KR101454359B1 - Pharmaceutical composition and health functional food for prevention or treatment of cancer comprising compound from dendropanax morbifera lev. extract as effective component - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating cancer and a health functional food for preventing or ameliorating cancer, which comprises a compound isolated from the extract of Hexachia bitera as an active ingredient.
In addition, the present invention provides a method for producing an extract of Hwangcholgulchiae japonica, which comprises adding water containing purified water, C1 to C4 lower alcohol, hexane or a mixed solvent thereof, A step 2 of preparing the Fusarium oxysporum fructus fractions by carrying out a phylogenetic fraction on the Fusarium oxysporum extract prepared in step 1; And separating and purifying the fragrant wood fractions produced in the step 2 by chromatography to prepare beta-amylin, alpha-amylin, dendropanic acid, beta-sitosterol or stigmasterol, There is provided a process for producing a nodal compound.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a compound isolated from the extract of Horticultura, as an active ingredient, and a health functional food for cancer prevention or improvement. [0002] PHARMACEUTICAL COMPOSITION AND HEALTH FUNCTIONAL FOOD FOR PREVENTION OR TREATMENT OF CANCER COMPRISING COMPOUND FROM DENDROPANAX MORBIFERA LEV. EXTRACT AS EFFECTIVE COMPONENT}

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a compound isolated from the extract of Hwangcholgulgi, as an active ingredient, and a health functional food for cancer prevention or improvement.

It is an evergreen broad-leaved arboreous tree native to South Jeolla Province and Jeju Island in Korea. Yellowish lacquer is called yellow lacquer. Yellow lacquer is used as paint for furniture. According to recent research results, it has been reported that U. pergolium has antioxidant activity, immunity enhancement, nerve stabilization, antibacterial activity, and anticancer activity.

Chemotherapy to treat cancer induces apoptosis of cancer cells, killing cancer cells. In cancer cells, once apoptosis is caused, cancer cells are destroyed and converted to apoptotic bodies. Subsequently, the epoprotein body is cleared by surrounding phagocytes, so that it does not involve side effects such as an inflammatory reaction. Therefore, induction of apoptosis in cancer cells is an effective method for removing malignant tumor cells by chemotherapeutic drugs (Hannun, 1997; Kaufman and Earnshaw, 2000).

Accordingly, the present inventors have conducted studies on natural products having excellent anticancer effects in order to develop pharmaceutically safe anticancer drugs capable of inducing apoptosis, and as a result, it has been found that compounds isolated from Hwangchil-myeon, especially triterpenoid compounds, And thus the present invention has been completed.

[Patent Literature]
KR1020120101743A

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer and a health functional food for preventing or ameliorating cancer, which comprises a compound isolated from Hwangcholguk extract as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient,

The present invention provides a health functional food for preventing or ameliorating cancer which contains, as an active ingredient, a compound which is isolated from an extract of Hokkaido, Japan.

In the present invention,

Wherein the compound isolated from the extract is one or more triterpenoid compounds selected from the group consisting of beta-amylin, alpha-amylin, dendropanoxide, beta-sitosterol, and stigmasterol, Is lung cancer or breast cancer.

The present invention relates to a method for producing an extract of Hwangcholgulchiae japonica by adding water containing purified water, C1 to C4 lower alcohol, hexane or a mixed solvent thereof, A step 2 of preparing the Fusarium oxysporum fructus fractions by carrying out a phylogenetic fraction on the Fusarium oxysporum extract prepared in step 1; And separating and purifying the fragrant wood fractions produced in the step 2 by chromatography to prepare beta-amylin, alpha-amylin, dendropanic acid, beta-sitosterol or stigmasterol, There is provided a process for producing a nodal compound.

In the present invention,

The systematic fraction of the second step is characterized by suspending the extract of Wuchungchuang prepared in the above step 1 in water, followed by fractionation with n-hexane, chloroform, ethyl acetate and n-butanol.

The pharmaceutical composition for prevention or treatment of cancer, which contains the compound isolated from the extract of Quercus acutiloba of the present invention, in particular, the triterpenoid compound as an active ingredient, and the health functional food for cancer prevention or improvement are used for the apoptosis of cancer cells And inhibits the growth of cancer cells, and thus can be usefully used for prevention or treatment of cancer.

Figure 1 shows the growth of cancer cells (A-549 and MCF-7) of beta-amyrin, alpha-aminoline and olein-12-ene-3,24- Inhibitory effect.
FIG. 2 shows the effect of inhibiting the growth of cancer cells (A-549 and MCF-7) of dendropanic acid, beta-sitosterol and stigmasterol among triterpenoid compounds isolated from P. forest.
FIG. 3 shows the DNA content and cell number of each cell cycle of cancer cells (A-549 and MCF-7) treated with triterpenoid compounds isolated from P. forest.
FIG. 4 shows the results of the expression of Bax and Bcl-2, which regulate epopotisis by Western blotting after treatment of cancer cells (A-549 and MCF-7) .

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition for preventing or treating cancer, the health functional food for cancer prevention or improvement according to the present invention contains a compound isolated from Dendropanax morbifera Lev. Extract as an active ingredient. In the following Examples of the present invention, triterpenoid compounds will be described as examples of the compounds isolated from Horticultural Extract, but the present invention is not limited thereto.

The triterpenoid compound induces apoptosis of cancer cells to inhibit the growth of cancer cells. G0 / G1, S, and G2 / M phase DNA contents were reduced by stopping cell cycle progression in human lung cancer cells and human breast cancer cells, and apoptotic Sub-G1 phage DNA (killed by apoptosis Cancer cell DNA). This cancer cell apoptosis is induced by a pathway that reduces the expression of anti-apoptotic Bcl-2 protein and increases the expression of pro-apoptotic Bax protein.

In the present invention, the triterpenoid compound is preferably selected from the group consisting of β-amyrin represented by the following formula (1), α-amyrin represented by the following formula (2), dendro Dendropanoxide, β-sitosterol represented by the following formula (4), and stigmasterol represented by the following formula (5).

[Chemical Formula 1]

(Wherein R < ₁ > is H)

(2)

(Wherein, R ₁ is CH ₃₎

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The triterpenoid compound can be obtained from the wood from three trees through the following three steps.

The present invention relates to a method for producing an extract of Hwangcholgulchiae japonica by adding water containing purified water, C1 to C4 lower alcohol, hexane or a mixed solvent thereof, A step 2 of preparing the Fusarium oxysporum fructus fractions by carrying out a phylogenetic fraction on the Fusarium oxysporum extract prepared in step 1; And isolating and purifying the fragrant wood fractions prepared in step 2 above by chromatography to obtain beta-amylin, alpha-amylin, dendropanic acid, beta-sitosterol or stigmasterol.

Hereinafter, the first step of preparing the extract of Huangchu tree from the above-mentioned Huangchu tree will be described in detail.

a) Weigh accurately 500 to 3000 g of green leaf oil, b) extract with water containing purified water, lower alcohols of C1 to C4, hexane or a mixed solvent thereof, and shaking at 10 to 200 DEG C for 1 to 20 hours After filtration, c) Concentrated solution can be obtained by concentration under reduced pressure to increase the component content of the filtered extract.

Hereinafter, the second step of preparing the fractions of Wuchulia chinensis by carrying out the fractionation of the phylloquinone tree is described below.

First, the Yellowish cherry tree concentrate obtained in step 1 is suspended in water, and then subjected to a first fractionation with n-hexane to be fractionated into a water layer and a n-hexane layer (Hexane ext.). The fraction of the water layer is fractionated with chloroform into a water layer and a chloroform layer (CHCl ₃ ext). The fraction of the water layer is fractionated with ethyl acetate to a water layer and an ethyl acetate layer (EtOAc ext.). Finally, the water layer is fractionated into a water layer and an n-butanol layer using n-butanol (n-butanol).

The three steps of preparing the triterpenoid compound by separating and purifying the fragrant wood fractions by chromatography will be described in detail as follows.

The n-hexane layer obtained in the above step 2 was loaded on chromatography packed with silica gel, and a solvent mixture of hexane: acetone at a ratio of 100: 1 to 1: 1 (W / V) was used A number of small fractions eluted by these solvents are obtained. The resulting small fractions were purified by column chromatography on silica gel using hexane (Hexane): Acetone in an open-column silica gel chromatography chromatograph (100: 1 to 80: 1 (W / V) or 100: 1 to 1: 1 (W / V) Or LiChroprep RP-18 column chromatography to obtain the above compounds. The molecular structure of these compounds can be determined by measuring ¹ FT-IR, ¹ H-NMR, ¹³ C-NMR and EI-MS spectra.

The pharmaceutical composition for preventing or treating cancer, which contains the triterpenoid compound isolated from the above-mentioned Houttuynia forest extract as an active ingredient, can be administered orally or parenterally, and can be administered orally, parenterally or intraperitoneally , Subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection.

The pharmaceutical composition may further contain conventionally used excipients, disintegrants, sweeteners, lubricants, flavors, and the like. Examples of the disintegrant include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, chitosan, guar gum, magnesium aluminum silicate, and polacrilin potassium.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate Starch glycolate, sodium starch glycolate, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, starch, sodium carboxymethylcellulose, Dextrose, sorbitol, talc, and the like may be used. The pharmaceutically acceptable additives according to the present invention are preferably contained in an amount of 0.1 to 90 parts by weight based on the pharmaceutical composition.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, syrups and aerosols. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives .

As the agent for parenteral administration, the external preparation such as powders, granules, tablets, capsules, sterilized aqueous solutions, liquid preparations, non-aqueous solutions, suspensions, emulsions, syrups, suppositories and aerosols, The composition may be formulated in the form of a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma, , But is not limited thereto. 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. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, age, sex and condition of the patient, and severity of the disease to be treated. For the desired effect, however, in the case of an oral administration, administration of the composition of the present invention at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per 1 kg of body weight per day to an adult good. The administration may be carried out once a day or divided into several times.

In addition, the health functional food for preventing or ameliorating cancer, which contains the compound isolated from the extract of Echinochloa crus-galli according to the present invention as an active ingredient, can be used together with other foods or food components and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or hygiene).

There is no particular limitation on the form and kind of the health functional food. The form of the health functional food to which the substance can be added may be tablets, capsules, powders, granules, liquids, and rings. The types of health functional foods include dairy products including butter, yogurt, cheese, dairy products including ice cream, Lactic acid bacteria preparation, fermented milk, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, various soups, drinks, tea, drink, alcoholic drinks and vitamin complex.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient such as a normal health functional food. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, polysaccharides such as disaccharide such as maltose and sucrose, dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, Glycerin, alcohols, carbonating agents used in carbonated beverages, and the like.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

< Example  1> Manufacture of woodwort extract Triterpenoid  Separation of compounds

1-1. Manufacture of methanol extract of yellowtail

1.3 kg of green leaf oil was extracted with 100% MeOH at 60 ° C for 3 hours. The extracted methanol extract was concentrated under reduced pressure to obtain 90 g of methanol extract of Huaculi wood.

1-2. Woodwort Fraction  Produce

The concentrate obtained in Example 1-1 was sufficiently suspended in water and the first solvent was transferred three times with two times volume of n-hexane to separate the water layer and the n-hexane layer (Hexane ext. 35.2 g). The water layer was transferred to the second solvent three times with twice the volume of chloroform (CHCl ₃₎ to obtain a water layer and a chloroform layer (CHCl ₃ ext. 8.1 g). The water layer was further subjected to a third solvent transfer three times with twice the volume of ethyl acetate (EtOAc) to obtain a water layer and ethyl acetate layer (EtOAc ext. 810 mg). Finally, the water layer was subjected to the fourth solvent transfer three times with n-butanol to obtain a butanol layer (n-BuOH ext. 17.44 g).

1-3. Burrows In the hexane layer  Isolation of active ingredient

6 g of the hexane layer (Hexane ext. 35.2 g) of Example 1-2 was taken out and subjected to open column silica gel chromatography to obtain 10 subfractions (H1 to H10) . The solvent used was hexane (Hexane: Acetone) at a ratio of 100: 1 to 1: 1 (W / V).

H-1 (146.0 mg), the first fraction of 10 small fractions, was dissolved in hexane (Hexane): Acetone in a solvent of 100: 1 to 80: 1 (W / V) 12-ene-3, 24-diol (3.0 mg) was obtained by carrying out torque cycling to obtain β-amyrin (10.0 mg) and oleyl-12-ene-24,2-diol

H-4 (80.7 mg), the fourth fraction of 10 small fractions, was dissolved in hexane (Hexane): Acetone in a solvent of 100: 1 to 1: 1 (W / V) After torque-cycling, open-column silica gel chromatography was further performed to obtain alpha-amyrin.

H-6 (98.6 mg), the sixth fraction of 10 small fractions, was dissolved in hexane (Hexane): Acetone in a solvent of 100: 1 to 1: 1 (W / V) After conducting the torque peptides, open-column silica gel chromatography chromatography with hexane: Acetone in a solvent of 10: 1 yielded β-sitosterol (10 mg), stigmasterol (4.3 mg, ).

H-7 (654 mg), the 7th fraction of 10 small fractions, was dissolved in hexane: Acetone in a solvent of 100: 1 to 1: 1 (W / V) After lapping, LiChroprep RP-18 column chromatography was performed to obtain dendropanoxide.

1-4. NMR Structure identification using

The substance obtained in Example 1-3,? -Amyrin,? -Amyrin, olein-12-ene-24-β-diol , 24 β-diol), dendropanoxide, β-sitosterol and stigmasterol were measured by FT-IR, ¹ H-NMR, ¹³ C-NMR and EI-MS spectra The structure of these materials was identified.

1) beta-amyrin &lt; / RTI &gt;

White amorphous powder; _{[α] D: + 88.3 °} (c 0.01, CHCl 3); IR (KBr): V _max 3300, 2950 cm ^-1 ; FAB-MS: m / z 427 [M + H] &lt; ⁺ &gt;.

2) alpha-amyrin &lt; RTI ID = 0.0 &gt;

White amorphous powder; _{[α] D: + 92.0 °} (c 0.005, CHCl 3); IR (KBr): V _max 3300, 2950 cm ^-1 ; FAB-MS: m / z 427 [M + H] &lt; ⁺ &gt;.

3) Olean-12-en-3, 24-diol

Colorless needles; [[alpha]] _D : +84.7 [deg.] (c 0.46); IR (KBr): V _max 3600-3100, 1630, 1038, 1020, 810 cm ^-1 ; EI-MS: m / z 442 [M] ^&lt; ⁺ & gt ^; .

4) Dendropanoxide

Colorless needles; [?] _D : + 45.7 ° (c 0.01, CHCl ₃ ); _{IR (KBr): V max 2950} cm -1; FAB-MS: m / z 427 [M + H] &lt; ⁺ &gt;, 44 [M + Na] &lt; ⁺ &gt;.

5) Beta-sitosterol

White amorphous powder; [?] _D : -40.7 ° (c 0.05, CHCl ₃ ); IR (KBr): V _max 3400, 2930 cm &lt; ^{-1 &} gt ;; FAB-MS: m / z 297 [M + H] &lt; ⁺ &gt;.

6) Stigmasterol

Colorless crystals; _{[α] D: -51 ° (} c 0.1, CHCl 3); IR (KBr): V _max 3494 (OH), 1053, 1020 (OH) cm ^-1 ; EI-MS: m / z 412 [M] ⁺ .

< Experimental Example  1> Inhibitory effect of growth of cancer cells

Cell survival rates of A-549 (human lung cancer cells) and MCF-7 (human breast cancer cells) were examined by MTT assay to analyze the effect of triterpenoid compound on cancer cell growth.

A-549 (human lung cancer cells) and MCF-7 (human breast cancer cell) cells were plated at a density of 4 × 10 ⁵ cells / well in a 96-well plate. After 24 hours, each sample (β-amylin, (10, 30, 50, 100 and 200 μM) were cultured for 24 hours. The cells were treated with various concentrations (10, 30, 50, 100 and 200 μM) After that, MTT solution was added, and after 4 hours, the medium was removed and washed once with PBS. After the DMSO was treated and allowed to stand at room temperature for 30 minutes, the absorbance was measured at 570 nm.

As a result, beta -amyrin and alpha-aminin inhibited the growth of A-549 cells and MCF-7 cells in a concentration-dependent manner (FIG. 1A, FIG. 1B). In addition, dendronaxone, beta-sitosterol and stigmasterol also inhibited the growth of A-549 cells and MCF-7 cells in a concentration-dependent manner (FIGS. 2A and 2B). These results indicate that these five triterpenoid compounds except oline-12-ene-3, 24-diol inhibit the growth of human lung cancer cells and human breast cancer cells.

< Experimental Example  2> Flow cell count ( Flow cytometry ) Experiment

A-549 cells and MCF-7 cells were treated with beta-amylin, alpha-amylin, dendropanoxide, beta-sitosterol and stigmasterol, And cultured for an hour to induce apoptosis. After washing with PBS (phosphate buffered saline), the cells were cultured in PI solution containing RNAase A and propidium iodide (PI).

Using a triterpenoid compound flow cytometer (FACSCalibur ^TM, Beckton Dickinson, GmbH, Heidelberg, GermanyBecton DICKINSON) to analyze popto sheath (apoptosis) in cancer cells by, the DNA corresponding to each of the cell cycle The content and cell number were measured, and the results are shown in Fig.

The content of G0 / G1, S, and G2 / M phase DNA was decreased in A-549 cells treated with triterpenoid compound, but the content of sub-G1 phage DNA (DNA of cells killed by epopotisis) (Fig. 3A). These results were also observed in MCF-7 cells (Fig. 3B). Therefore, it was found that these compounds stop cell cycle progression and induce apoptosis in human lung cancer cells and breast cancer cells.

< Experimental Example  3> Western Blasting ( Western Blotting ) Experiment

A-549 cells and MCF-7 cells were treated with beta-amylin, alpha-amylin, dendropanic acid, beta-sitosterol and stigmasterol for 24 hours and then subjected to Western blotting.

The cells were treated with a lysis buffer (50 mM Tris / HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 10% glycerol, 10 μg / mL leupeptin (10 μg / mL), 2 mM PMSF) After dissolution, proteins were extracted by centrifugation, proteins were separated by SDS PAGE and transferred to nitrocellulose membrane. Then, to confirm the expression of Bax and Bcl-2 protein that regulate apoptosis, each antibody was attached to the membrane, secondary antibody connected with horseradish peroxide (HRP) was bound, and ECL Western blotting was performed using a reagent, and the results are shown in Fig.

As can be seen in FIG. 4, the expression of Bcl-2 protein was decreased in A-549 cells and MCF-7 cells (FIGS. 4A and 4B). On the other hand, the expression of Bax protein was increased in A-549 cells and MCF-7 cells (Figs. 4A and 4B). These results suggest that these compounds induce apoptosis of cancer cells through decreased Bcl-2 expression and increased Bax expression.

< Manufacturing example  1> Preparation of pharmaceutical preparations

1-1. Sanje  Produce

200 mg of the compound of the formula (1)

Lactose 100 mg

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

100 mg of the compound of the formula (1)

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

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

1-3. Preparation of capsules

100 mg of the compound of the formula (1)

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

1-4. Manufacture of rings

100 mg of the compound of formula (I) of the present invention

Lactose 150 mg

100 mg of glycerin

Xylitol 50 mg

After mixing the above components, they were prepared to be 4 g per one ring by a conventional method.

1-5. Manufacture of granules

150 mg of the compound of formula (I) of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacture of Health Functional Foods

2-1. Manufacture of flour food products

0.5-5.0 parts by weight of the compound of formula (I) of the present invention was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

2-2. soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of the compound of the formula (1) of the present invention was added to the soup and the juice to prepare health promotion soup and juice.

2-3. ground Beef  Produce

10 parts by weight of the compound of formula (1) of the present invention was added to ground beef to prepare ground beef for health promotion.

2-4. dairy product( dairy products )

5 to 10 parts by weight of the compound of formula (I) of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Claims (8)

delete A pharmaceutical composition for preventing or treating cancer, which comprises dendropanic acid isolated from a Horticulte extract, as an active ingredient.
3. The method of claim 2,
The pharmaceutical composition for preventing or treating cancer, wherein the cancer is lung cancer or breast cancer.
delete A health functional food for preventing or ameliorating cancer, which contains dendrofenoxide as an active ingredient, which is isolated from a Horticultural extract.
6. The method of claim 5,
Wherein the cancer is lung cancer or breast cancer. delete delete

Images