KR101090196B1 - Anti-cancer composition comprising Ergosta-7,22-dien-3?,5?,6?-triol - Google Patents

Abstract

The present invention uses Ganoderma lucidum-derived compounds extracted with an organic solvent as an active ingredient and induces apoptosis of cancer cells by activating the cleavage cleavage of caspase-3 and poly adp ribose polymerase (PARP). It relates to a composition for preventing or treating cancer. The compound of the present invention is excellent in cancer cell killing effect, it was confirmed that the DNA fragmentation accompanying the cell killing effect, the cleavage cleavage and activity of caspase-3, PARP cleavage cleavage activity and the like. In addition, the compound may be usefully used as a pharmaceutical composition and health functional food for treating, preventing or improving cancer with a safe substance having low cytotoxicity.

Description

Anti-cancer composition comprising Ergosta-7,22-dien-3β, 5α, 6α-triol, including ergosta-7,22-diene-3beta, 5alpha, 6alpha-triol

The present invention relates to Ergosta-7,22-dien-3β, 5α, 6α-triol, which is a compound derived from Ganoderma lucidum extracted with an organic solvent, and a composition for preventing or treating cancer using the same as an active ingredient. More specifically, the compound derived from Ganoderma lucidum extracted with an organic solvent as an active ingredient, and activated by the cleavage cleavage of caspase-3 and PARP (Poly ADP ribose polymerase) to induce cancer cell death (apoptosis) of cancer cells Or to a therapeutic composition.

Cancer is a malignant cell population that autonomously proliferates out of the host's regulatory mechanisms, and its cause and mechanism are still unclear. However, since it has been recently discovered that the mechanism of action of anticancer drugs depends on the mechanism of apoptosis, there has been a great interest in researches to improve the efficacy of chemotherapy by changing the function of apoptosis signaling system.

Apoptosis is an important mechanism for maintaining homeostasis of cells and organs. In particular, apoptosis plays an important role in the generation, differentiation and function expression of cells of the nervous and immune systems. Abnormalities in such apoptosis mechanism are known to be an important factor in pathologies such as cancer, autoimmune diseases, degenerative diseases and HIV.

The mechanism of apoptosis includes Fas / Fas-L system, caspase family cysteine protease, a system that amplifies apoptosis signaling via mitochondria, and endonugen that causes DNA fragmentation. Clease and the like have been reported to be involved.

To date, a subfamily of about 15 caspase family cysteine proteases has been identified. These genes recognize specific protein sequences of substrates and then selectively cleave cysteine groups. It encodes an enzyme. Caspase exists in the form of an enzyme precursor (proenzyme) in the cytoplasm, activated by para-lysis and auto-lysis by apoptosis signaling. By apoptosis stimulation, caspases above the signaling activate other caspases below and induce apoptosis by decomposing or activating substrates important for apoptosis. As a representative example, caspase-3 is an intracellular target known as apoptosis, and known as protein kinase-C (PKC), lamin and poly ADP ribose polymerase (PARP). The activation of (ICAD: caspase-activated DNase) is also known to be dependent on the activity of caspase-3.

On the other hand, Ganoderma lucidum (Fr. Karst) is one of the most widely known medicinal mushrooms, mushrooms of the basidiomycetes fungi, and are distributed throughout the world. Ganoderma lucidum is nicknamed Bulchocho, and it occurs on the ground at the root of the hardwood root or stump in summer. It is a year-round mushroom with a gloss like lacquer on mushroom shade and mushroom stand. Mushroom shade is 5 ~ 15㎝ in diameter, 1 ~ 1.5㎝ in thickness, semicircular, kidney-shaped, fan-shaped, and has flat and concentric annular grooves. The surface of the mushroom shade is initially yellowish white and then turns yellowish brown or reddish brown and turns brown at age. The flesh is cork, and the upper and lower two layers, the upper layer is almost white, and the lower layer of the tube is light orange. The underside of the shade is yellowish white with 5 ~ 10mm long holes lined in one layer and the tool is round. Mushroom stand is 3 ~ 15 × 1 ~ 2㎝, reddish brown to black brown, wrapped in hard cuticle, slightly bent. The hole is double-layered and the hole pattern is light brown. The mushroom stand is wrapped in hard skin and has a varnish like varnish. It is used as an herbal medicine in Japan, and it is used as an herbal medicine in Japan. Among them, traditionally, it has been widely used as a medicinal herb in China, Japan, Korea, and the like, and because of its high medical value and safety as an edible use, interest in its efficacy and use is increasing recently.

Ganoderma lucidum has the effects of tonic, Jinhae, and small sarcoma, and has been used for nervous breakdown, heart disease, hypertension and various carcinomas. In addition, it has been mainly used for preventing and treating liver disease, chronic hepatitis, nephritis, gastric ulcer, arthritis, insomnia, asthma, acute and chronic bronchitis, leukemia, and has been widely used as a health food to improve it.

Ganoderma lucidum is characterized by a bitter taste unlike other mushroom types. The ganoderma lucidum's bitter taste is mainly derived from triterpene. Triterpenes for medical and biological use have made great strides in recent decades, and a variety of related substances, such as triterpenes and their derivatives, have been identified mainly in mushroom bodies, mycelia and spores.

Several types of polysaccharides, the main components of the water-soluble substance of Ganoderma lucidum, were the most well known anti-cancer-related substances in Ganoderma lucidum, but it was difficult to determine the chemical structure. Therefore, the inventor of the present invention extracted the ganoderma lucidum mushroom using an organic solvent and found a substance with anticancer activity among them.

Ganoderma lucidum triterpene is a lanostane-type triterpene and is one of the main substances that are dissolved when Ganoderma lucidum is extracted from an organic solvent. Ganoderma lucidum triterpenes are classified into two types: ganoderic alcohol and ganoderic acid. Among them, ganoderic alcohol is known to have stronger cytotoxicity than ganoderic acid.

Based on the above results, in the present invention, the compounds of triterpenes contained in Ganoderma lucidum extract were confirmed to have anticancer activity in the compounds.

An object of the present invention Ergosta-7,22-dien-3β, 5α, 6α-triol derived from Ganoderma lucidum that induces apoptosis due to the action of caspase-3 and PARP, and comprising the same as an active ingredient It is to provide a composition.

Another object of the present invention is Ergosta-7,22-dien-3β, 5α, 6α-triol, a compound derived from Ganoderma lucidum for preventing or treating diseases related to cancer, and a pharmaceutical composition and health functional food comprising the same as an active ingredient. To provide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the compound Ergosta-7,22-dien-3β, 5α, 6α-triol represented by the following formula (1) as an active ingredient.

[Formula 1]

The compound according to the present invention can be easily separated into Ganoderma lucidum, and can be used as an additive of food and medicine because of its high stability.

Confirmation of the anticancer effect of the compound of the present invention, confirming the cytotoxicity to cancer cells;

Confirming caspase-3 activity; Confirming DNA fragmentation; Confirming cleavage of caspase-3 and PARP protein; In vivo anticancer activity confirmation step; can be confirmed to include.

The compound of the present invention can be extracted from Ganoderma lucidum with a solvent at room temperature or warm conditions using an alcohol (methanol, ethanol, isopropanol, butanol), an organic solvent or a mixed solvent of these and purified water.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated and the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

The present invention also provides a dietary supplement for cancer prevention and improvement comprising the compound of the present invention and a food supplement acceptable food supplement. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. And health functional foods. In detail, the present invention provides a dietary supplement for cancer prevention and improvement comprising an extract containing the licorice extract as an active ingredient and a food supplement acceptable food supplement additive.

While the present inventors studied anticancer activity using a compound derived from Ganoderma lucidum, the compound Ergosta-7,22-dien-3β, 5α, 6α-triol of the present invention extracted from Ganoderma lucidum has apoptosis effect for cancer cell death. The present invention has been completed by confirming to actively induce.

The compound of the present invention has been shown to be excellent in cancer cell killing effect, thereby resulting in DNA fragmentation, caspase-3 activity and caspase-3 and PARP cleavage cleavage activity accompanying the apoptosis effect. In addition, the compound may be usefully used as a pharmaceutical composition and health functional food for treating, preventing or improving cancer with a safe substance having low cytotoxicity.

1 is a ¹ H NMR spectrum result of Compound 1 of the present invention.
2 is a ¹³ C NMR spectrum of Compound 1 of the present invention.
3 is a graph showing the caspase-3 activity of compound 1 of the present invention.
4 is a photograph showing DNA fragmentation of cancer cells of Compound 1 of the present invention.
5 is a Western blot showing the proteolytic cleavage of PARP and caspase 3.
6 is a graph showing tumor size generated by injecting cancer cells into female BDF-1 mice.
7 is a graph showing tumor weight generated by injecting cancer cells into female BDF-1 mice.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the contents introduced herein are thorough and complete, and that the spirit of the present invention to those skilled in the art can fully convey.

Example 1 Separation and Physicochemical Properties of Compounds from Ganoderma lucidum

In order to extract anticancer compounds from Ganoderma lucidum, 10 kg dried Ganoderma lucidum body was first extracted by heating three times in 50 l of methanol. After distilling the methanol of the methanol extract under reduced pressure, 390 g of the remaining extract was dissolved in 5 L of water and fractionated using 5 L of hexane and 5 L of chloroform (CHCl ₃ ).

The chloroform fraction was eluted in a silica gel column under the same conditions as CHCl ₃ -MeOH (200: 1 to 1: 1) and fractionated into 9 subfractions (Subfraction C1-C9). Among them, the subfraction C9 showing activity was silica gel. Fractionation into four subfractions (C91-C94) was carried out using a column (60: 1 to 40: 1). Among the compounds showing the highest activity belong to the (C94) fraction and the active compound was identified as Ergosta-7,22-dien-3β, 5α, 6α-triol by analysis using ¹ H NMR and ¹³ C NMR. (Hereinafter referred to as 'Compound 1'). The physicochemical properties of Compound 1 are as follows, and ¹ H NMR and ¹³ C NMR spectral graphs are shown in FIGS. 1 and 2.

Compound 1 [Ergosta-7,22-dien-3β, 5α, 6α-triol]: white powder; [α] 25 D 2.0 ° ( c = 0.1, MeOH); IR (KBr) ν max cm ^-1 : 3400, 2960, 1650, 1460, 1385;

1 H NMR (CDCl 3, 300 MHz): δ 5.25 (1H, dd, J = 16.0, 8.0 Hz, H-23), 5.15 (1H, dd, J = 16.0, 7.5 Hz, H-22), 5.03 (1H, d, J = 1.5 Hz, H-7), 4.05 (1H, m, H-3), 1.03 (3H, d, J = 6.6 Hz, H-21), 0.98 (3H, s, H-19), 0.93 (3H, d, J = 6.9 Hz, H-28), 0.85 (3H, d, J = 6.9 Hz, H-26), 0.83 (3H, d, J = 6.9 Hz, H-27), 0.57 ( 3H, s, H-18);

13 C NMR (CDCl 3, 75 MHz): 32.4 (C-1), 31.4 (C-2), 68.9 (C-3), 39.8 (C-4), 68.0 (C-5), 61.6 (C-6) , 65.3 (C-7), 125.4 (C-8), 39.4 (C-9), 36.0 (C-10), 19.2 (C-11), 36.8 (C-12), 43.2 (C-13), 152.8 (C-14), 25.2 (C-15), 27.4 (C-16), 57.1 (C-17), 18.3 (C-18), 16.7 (C-19), 40.5 (C-20), 21.4 (C-21), 135.7 (C-22), 132.5 (C-23), 43.0 (C-24), 33.3 (C-25), 20.1 (C-26), 21.1 (C-27), 18.3 ( C-28).

<Example 2. Cytotoxicity>

Human promyelocytic leukemia (HL-60) and mouse Lewis lung carcinoma (LLC) cell lines were used to confirm cytotoxicity of cancer cells of Compound 1 of the present invention. The cells were maintained in RPMI 1640 with 10% bovine serum.

Cytotoxicity was measured using MTT assay and for this purpose, 1 × 10 ⁴ cells per well were first dispensed into 96 well cell culture plates. Compound 1 of the present invention was dissolved in DMSO at a concentration of 5-100 µg / ml in cell culture medium, and after about 2 hours of cell division, 20 µl was added to each well and incubated for 48 hours. Thereafter, 20 µl of 5 µg / ml MTT solution was added to each well. After two hours, the cell culture plate was centrifuged at 1500 rpm for 5 minutes, the cultures were removed, 150 μl of formazan crystals solution was added, and the Titertek microplate reader (Multiskan MCC / 340) at 570 nm. , Flow) was used to determine the value of IC50 (Inhibitory concentration 50%) by measuring the optical density (OD). Cytotoxicity confirmed through the MTT assay, as shown in Table 1, Compound 1 of the present invention has a superior anticancer activity corresponding to Adrianmycin (Adrimycin).

Condition
IC50 (μg / ml)
HL-60
LLC
Compound 1 of the Invention
12.7
15.2
Adriamycin
3.0
2.7

<Example 3. Caspase-3 activity confirmation>

Caspase-3 enzyme activity was measured using the proteolytic cleavage of caspase-3. For this purpose, HL-60 cells were dispensed in 96-well plates at 1 × 10 ⁵ per well, 5 to 100 Μg / ml of Compound 1 of the present invention were treated and incubated for 24 hours. After 24 hours, cells were washed with 4 ° C. PBS, and cells were dissolved in 15 μl of lysis buffer [10 mM Tris-HCL (pH 8.0), 10 mM EDTA, 0.5% Triton X-100] at 25 ° C. for 10 minutes. Leave on and then put on ice. Here, 100 μl of caspase-3 activity measuring buffer [100 mM Hepes (pH 7.5), 10 mM dithiothreitol, 10% (w / v) sucrose, 0.1% (v / v) Chaps, 0.1% (v / v). ) BSA] and 10 μl of a fluorogenic substrate (200 μM in assay buffer) were added and left at 37 ° C. for 1 hour. Subsequently, measurements were performed at 370 nm (excitation) and 505 nm (emission) using a fluorescence plate reader (Twinkle LB970 microplate fluorometer, Berthold Technologies, Germany) and the results are shown in FIG. 3. In cells treated with Compound 1 of the present invention, caspase-3 activity was significantly increased in a concentration and time dependent manner as shown in FIG. 3.

<Example 4. DNA fragmentation for confirmation of cell death>

In order to confirm how Compound 1 of the present invention affects DNA of cancer cells due to cell death, the DNA status of Compound 1 treated cancer cells was confirmed by electrophoresis. To this end, compound 1 of the present invention was reacted with ⁵ × 10 ⁵ HL-60 cells for 24 hours. As a positive control group, Campthothecin with anticancer activity was used, and only negative amount of DMSO, a solvent of Compound 1, was added in an equal amount (less than 0.1% of the total cell medium). After 24 hours, the cells were centrifuged at 1,500 rpm and 4 ° C, and the supernatant was removed and washed with PBS. Subsequently, the cells were allowed to settle by centrifugation at 3000 rpm for 10 minutes at 4 ° C. When the DNA was purified from the cells by this reaction, Apoptotic DNA Ladder Kit (Roche, Germany) and 1% agarose gel (1% w / v) were used. DNA status was confirmed using agarose gel). The results are shown in FIG. 4, which shows that Compound 1 of the present invention induced DNA fragmentation of typical apoptosis similar to Campthothecin with anticancer activity.

<Example 5. Proteolytic cleavage confirmation of caspase-3 and PARP>

Next, proteolytic cleavage of caspase-3 and PARP was confirmed, indicating that apoptosis occurred. After treating Compound 1 of the present invention with 5x10 ⁵ HL60 cells for 24 hours, the cells were lysed with a cell lysis buffer. . Cells that did not lyse were removed by centrifugation at 14,000 rpm for 10 minutes, and 50 μg of proteins were electrophoresed by SDS-PAGE and transferred to PVDF membranes. Confirmation of protein expression was confirmed using Western blot and shown in FIG. 5. As a positive control group, Campthothecin with anticancer activity was used, and the negative control group was given the same amount of DMSO as the solvent of the compound (less than 0.1% of the total amount of the cell medium). As a result of apoptosis due to the caspase-3 activity, it can be seen that PARP proteolytic cleavage is increased to activate the proteolytic cleavage of caspase-3. As a result of FIG. Due to the proteolytic cleavage of PARP and caspase-3 activity can be confirmed.

Example 6 in vivo anticancer activity

Next, LLC cells were injected into the skin of five female BDF-1 mice to confirm in vivo anticancer activity. LLC cells were maintained in 10% bovine serum and RPMI medium. To this end, 5 × 10 ⁶ LLC cells were injected into the skin of female BDF-1 mice, followed by dissolving Compound 1 of the present invention in 0.2 ml per 20 g of body weight in 2% (w / v) acacia (gum arabic). It was. Thereafter, 1 mg / kg and 10 mg / kg of Compound 1 of the present invention and 2 mg / kg of adriamycin (adriamycin) were administered daily for 14 days and the tumor volume and body weight were measured on the 21st day. Was measured. 6 and 7 show that Compound 1 of the present invention has an excellent anticancer effect similarly to adriamycin, a positive control group.

Example 7 Preparation of Pharmaceuticals

7-1. Manufacture of powder

Compound 1 of the present invention ............... 300 mg

Lactose ... 100mg

Talc ........................ 10mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

7-2. Manufacture of tablets

Compound 1 of the present invention ............... 300 mg

Corn starch ........................... 100 mg

Lactose ... .. 100mg

Magnesium Stearate ......................................................... 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

7-3. Manufacture of capsules

Compound 1 of the present invention ............... 300 mg

Corn starch ........................... 100 mg

Lactose ... .. 100mg

Magnesium Stearate ......................................... 2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

7-4. Injection preparation

Compound 1 of the present invention ............... 300 mg

Sterile Distilled Water for Injection ...

pH adjuster ...............

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

7-5. Preparation of liquid

Compound 1 of the present invention ......................................... 0.5g

Isomerization sugar ... 10 g

Mannitol ... 5 g

Purified water ................................................. ..

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Example 8 Preparation of Health Foods

8-1. Manufacture of health food

Compound 1 of the present invention ......................................... 1000 mg

Vitamin Blend ...........

Vitamin A Acetate ............... 70 μg

Vitamin E ........................................ 1.0 mg

Vitamin B1 ......................................... 0.13mg

Vitamin B2 ........................................ 0.15mg

Vitamin B6 ......................................... 0.5 mg

Vitamin B12 ......................... 0.2 μg

Vitamin C ......................................... 10 mg

Biotin ......................................................... 10 ㎍

Nicotinamide ................................... 1.7 mg

Folic acid ......................................... 50㎍

Calcium Pantothenate ......................................... 0.5mg

Mineral mixture ...........

Ferrous Sulfate ............... 1.75mg

Zinc Oxide ......................................... 0.82mg

Magnesium Carbonate ......................................... 25.3mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate Dioxide ............... 55mg

Potassium citrate ............... 90 mg

Calcium Carbonate ......................................... 100 mg

Magnesium Chloride ......................................... 24.8mg

The composition ratio of the above-mentioned vitamin and mineral mixtures is composed of relatively suitable ingredients for health foods as a preferred formulation, but the formulation ratio may be arbitrarily modified, and the above ingredients may be mixed according to a general health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Although the composition ratio is mixed with a relatively suitable component for a preferred beverage in the preferred formulation example, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demand country, and use purpose.

Claims (4)

Ergosta-7,22-dien-3β, 5α, 6α-triol of the formula (1) as a active ingredient for cancer prevention and treatment pharmaceutical composition.
[Formula 1]

The method of claim 1,
The composition is a pharmaceutical composition for preventing and treating cancer, characterized in that to induce cleavage and activity of caspase-3 (Caspase-3). The method of claim 1,
The composition is a pharmaceutical composition for preventing and treating cancer, characterized in that to induce the cleavage (cleavage) of PARP (Poly ADP ribose polymerase). Ergosta-7,22-dien-3β, 5α, 6α-triol of the formula (1) and a food supplement for food supplements, characterized in that it comprises a food supplement acceptable food supplement.
[Formula 1]

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