JPS60126225A - Novel polyphenol compound and carcinostatic agent - Google Patents

Abstract

NEW MATERIAL:A polyphenol compound having the following characteristics. Appearance, brownish powder; melting point, 185-190 deg.C (decomposition); molecular weight, 20,400; elemental analysis, C 58.19, H 4.53, N 0.00, ash 1.33 (Na); peak of UV absorption spectrum, 280nm in methanol. USE:A carcinostatic agent. Useful as an excellent chemotherapeutic agent for cancer of warm-blooded animal such as man and domestic animals. PREPARATION:The dry powder of bark of the twig of Pterocarpus indicus willd is extracted by immersing in hydrated methanol for a night under stirring, filtered, extracted again with hydrated methanol for a night, and filtered. Both filtrates are joined together to obtain the extract liquid, which is concentrated under reduced pressure to remove methanol. The concentrated product is adsorbed with a column packed with Sephadex HL200, washed with water, and eluted with dioxane. The eluted active component is collected, concentrated under reduced pressure, and purifid to obtain the objective polyphenol compound.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anticancer agent characterized by containing a compound as an active ingredient.

Traditionally, it has been used as a pain reliever, alkylating agents (nitrodiene mustards, ethyleneimines, sulfone esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), and plant branches. % poison (colcemid,
vinblastine, etc.), antibiotics (pleomycin, daunomycin, mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, C0PP), etc. are used.

However, most of them are cytotoxic substances;
At present, there is a strong desire to develop anticancer agents that exhibit low toxicity and excellent anticancer activity without causing serious side effects.

In order to search for substances with low toxicity and anticancer activity, the present inventors conducted intensive research on physiologically active substances of plant origin, and as a result, succeeded in extracting a new polyphenol compound from the bark of Indocalin. We have also discovered that this compound has excellent anticancer activity, and have obtained new knowledge that it can exert a remarkable effect on cancer treatment, leading to the completion of the present invention. The compound of the present invention is used as an active ingredient of an anticancer agent, and the anticancer agent can serve as an excellent cancer chemotherapeutic agent for warm-blooded animals such as humans, livestock, dogs, and cats.

First, the compound of the present invention will be explained below.

The plant used as a starting material has the common name Indocarin (or Yaeyamacarin) (scientific name pte-rocarp).
It is a tropical tree of the leguminous family native to Malai, called U.S. 1ndicus Will.

The bark of this Indian quince twig
ing, Angsana) was used as a raw material, immersed in water-containing methanol under stirring, extracted overnight, separated by F, extracted again overnight with water-containing methanol, and after separated by F, the two were combined to form an extract solution. This is concentrated under reduced pressure to remove methanol, and the aqueous solution is adsorbed on a Sephadex LH, 2θ column.

The active fraction does not elute with water. After thoroughly washing the column with water, the active fraction was eluted with 9θ thidioxane, concentrated under reduced pressure, dialyzed day and night, and the dialyzed solution was concentrated to dryness to obtain a brown solid. This is subjected to high-performance liquid chromatography to separate the main component at a retention time of 4 minutes, and the mixture is again dialyzed overnight and concentrated to obtain a pure colored powder.

The physicochemical properties of the substance obtained in this way are as follows: 2 in the ultraviolet absorption spectrum (in methanol)
It was confirmed that it is a new polyphenol-based substance because it has many hydroxyl groups, as seen in the riatists' pattern showing a maximum in gOnm and the U external radiation absorption spectrum (Figure 1).

(1) Elemental analysis value ((6): C: 5g, /?, H: 'A33.N: θθ0° Ash content: /
33(Na) (to) Molecule 9: (by ultracentrifugation) The molecular weight was estimated to be approximately λo, poθ by high performance liquid chromatography (HPLC).

■ Melting point: /g! ;-/? θ℃ (decomposition) ■ Ultraviolet absorption spectrum: It shows maximum absorption at 2gOnm in methanol.

(a) Infrared absorption spectrum = (main maximum values in potassium bromide) (Figure 1) 335θ, /6θθ, /S/left, /'lJ, t, /
Kogθ.

/10θ low! (4> “CN MR spectrum (DMSO), 6th
Figure (7) Shape: Polyphenol-based brownish powder Next, the compound of the present invention (hereinafter referred to as "the compound").

) is described below.

(1) Effect on enzymes/) f? Effect on sumin activity Plasmin is highly active in cancer cells and is an important enzyme system associated with cancer. p+asmjnogen) (manufactured by Sigma, USA) and urokinase (
A method of determining the degree of decomposition of P-1 syl-L-arginine by coloring with the addition of ferric chloride (Snyder, F, and N, 5teph).
ens, 8iochim, 8iophys, Ac
ta 34t 21111-21I! ; (/9!;9
)).

The results are shown in Figure 12, and the compound is 2, Arp
f/m1. At a low concentration of about 3θ, /θp f, Int
in? The grasmin activities of g and 4′ were inhibited, respectively.

2) Effect of ornithine decarboxylase on ornithine decarboxylase? Xyler < or n it
In order to examine the effect on 14C-ornithine decarboxylase), the test involves measuring the radioactivity of 14CO1, which is decarboxylated by 14C-ornithine (T, G).

0' Bren 6t 61: Cancer Res,
GA;, /A Otsuko/1.70 C/97! r)].

The results are shown in Figure 3, and the compound has 2 kpt.
/ml about A O%, A; Opt7ml about go
% inhibition rate is shown respectively.

Q) Effect on proliferation of cultured animal cells FM-3A, floating cells derived from mouse breast cancer as malignant cells
and chemically converted Kyushu cells Cu/bC/R/ derived from rat liver.
Looking at the effect of the compound on the in vitro proliferation of TC, both cells showed a growth rate of 23μ//.
Up to mt, there is weak growth inhibition (cell number = 6×/θ'), but 3θμ? A remarkable suppressive effect (cell number: 3 x 10'') was observed at cells of /m1 or higher.

■ Ehrlich ascites tumor (ascites Ehrl)
Antitumor activity test against Ehrlich ascites tumor obtained from ascites of mice with Ehrlich ascites tumor
The cells were inoculated into the peritoneal cavity of a CD-/mouse (currently female), and the compound was intraperitoneally administered twice, 29 hours later and 72 hours later, to measure the survival rate.

As a result, the lower dose of 1.2 mf/Kg in Fig.
%,! A survival effect of 30-J-2% was observed when administered to 0, 8, and 2 patients.

In Figure ≠, the compound is -2tr9/K9.1
0m)/L9 and jOmp/1'4, and 0.0m)/L9 and jOmp/1'4 for the control. The j
% physiological saline, and the numbers within 0 for each sample represent the average survival rate (A) of 6 test mice. From the above experimental results, it was confirmed that the above compound can be used as an active ingredient of an anticancer drug. Ta.

(4) Anti-1111'll against P3ff and ascites leukemia
Activity test P3♂♂ Ascites leukemia vc-affected mice were intraperitoneally administered to mice 2≠hours after ascites, 7 times on 7 days, and the survival rate was measured.

As a result, after 9 administrations at 1.2W9/, a survival effect of /2.13% was observed on average for 7 mice compared to control mice.

, (5) Acute toxicity The acute maternal toxicity of the compound was determined by oral administration to mice.
D50>300t-y/19'''Ch!l,s.

``No toxicity was observed in continuous administration for 10 days from 9 to 9 in 1997.

Next, the details of the compound and its administration method will be described.

The drug of the present invention is administered as an oral preparation such as a tablet, capsule, or aerosol, or as a parenteral preparation such as an injection (intravenous or subcutaneous), infusion preparation, or suppository. Although it may vary depending on the route of administration, dosage form, number of administrations, etc., the effective dose when used for the treatment of adults is usually:
For oral administration, the compound as an active ingredient is preferably y θ 0 s to 100 s/θ type 0 s to 100 s per 7 days, preferably θ 0 kg/9. More preferably θ-10
■ / Body weight is appropriate, and for parenteral administration, / per day is appropriate, and the compound as an active ingredient is θ002~2θθ
■/Body weight in the range of 2, preferably θθ ~ 10θ■/
Body weight Kf, more preferably 02 to 2θ■/body weight, is preferably 9.

When the compound is formulated for injection or infusion, it can be provided in a unit dosage form or in a dosage container together with additives. The preparations may take such forms as suspensions, solutions, emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing, or dispersing agents.

Suspending agents include glucose, gelatin, aluminum stearate, etc. Stabilizing agents include lecithin,
linoleic acid, monooleate, rubitan, etc., as a non-aqueous vehicle, almond oil, coconut oil, refined sesame oil,
As preservatives such as oily esters, P-hydroxybenzoate t1'2 methyl, rubic acid, etc. are used.

When preparing the compound for oral administration, it is preferably administered in a form suitable for absorption from the intestinal tract, such as a capsule.

In capsules for oral administration, binders such as gelatin,
Excipients such as sorbitol, such as lactose, sugar, starch, glycine, etc., stabilizers, such as magnesium stearate, ascorbic acid, etc., and disintegrants, such as potato starch, can be appropriately contained.

For the preparation of oral administration of the above compound: J, 'J,
Additionally, coatings can be applied using pharmaceutically acceptable enteric coating materials. The film-forming substances include cellulose, cellulose acetate phthalate (CAP) as a carbohydrate derivative such as saccharides, acrylic acid copolymers, dibasic acid monoesters, etc. Examples of ribinyl derivatives include methyl acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In order to improve disintegration and dissolution of the active ingredient of the present invention, surfactants such as alcohols, esters, 7+?
Seven or more types of reengineered tylene glycol derivatives, sorbitan fatty acid esters, etc. can be added. In addition, as excipients, for example, 7 or 2 types of excipients such as sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, magnesium aluminate metasilicate, calcium carbonate, hydrogen carbonate, etc. A combination of the above can be added. As a lubricant, for example, seven or more types of magnesium stearate, talc, hydrogenated oil, etc. can be added, and as a flavoring or flavoring agent, salt, saccharin, sugar, mannitol, orange oil, licorice, etc. can be added. Extract, citric acid, glucose, menthol, eucalyptus oil, sweeteners such as malic acid, fragrances, coloring agents,
It may also contain a preservative or the like. Adjuvants such as suspending agents and wetting agents may include, for example, coconut oil, olive oil, sesame oil, peanut oil, calcium lactate, safflower oil, soybean phospholipid, and the like.

When the drug of the present invention is administered orally, it is administered as a suspension, soft/hard capsule, tablet, granule, fine granule, powder, or aerosol; when administered parenterally, it is administered in the form of a solid or suspension. It is administered as a viscous liquid to maintain sustained mucosal absorption.

Next, examples of the above-mentioned compounds and formulation examples of the anticancer agent of the present invention will be given.

Example Indonesian Indian quince bark (KulitRan)
The starting material was 500 t of dry powder of Angsana, which was mixed with 3 t of methanol/water (70/3
θ, V/V) overnight, and then extracted overnight using 3 tons of the same solvent, extracted overnight, and combined the two to form an extract. The extract was concentrated under reduced pressure to remove methanol, and the aqueous solution was poured into Sephadex LH2θ (Sephadex LH2θ).
Adsorb onto LH, 2 column (4tX7 (7 crn). The active fraction is insoluble in water, so after washing the column thoroughly with water, elute with dioxane/water (R0//θ, V/v).Activity The fractions are collected, concentrated under reduced pressure to 3θ, and dialyzed for a day and night. The dialyzed solution is concentrated to dryness, yielding a brown solid of approximately 70θwy #'J. At this stage, high-performance liquid chromatography (HPLC) is performed. , as shown in Figure S, J/'I minute C shorts polymer/4 for holding.
'Tsuku (Sh.

dexpo! ymerpak) ρ--〇/l,,2
The main component was found in X left θα, elution rate gmlZ min, 90 timetanol]. This fraction is separated, dialyzed again overnight, and concentrated to obtain a pure product of warm-colored powder. ; Obtained Ovq. In order to confirm the purity, HPLC was performed again under the above conditions, and it was confirmed that the product was pure.

Formulation example/(injection/infusion) Powdered glucose jf to contain the above compound θ0■
Aseptically dispense into vials, seal and store. Before use, add 00 ml of physiological saline to make an intravenous injection, and 7 days later, θθmI! Administer by intravenous injection or drip depending on the symptoms.

Formulation example, 2 (tablet) Seven hundred adult tablets were prepared with the following ingredient composition.

CA) The above compound iot lactose 9 q lf t hydro-2yf lobil cellulose o, otv stearin magnesium 0.27 [B] Cellulose acetate phthalate θ Otsu 1 Take the components of A, mix well, and add this to (substituent). Contact force (1 pressure or after mixing well, extrude f!l ?4 R
(Granule molding was carried out through a 1% screen, and after thorough examination, tablets were prepared by pressurization.

Furthermore, if necessary, the molded tablets are coated with a well-dissolved base material to form enteric-coated tablets.

Formulation example 2 (capsule) Seven hundred capsules for adults were prepared with the following ingredients.

[A]

Said compound /θ2 lactose,? 61 Hydroxypropyl cellulose θθ Rita CB) 6″ Keftal I Cellulose / 2 Hydroxyzorobyl methyl / f Pour 1 ml of component A on cellulose phthalate, mix well, and form into 4K particles according to the usual method. Dry thoroughly and sieve to make 7! neck granules suitable for capsules, and add 1+- to capsules.
R, SL - capsules were prepared. Furthermore, if necessary, the tq granules are mixed with the dissolved B while being suspended in a floating flow, and then covered with (#) to form enteric-coated granules, which are then filled into capsules to obtain enteric-coated capsules. .

[Brief explanation of drawings]

Figure 1 is a graph showing the infrared absorption spectrum of the compound of the present invention, Figure 2 is a graph showing the action (inhibitory activity) of the compound of the present invention on enzyme threads (plasmin), and Figure 3 is a graph showing the infrared absorption spectrum of the compound of the present invention.
The figure is a graph showing the action (1q1 harmful activity) of the compound of the present invention on the enzyme (ornithine decarboxylase). And, in Figure 5, the 6th ? This is a high-speed tube chromatograph of the ancient classification. Patent applicant RIKEN

Claims (1)

[Claims] (1) A polyphenol compound having the following physical and chemical properties. /) Shape: Polyphenol-based brownish powder, 2) Melting point: /
g! ;-/90℃ (decomposition) 3) Molecular weight: approx. θ, 4! θ
04t) Elemental analysis value %: C: A-g, /9. H: 41. ! F3. N:
θθ0° Ash content: 133 (Na) S) Ultraviolet absorption spectrum: 2gOnm in methanol
Maximum absorption 2 6) Infrared absorption spectrum: (main maximum values in potassium bromide) 33! ;0. /Otsuθ0. /,! ;/! r, /4'
3 left, /2gθ. //θθcrn-1 Q) An anticancer agent characterized by containing a polyphenol compound having the following physical and chemical properties as an active ingredient. /) Shape: Polyphenol brownish powder 2) Melting point: 1
g5-i'yθ°C (decomposition) 3) Molecular weight: approximately λθ, t00 1I) Elemental analysis value %: C: &ff, /q, I-1: 11. ,t,? , N:
0.00゜Ash content: 13j (Na>) Ultraviolet absorption spectrum: In methanol, maximum absorption at 2gθ nm Infrared absorption spectrum = (main maximum value in potassium bromide) 33 left 0./Oθ, /left/A ;, /'73!;,
/tsugθ. /10θ uniformity! (■ The anticancer agent according to claim 1 in a parenteral administration form. ■ The anticancer agent according to claim 2 in an oral administration form.