (I H,e), 6
．． 655 (I H, s).

6.614 (I H,”), 6.607 (L
H,S), 6.57(])(,d,J=4RoZ)
, 6.56 (I H, θ ), 6.55 (I H, d
, J=4Hz), 6.44 (11(,B),
6.36 (IH, S), 6.35 (IH, 8), 5.6
1(11(,t.

J = 10 mouths z ), 5.50 (l H,t, J = 1
0Hz), 5.37(lH, dd, J=10/4H
z), 5.36 (l H, dd.

J=lO/4Hz), 5.25 (I H,t, J
=IO[1z), 5.17(IH,t, J=IOH
z), 5.11-5.27 (2H, m).

4.64 (lH, dd, J=lO/6Hz),
4.49 (IH.

dd, J=10/6Hz)+3.79(lH
, d, J=14Hz).

3.70 (11(,d, J = 14Hz) carbon nuclear magnetic resonance spectrum, -=kuh ("'C-NMRSpect
rum)δI)Pm (200MHz, methanol-
d, ): 170.6 (11ri.

170.3(8), 169.8(8), 169.7
(θ),], 69.5(S), 169.1(8),
168.9 (il+), 165.6 (8).

165.4 (8), 148.3 (θ), 147
．． 3(8), 145.8(s), 144.9(θ)
, 144.7(θ), 144.2(8).

142.3 (su, 141.1 CB), 140.
3 (θ), 137.6 (s), 137.4 (S)
', 137.2(8), 126.7(θ).

126.4 (8), 126.3 (θ), 126
．． 1(8), 125.8(Il+), 125.5(
il+), 120.1 (θ), 116.8 (S)
.

116.5 (Illi, 116.3(8), 115.
9(8), 115.54(s), 115.45(8
), 115.1 (e), 114. q(s).

112.7(d), 110.4(d), 108.9
(d), 108.7(d), 108.3(d),
107.9(d), 91.9(d).

91.4(d), 76.6(a), 76.4(d
), 75.0(d).

71.5(d), 71.4(d), 69.6(a)
, 69.1(d).

63.7(t).

Next, the antitumor effect and safety of the drug of the present invention will be explained using experimental examples.

Experimental Example I C31 (/He) MM2 mouse mammary cancer was intraperitoneally transplanted into a mouse, one week later, ascites was collected, and lXl0' MM2 mice were intraperitoneally transplanted into 6-7 week old female mice. Breast milk cells were transplanted. A group of 6 animals was given a single intraperitoneal administration of agrimoniin 4 days before transplantation, and the survival rate was examined for 2 days. The results are shown in Table 1.

Table 1 In this experiment, all cases in the control group died within 40 days.

Experimental Example 2 MM2 mouse mammary cancer was transplanted into the peritoneal cavity of a C3H/He mouse, and one week later, the ascites was collected, and 1×103 MM2 mouse mammary cancer cells were implanted into the peritoneal cavity of 6-7 week old female mice per mouse. was transplanted. Agrimoniin was administered intraperitoneally to 6 mice per group on the 1st, 4th, and 7th day after transplantation, and the ω-day survival rate was examined. The results are shown in Table 2.

Table 2 In this experiment, all cases in the control group died within 5 days.

Experimental Example 3 Eaglθ-MEM medium to which 5 ml/serum was added was used as a maintenance medium.

1X10' HeLa53 cells were placed in a 351+1JI plastic plate, and after 24 hours, agrimoniin was dissolved in Eaglθ~IIIKM medium and left in contact with the cells for 2 hours.1 After this, the cells were washed and treated with maintenance medium; After culturing for 3 days, the formed colonies were stained with crystal violet and counted.

The number of black formation in cells treated with agrimoniin was divided by the number of black formation in the control, and the ratio (colony formation rate, CFR) is shown in Table 3.

Table 3 Experimental Example 1 shows that the drug of the present invention has a preventive effect on tumor cell proliferation, Experimental Example 2 shows that the drug of the present invention has a therapeutic effect on tumor-prone tumors, and Experimental Example 3 shows that the drug of the present invention has a tumor cell growth prevention effect. It has been shown that it has a direct inhibitory effect on cells.

Experimental Example 4 50% lethal It (LD5
The results are shown in Table 4.

Table 4 LD5o was calculated using the probit method from the number of deaths after one week of observation.

From the above results, the dosage of the active ingredient in the anticancer agent of the present invention varies depending on the patient's age, weight, and degree of disease, but is usually 100 to 500 μg per dose for adults, 300 μg per day.
Oral administration of ~1,500■ is considered appropriate;
Intravenous infusion or intramuscular injection with El 500 or less is considered appropriate.

In the system of the present invention, agrimoniin, which is the main component of the I'l drug, can be prepared into liquid, powder, granule, Formulations such as tablets, enteric coated tablets and capsules can be made.

In the formulation, it can also be combined with other pharmaceutically active ingredients.

For oral administration, K can be formulated into tablets, pills (capsules, powders, granules, etc.) using at least one excipient, such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, etc. .

In addition to the above-mentioned excipients, this type of preparation may contain, for example, magnesium stearate, sodium lauryl sulfate, lubricants such as talc, dextrin, crystalline cellulose, polyvinylpyrrolidone, gum arabic, corn starch, gelatin, etc. Disintegrants such as binders, potato starch, and calhodic methyl cellulose can be used. In addition, the drug of the present invention can be used in suspensions, emulsions,
70 tablets and elixirs can also be administered, and these various dosage forms may contain flavoring agents and coloring agents.

When producing an injection, diluents that can generally be used include distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol, polyethylene glycol, and the like. Furthermore, an isotonizing agent, a stabilizer, a preservative, a soothing agent, etc. may be added as appropriate. In addition, in the case of this type of dosage form, it is desirable to dissolve it in a sterile injection medium.

Next, the present invention will be explained in more detail by showing Examples, but the present invention is not limited thereto.

Example 1 Agrimoniin 6O produced according to the above production example
f was made into a fine powder, mixed with lactose 2202 and magnesium stearate 207, and this mixture was compressed into tablets using a single-shot slug tablet machine to give a diameter of 201+IM and a weight of 2.
No. 37 slug tablets were prepared, crushed with an oscillator, sized, and sieved to obtain good granules with particles of 20 to 50 mesh.

This granule contains agrimoniin 2001n9 in 1 gram, and the dosage is 0.5 to 2.52 at a time, depending on the symptoms.

Example 2 Agrimoniin 10 produced according to the above production example
Microcrystalline cellulose 90F and magnesium stearate 10F were added and mixed to 0V, and the mixture was compressed using a single-shot tablet machine to produce tablets of 9 diameters and 200cm in weight.

Each tablet contains agrimoniin 1OO■, and 1 to 5 tablets should be taken at a time depending on the symptoms.

Example 3 Agrimoniin 20 produced according to the above production example
01 was made into a fine powder, and each 100 square meters of this powder was filled into suction capsules to obtain capsules.

This capsule contains 100 agrimoniin in one capsule.
(1) Contains 1 to 5 capsules at a time, depending on the symptoms.

Example 4 Agrimoniin 20 produced according to the above production example
2 was dissolved in 1 ton of distilled water for injection heated to ω゛C according to a conventional method for manufacturing injections, and after making the solution isotonic with sodium chloride, the solution was sealed in an ampoule.

This injection 1 xi contains Agrimony 720■. This injection can be administered up to 25 ml per day by subcutaneous injection, intravenous injection, or intramuscular injection depending on the symptoms.

[Brief explanation of drawings]

The attached drawing shows the chemical structure of agrimoniin contained in the anticancer agent of the present invention using a formula. Patent Applicant Ryozo Koshiura Patent Applicant Jun Tsumura Co., Ltd. Tendo Agent Patent Attorney Takao Minami H,,,"H,'+, I S121.,"

Claims (1)

[Claims] An anticancer agent characterized by containing agrimoniin.