KR0136447B1 - Novel activity polyozellus having anti-cancer activity produced by a strain of polyozellus multiplex and a process for producing them - Google Patents

Abstract

The present invention provides a novel anti-cancer active material polyozellin of the following formula (I) extracted from the mushroom (Polyozellus multiplex) belonging to the mushrooms and a method for producing the same.

Description

New anti-cancer active material polygel produced by Polyozellus multiplex strain and its preparation method.

1 shows the ultraviolet absorption spectrum of polygelins,

2 shows the infrared absorption spectrum of polygelline,

3 shows the carbon nuclear magnetic resonance spectra of polyjoliline,

4 shows the hydrogen nuclear magnetic resonance spectra of polyoline.

The present invention relates to a novel anticancer active material and a method for producing the same, and more particularly, to a new anticancer active material polyozellin material extracted from a black mushroom (Polyozellus multiplex) belonging to basidiomycetes and a method for producing the same.

Magpie mushroom is a kind of fungi belonging to the Democratic Mushroom chimney and Aphylloporalles Teleporaceae polyozellus (published by Rural Development Administration, Korea Mushroom Book 223, 1987) .In late summer or early autumn, conifers and spruce such as pine and fir It can be harvested from humus under hardwoods such as trees. In Gangwon-do, it has long been used as a commercial medicinal mushroom and health food.

The present inventors discovered a new anti-cancer active substance from the magpie mushroom, which is a medicinal mushroom of folk medicine, while searching for a new bioactive substance from mushrooms growing in Korea, and investigated the chemical structure and physicochemical properties of the substance. And identified as polygelline.

The polygelline substance of the present invention is a novel substance that has not been previously identified in its structure and physicochemical properties, and is used not only for inhibiting lipid peroxidation and prolyl endopeptidase enzyme as an anti-aging and dementia treatment agent, but also for tumors, especially The inventors have found for the first time a substance that exhibits strong anticancer activity against various cancer cell lines such as breast cancer, lung cancer, colon cancer, skin cancer, central nervous system cancer, and prostate cancer cells.

The biological characteristics of the blackcurrant are as follows.

Habitat: Native to Odaesan, Gangwon-do

Fruiting body: leaf mushroom of 10 to 20 cm in height and 10 to 30 cm in width: blue black to indigo black;

Large: Size 3-5cm, thickness 1-2cm: Split into small branches: Form the ripples of the wavy surface at the tip of each basin and form spatula or fan shape as a whole: The surface of the gilt is indigo black or grayish gray Radial shallow wrinkles: Terminal cells in the epidermal layer are long, flexible, cylindrical, 42.9-119.3 × 2.9-3.7 μm, with narrowings in the septum:

Spores: White oval to spherical in size of 5.6 to 6.9 × 4.6 to 5.6 μm. Spores: Surface of spores

Basidia: 40.2 to 76.3 × 6.0 to 7.7 μm in size 4 polarity

Cultivation: Magpie mushroom is a kind of exocytosis and is a fungus that cannot be artificially cultured and must be collected directly.

It is an object of the present invention to provide a stable nontoxic natural anticancer substance having anticancer activity against various cancer cell lines as described above and a purification method thereof.

Accordingly, the present invention provides a polygel jelly substance or a pharmaceutically acceptable salt of the following structural formula (1).

In another aspect, the present invention provides a method for producing a polygel jelly material of the formula (1) comprising extracting the magpie mushroom with a solvent.

The present invention also provides a pharmaceutical composition comprising the compound of formula (1) or a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier as an active ingredient.

Hereinafter, the present invention will be described in detail.

The preparation of the novel polygelline material is as follows.

After extracting the blackcurrant fruiting body with methanol solution, the extract is concentrated and extracted again with benzene solution. The active material layer was extracted three times with chloroform, extracted with a solution of ethyl acetate, dichloromethane, tetrahydrofuran, acetone, dioxane, acetonitrile, concentrated under reduced pressure, and neutralized by adding a small amount of 0.5N ammonia water. Obtain an alkaline active fraction. This fraction is concentrated in vacuo again, followed by the addition of a small amount of methanol, followed by centrifugation and removal of the precipitated methanol soluble impurities to obtain pure polygelline material.

Purity was confirmed by high performance liquid chromatography on the obtained polygelline material on an ODS (octadodesyl) reversed phase column using 65% methanol containing 1% acetic acid as a solvent.

The active substance according to the present invention purifies the antioxidant active substance by measuring the amount of malondialdehyde produced by lipid peroxidation of microsomes isolated from livers of rats (SpragueDawley, male) [Hogeboom, GH, Fractionation of cell Components of Animal Tissues, Method of Enzymology, Vol. 116­19. (1955)] The physicochemical properties of the purified polygelline are as follows.

1. Appearance of substance: green powder

2. Molecular Weight: 438

3. Molecular Formula: C ₂₂ H ₁₄ O ₁₀

4. Mass spectrometry (M + H ⁺ ): 439 m / z

5. Melting Point: 245 ℃

6. Ultraviolet absorption spectrum (UV lambda ^MeOH max nm): The ultraviolet absorption spectrum measured in methanol solution is shown in FIG.

7. Infrared spectrum (IR lambda ^KBr max cm- ¹ ): The infrared absorption spectrum measured by the KBr method is shown in FIG.

8. Nuclear Magnetic Resonance (NMR) Absorption Spectrum: Carbon nuclear magnetic resonance (1 ₃ CNMR) spectrum and hydrogen nuclear magnetic spectrum measured using DMSO: chloroform (1: 4) as a solvent and tetramethylsilane (TMS) as a standard Resonance ( ₁ H-NMR) spectra are shown in FIGS. 3 and 4.

9. Solubility

Sun: DMSO, Pyradine, Ethyl Acetate

Slightly soluble: Ethylene glycol, methanol, ethanol

Insoluble: Chloroform, Benzene, Hexane, Water

10. RF value of thin layer chromatography (TLC):

Adsorbent: Silica TLC. Merck 5554

Developing solvent: Cloform: Methanol: Acetic acid (13: 1: 0.5)

RF = 0.18

Ethyl Acetate: Methanol: Acetic Acid

(25: 1: 0.2) RF = 0.66

Benzene: Methanol: Acetic Acid (1: 2: 0.2) RF = 0.54

11. Chemical structure:

The polygelline material of the present invention can be found in other kinds of mushrooms and fungi as well as the blackcurrant having biological properties as described above. Thus, the polygelline manufacturing method of the present invention is particularly limited to the method using the blackcurrant. It is not.

In addition, the present invention includes a polygel jelly having the above structural formula as well as pharmaceutically acceptable salts thereof.

The polygelins of the present invention react with many non-toxic inorganic bases and non-toxic organic acids to form pharmaceutically acceptable salts.

Pharmaceutically acceptable salts refer to derivatives of the disclosed compounds in which the functional groups present in the compound are cleaved in vivo or by conventional manipulation to form the desired compound, or modified with acid or alkali salts . Examples include, but are not limited to, organic or inorganic acid salts of basic residues such as amines: alkali or organic salts of residues such as carboxylic acids: acetates, formate and benzoate derivatives of alcohols and amines, and the like.

Acids commonly used to form pharmaceutically acceptable acid addition salts are inorganic acids such as hydrochloric acid, bromic acid, iodic acid, phosphoric acid sulfate, and the like and ptoluenesulfonic acid, methanesulfonic acid, pbromophenylsulfonic acid, carbonic acid, succinic acid And organic acids such as citric acid, benzoic acid and acetic acid.

Pharmaceutically acceptable salts of the compounds of this invention may be prepared by reacting a stoichiometric amount of a suitable base with the free acid or base form of a compound of the invention in water or an organic solvent or mixture thereof. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

The anti-cancer composition can be prepared by mixing with a pharmaceutically acceptable carrier as the active ingredient polygelline of the present invention. The anticancer composition may further include conventionally used excipients, disintegrants, sweeteners, glidants, flavoring agents and the like, and may be administered in unit dosage form or multiple doses such as oral or parenteral administration by conventional methods. It may be formulated into a pharmaceutical formulation.

The anticancer agent containing the polygelline of the present invention as an active ingredient may be parenterally or orally administered by subcutaneous administration, intravenous administration, transdermal administration, nasal inhalation, infusion pump, gastrointestinal administration, etc. as desired. An amount of 0.1 to 0.5 mg / kg body weight can be administered in one to several times a day. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, excretion rate, severity of the disease, and the like.

Through the following examples will be described the present invention in more detail. These examples are merely illustrative and are not intended to limit the scope of the invention in any sense.

Example 1 Preparation of Polyolineline Materials

After drying about 1 kg of Magpie mushroom (Polyozellus multiplex), finely cut into 2 to 3 cm in size, and leaching overnight by adding about 3 L of methanol to extract sulfurous material. The extracted solvent was recovered, and the residue was extracted six more times using about 3 L of methanol, and the extracts were collected and concentrated under reduced pressure to remove methanol. 1.5 liters of water was added to the concentrated active fraction and redispersed. The mixture was extracted three times with the same amount of benzene. The benzene layer was removed. The aqueous layer was collected and extracted three times with 1.5 L of chloroform. The chloroform layer was then removed. It was. The aqueous layer, which was the active fraction, was extracted again with 1.5 L of ethyl acetate, and then the ethyl acetate layer was concentrated to dryness under reduced pressure to obtain 1.63 kg of crude extract.

The crude extract was again treated sequentially with about 100 mL of ethyl acetate and methanol to remove impurities and then the active material was completely dried and then extracted eight times with 100 mL of ethyl acetate again under alkaline conditions. The ethyl acetate layer, which is the fractionated layer of the active substance, was completely dried to obtain about 847 mg of green powder. After adding 300 ml of methanol, the precipitated methanol-soluble impurities were removed by centrifugation at 3000 rpm for 5 minutes, and concentrated under reduced pressure to obtain 554 mg of pure polyoline.

High-performance liquid chromatography (HPLC) analysis was performed on an ODS reversed phase column using 65% methanol containing 1% acetic acid as a solvent to confirm the purity of the final target material (analysis showed a single peak at 9.5 minutes residence time). ).

Example 2. Anticancer Activity of Polygonin Substances on Breast Cancer Cell Lines

Anti-cancer activity of the polygline material obtained in Example 1 on the human breast cancer cell line [hman breast tumor cel line, MCF7: obtained from the American National Cancer Institute], SRB Methods, Monks, A. et al., J. Natl, Cancer Inst., 83: 757, 1991). The results are shown in Table 1 below, and as can be seen from the table, the effective concentration of anticancer activity against human breast cancer cell line MCF7 (ED ₅₀ ) was 15.8 ppm.

Table 1. Anticancer Activity of Breast Cancer Cell Lines of Polygelline Substances

Concentration (ppm) Anticancer activity (%)

25.019.7 ± 6.0

2.576.8 ± 2.4

0.2599.7 ± 2.5

0.02598.6 ± 4.2

0.002599.2 ± 4.2

Effective concentration (ED50) 15.8 ppm

Example 3 Anticancer Activity of Polycarolin Agents on Each Cancer Cell Line

Anticancer activity against each cancer cell line was carried out in the same manner as in Example 1 and the results are shown in Table 2 below.

Table 2.

Cancer cell line effective concentration (ED50)

Kidney cancer cell line (human renal4.9 ppm

tumor cel line, U0­31)

Human central nervous system tumor cell

line, SNB­19) 0.5ppm

Human skin tumor cell line (CRL 1579)

.2.3 ppm

Human leukemia tumor cell line (MOLT'4F)

.1.1ppm

Human lung tumor cell line (NCI­H522)

.0.2ppm

Human colon tumor cell line (KM­12)

.0.3 ppm

Each cell line was obtained from the Institute of Genetic Engineering, Korea Advanced Institute of Science and Technology.

Example 4. Lipid Peroxidation Inhibitory Activity of Polygones

Lipid peroxidation inhibitory activity of the polygel jelly was evaluated according to the Fe ++ / ascorbate method (Fe ++ / ascorbate Methods, Ohkawa, H. et all., Anal, Biochem, 95, 351 (1979)). . That is, after separating microsomes from the liver of 6 months old male spragueDawley, the amount of malondialdehyde produced by oxidation of microsomes of unsaturated fatty acids by Fe ++ / ascorbate is determined by TBA (thiobarbituric acid). After reacting with, the antioxidant activity was evaluated by measuring the absorbance at 530 nm. The results showed the relative inhibition in percentage (%) compared to the untreated control. The results are shown in Table 3 below. As can be seen in Table 3, the effective concentration of polyoline antioxidant activity (ED50) was 1.96 ppm.

Table 3 Antioxidant Activity of Polygones

Concentration (ppm) antioxidant activity (%)

0.52.2

1.08.3

2.052

4.090

8.098

Hazardous Concentration (ED ₅₀ ) 1.96ppm

Example 5 Pololyl Endopeptidase Inhibitory Activity of the Polygoneline Substance

The prolyl endopeptidase inhibitory activity of the polygel jelly was evaluated using an enzyme obtained from Flavoacterium (Yamakawa, N. et. Al., Chem. Pharm. Bull. 34: 256263, 1986). In other words, the amount of paranitroaniline produced after reacting prolyl endopeptidase purified from Flavoacterium meningosepticum with carbobenzyloxyglycylylprolylparanitroanilide at 410 nm The prolyl endopeptidase inhibitory activity was evaluated by measuring the absorbance at. Activity represents the percentage inhibition relative to the untreated control. The results are shown in Table 4 below, and as shown in Table 4, the noxious concentration (ED ₅₀ ) of the prolyl endopeptidase inhibitory activity of polygel jelly was 10.6 ppm.

Table 4. Prolyl endopeptidase inhibitory activity of polygelline substances

Concentration (ppm) Prolyl endopeptidase inhibitory activity (%)

123

225

435

846

1657

3259

6473

Effective concentration (ED ₅₀ ) 10.6 ppm

Example 6 Acute Toxicity of Polygonelin Material

Five or six weeks old ICR mice were orally administered with a polygelline substance at a concentration of 500 mg / kg for 14 days. After autopsy, abnormality of organ was observed, but no abnormality was found. From these results, the LD ₅₀ value of the polygel of the present invention was at least 500 mg / kg.

Claims (4)

The anticancer active material polyozelline of the formula (I) and a pharmaceutically acceptable salt thereof: The structural formula of claim 1, comprising extracting Ployozellus multiplex with a solvent selected from the group consisting of dimethyl sulfoxide (DMSO), pyridine, ethyl acetate, ethylene glycol, methanol, ethanol or a mixed solvent thereof. I) Polyoline manufacturing method. The method of claim 2, 1) Steps to dry and cut the blackcurrant mushrooms: 2) extracting the cutlet mushroom with methanol and concentrating under reduced pressure to remove methanol; 3) redispersing the concentrate in water, then extracting with benzene and removing the benzene layer; 4) extracting the aqueous layer with chloroform and then removing it with a chloroform layer; 5) extracting the aqueous layer with ethyl acetate and concentrating the ethyl acetate layer under reduced pressure: 6) adding ammonia water to the concentrate to make it neutral or alkaline, and then extracting with ethyl acetate; And 7) Concentrating the ethyl acetate layer under reduced pressure, and adding methanol to it and centrifugation to obtain a supernatant, the production method. An anticancer composition comprising the polygel jelly of the formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier as an active ingredient.