KR20190086132A - A pharmaceutical composition comprising fraction of Lespedeza cuneata extract for preventing or treating cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, containing a fraction of a Lespedeza cuneate extract as an active component. The fraction of the Lespedeza cuneate extract of the present invention has an effect of inducing apoptosis of cancer cells, and also has an advantage of little or no side effects by using ingredients derived from natural plants. In addition, the present invention confirms that a Lespedeza cuneate ethyl acetate fraction is particularly toxic to ovarian cancer cells and suggests the availability as an effective cancer treatment by separating a compound which has a great effect on reducing the viability of ovarian cancer cells from the ethyl acetate fraction.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancers,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a fraction of an extract from a yam door extract as an active ingredient.

" Lespedeza cuneata "is a perennial plant belonging to the soybean family. It is said to be a subspecies in our language, and as a Chinese character, it is called a 葉 추), 葉)), , Half-lightning, closed door grass, public grass grass, yin-yang grass, white guanmen grass, and wild grass grass. It is known. The physiologically active substance contained in the doorway contains pinitol, flavonoid, phenol, tannin and β-sitosterol, and among the flavonoids, quercetin ), Kaempferol, vitexin, orientin and the like, and the yamantown is excellent for bronchial asthma and bronchial asthma severely coughing and getting a lot of sputum, It is also well known that the effect of male sexual function improvement (Korean Registered Name) is improved, but it is also known to be effective in improving male sexual function. Patent Publication No. 10-1784503), and the therapeutic effect of cancer, particularly the therapeutic effect of ovarian cancer, is not known.

On the other hand, ovarian cancer is one of the worst prognosis of genital cancer in women, most of which is epithelial carcinoma. The incidence of ovarian cancer is 1.2%, but progressive disease of stage III or IV is seen in 60 to 70% of patients at the time of diagnosis and the 5-year survival rate is less than 50% of the total (Siegel R, Ward E, Brawley O, A. Cancer statistics, 2011). It is also the sixth most common and the most mortal among women worldwide (Gui & Shen, 2012; Romero & Bast, 2012).

The most common causes of ovarian cancer are BRCA1 and BRCA1 mutations. In addition, only the risk factors such as infertility, elderly, family history, breast cancer, endometrial cancer, obesity, and obesity are known. Intraperitoneal metastasis is most common, and vascular or lymphatic metastasis may occur.

To date, the classic treatment is to perform platinum-taxane-based chemotherapy after staging laparotomy and tumor debulking to minimize tumor residual lesions. However, despite this active treatment, about 70-80% of ovarian cancer patients recur after the first chemotherapy. In conclusion, ovarian cancer is a refractory disease with severe side effects and high recurrence rate. Therefore, effective drug development for ovarian cancer treatment and prevention is needed.

Under these circumstances, the present inventors have confirmed the activity of inducing apoptosis of ovarian cancer cells in fractions of the extracts of Yushenko extract, and separated the compounds contributing to these activities to complete the present invention.

It is an object of the present invention to provide a composition for preventing, treating or ameliorating a cancer comprising a fraction of a Yamazaki extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a fraction of an extract from a yam door extract as an active ingredient.

Further, the present invention provides a health functional food composition for preventing or ameliorating cancer, which comprises a fraction of a yam Kanto extract as an active ingredient.

In addition, the present invention provides a method for treating cancer, comprising treating a fraction of a Yamazaki extract with an individual.

In addition, the present invention provides the use of the fractions of the night-soil extract to treat cancer.

In one embodiment of the present invention, the yam cabbage extract may be a methanol extract.

In another embodiment of the present invention, the fraction may be the ethyl acetate fraction of the yam cabbage extract.

In another embodiment of the present invention, the fraction may contain a compound represented by the following formula (1) in a concentration of 0.01 to 10 μg / mg.

[Chemical Formula 1]

In another embodiment of the present invention, the fraction may contain a compound represented by the following formula (2) in a concentration of 0.01 to 10 μg / mg.

(2)

In another embodiment of the present invention, the cancer may be an ovarian cancer.

The fraction of the extract of the present invention of the present invention has an effect of inducing apoptosis of cancer cells, and has advantages of using little or no side effects by using ingredients derived from natural plants. In addition, the present invention confirms that the benzoyl ethane acetate fraction is particularly toxic to ovarian cancer cells, and separates compounds that greatly affect the viability of ovarian cancer cells from the ethyl acetate fraction and thus can be used as an effective cancer therapeutic agent present.

FIG. 1 is a graph showing the effect of the extract of the yam Kwan Mun extract and its fractions on the viability of the ovarian cancer cell line A2780 cells. Specifically, (A) is yagwanmun methanol extract, (B) is its n - butanol (n- butanol: BuOH) fraction, (C) hexane (hexane: HX) fraction, (D) ethyl acetate (ethyl acetate: EA ), And (E) shows the change in viability of A2780 according to the treatment of dichloromethane (MC) fraction.
2 is a diagram showing the structures of Compounds 1 to 9 isolated from ethyl acetate fraction of a methanol extract of a gazebo door.
FIG. 3 is a graph showing the change in A2780 cell viability according to the treatment with an active compound of the ethyl acetate fraction of the fieldbus.
FIG. 4 is a diagram showing the morphological changes of A2780 cells according to the treatment of Compound 3 with a phase contrast microscope (A) and a fluorescence microscope (B).
FIG. 5 is a graph showing that the expression of apoptosis-related protein is increased in A2780 cells according to the treatment with Compound 3. FIG.

The present inventors have searched for functional materials having excellent physiological activity derived from plant resources, and confirmed the anticancer effect of the fractions of the fractions of the present invention without any previous studies. Based on this finding, the present invention has been completed.

Accordingly, the present invention provides a composition for preventing, treating, or ameliorating cancer comprising a fraction of an extract of a yam door extract as an active ingredient.

In this specification, " Lespedeza cuneata "is a perennial plant belonging to the soybean family. It is said to be a subspecies in our language, and as a Chinese character, it is called a 葉 추), 葉)), , Half-lightning, closed door grass, public grass grass, yin-yang grass, white guanmen grass, and wild grass grass. It is known. The physiologically active substances contained in the doorway include pinitol, flavonoid, phenol, tannin, and β-sitosterol. Among the flavonoids, quercetin quercetin, kaempferol, vitexin, and orientin have been reported. Bacillus or bronchial asthma can cause severe coughing and sputum production. It is known that it is effective in treating acute gastritis, stomach ulcer, diarrhea, exfoliation, bruise, and boil. However, it is known that the therapeutic effect of cancer, especially the therapeutic effect of ovarian cancer Is not known.

In one embodiment of the present invention, the inventors have found that the methanol extracts of this yagwanmun n - butanol (n- butanol: BuOH), hexane (hexane: HX), ethyl acetate (ethyl acetate: EA: EtOAc), or dichloromethane ( dichloromethane: CH ₂ Cl _2: MC) to prepare four fractions (see Example 1-1), and then treated with A2780 cells, a human cancer cell line, to examine the toxicity of the extracts and their fractions thereof to cancer cells , The methanol extract of the field capillary was found to be very toxic to cancer cells and the cytotoxicity of ethyl acetate fraction was most active (see Example 2-1).

Also, in one embodiment of the present invention, the present inventors conducted HPLC and column chromatography in order to identify active ingredients that inhibit the growth of ovarian cancer cells in the ethyl acetate fraction and reduce their viability, (See Example 1-2), and the change of A2780 cell survival rate was examined according to the treatment of each compound. As a result, it was confirmed that Compounds 3 and 9 were most toxic to cancer cells (See Example 2-3).

The compound 3 contained in the fraction of the extract of the present invention is lyoniresinol () -9'- O- (? -L-rhamnopyranosyl).

[Chemical Formula 1]

The fraction of the yam Guanxi extract of the present invention may contain the compound 3 at a concentration of 0.01 to 10 占 퐂 / mg, preferably 6 to 9 占 퐂 / mg, more preferably 7.0 to 8.7 占 퐂 / Concentration, but is not limited thereto.

In one embodiment of the present invention, the inventors observed morphological changes of A2780 cells following treatment with Compound 3, and found that A2780 cells treated with Compound 3 changed into apoptotic cells (see Example 3) ).

Further, in one embodiment of the present invention, the present inventors conducted western blotting in order to more specifically confirm the mechanism of inducing apoptosis of ovarian cancer cells of Compound 3. The apoptotic effector caspase, caspase-3, is activated by caspase-8, an apoptotic initiator caspase, and activation of caspase-3 induces cleavage of PARP. Bid is known to be an activated (truncated) substrate of caspase-8. In the mitochondria, caspase-8 activated Bid migrates directly to change the membrane potential, while Bcl2 (anti-apoptotic) / Bax (pro- To induce cytochrome c release to cytochrome c, leading to apoptosis mechanism. Western blotting revealed that Compound 3 in A2780 human ovarian cancer cells induces mitochondria-dependent cell death through activation of caspase-8. In addition, it was confirmed that expression of pro-apoptotic Bcl-2 family member and anti-apoptotic Bcl-2 family member was increased in A2780 cell treated with Compound 3. As a result, Compound 3 showed apoptosis through mitochondrial pathway (See Example 4). Therefore, Compound 3 isolated from the ethyl acetate fraction of the methanol extract of the gazebo was effective in the treatment of ovarian cancer.

The compound 9, which is contained in the fraction of the yam Kanto extract of the present invention, is a syringic acid and has a structure represented by the following formula (2).

(2)

In the specific examples of the present invention, the effect of reducing the viability of the ovarian cancer cells by the compounds 1 to 9 isolated from the fractions of the ethyl acetate fraction of the extracts of the yamguram extract was found to be the best for reducing the viability of ovarian cancer cells. Compared with the decrease rate of viability of ovarian cancer cells according to the treatment of ethyl acetate fraction, Compound 3 and Compound 9 are considered to be toxic to ovarian cancer cells together. This means that each active ingredient alone or in combination thereof is excellent in the cancer treatment effect of the ethyl acetate fraction of the yuan guanyum.

The night scoop extract of the present invention can be extracted using a conventional solvent known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. For example, in the present invention, the yam cabinet extract is prepared by dissolving 1, 2 or 3 selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, Can be extracted using more than two kinds of solvents, preferably methanol, and more preferably 80% methanol. In addition, the method for extracting the extract from the doorway can be extracted by various methods such as hot water extraction, cold extraction, reflux extraction, and ultrasonic extraction, but the present invention is not limited thereto.

The extract thus prepared may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. For example, the filtration may be performed using a filter paper or a vacuum filter, the concentration may be carried out by a reduced pressure concentrator, the spraying may be carried out by a spray drying method or a freeze drying method.

Further, the extract extracted with the solvent is further subjected to fractionation with a solvent selected from the group consisting of n -butanol, hexane, dimethyl chloride, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, And may be selected from the group consisting of n -butanol, hexane, dimethyl chloride, ethyl acetate, and mixtures thereof. Ethyl acetate is more preferably used, but is not limited thereto.

As used herein, " prophylactic " means any action that inhibits cancer or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, " treatment " means any action that improves or alters the symptoms of cancer by administration of the pharmaceutical composition according to the present invention.

Accordingly, the present invention may provide a method for treating cancer, which comprises administering to a subject a pharmaceutical composition for preventing or treating cancer comprising a fraction of the extract from the yam instructor extract as an active ingredient. Preferably, the cancer is ovarian cancer But are not limited thereto.

As used herein, the term " individual " may be a mammal such as a mouse, a livestock, a mouse, a human, and the like, and may specifically be a dog, horse, human or the like which requires chemotherapy.

The pharmaceutical compositions for preventing or treating cancers containing the fractions of the insecticide extract according to the present invention as an active ingredient can be prepared by conventional methods such as powders, granules, tablets, capsules, external preparations such as suspensions, emulsions, syrups, aerosols, It may be formulated in the form of a sterile injectable solution and may preferably have formulations of cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma. Examples of carriers, excipients and diluents that can be included in the composition containing the fractions of the insect extract include lactose, dextrose, sucrose, oligosaccharide, 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

In addition, the present invention provides a health functional food composition for inhibiting or ameliorating cancer, which comprises a fraction of an extract from a yam door extract as an active ingredient. In addition, fractions of the yam instructor extract may be added to the food for the purpose of improving cancer. When the fraction of the extract of the present invention is used as a food additive, the fraction may be added as it is or may be used together with other food or food ingredients, and may be appropriately 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 therapeutic treatment). Generally, the fraction of the night scoop extract of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The health beverage composition according to the present invention may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as 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. The ratio of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

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 following examples.

[Example]

Example 1. Preparation and method of experiment

1-1. Production of yambaekman extract and its fractions

In October, 2016, the top part of the yuan gate was harvested and used in Mt. Bangtae (Inje, Gangwon Province, Korea), and the sample was stored in Sangkyunkwan University School of Pharmacy (Suwon, Korea) (YKM-2016).

The top portion (4.2 kg) of the harvested nightstand was dried and extracted three times with 80% methanol (MeOH) (4.2 LX 3 days each) at room temperature and then filtered. Subsequently, the filtrate was evaporated under reduced pressure to obtain 401.8 g of crude extract, which was then suspended in 2 L of distilled water. The suspension was subjected to solvent fractionation using hexane, CH ₂ Cl ₂ , EtOAc, and n- BuOH (2.0 LX 3 each) in the order of increasing polarity. As a result, hexane (20.6 g), CH ₂ Cl ₂ EtOAc (12.7 g) and n- BuOH (69.3 g).

1-2. Effective compound isolation of ethyl acetate fraction

Gradient of ethyl acetate (EtOAc) fraction (12.7 g) with _{MeOH (100% H 2 O,} 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH) prepared by the method of the 1-1 Six separate subfractions (A-F) of the Diaion HP-20 column were isolated in the solvent system. Fraction D (5.4 g) was separated by RP-C ₁₈ column chromatography using a gradient solvent system of MeOH (30-100% MeOH) to yield six lower fractions (D ₁ to D ₆ ).

The sub-fraction D ₃ (2.8 g) was dissolved in CH ₂ Cl ₂ -MeOH-H ₂ O [CH ₂ Cl ₂ -MeOH (15: 1, v / v) to CH ₂ Cl ₂ -MeOH- , v / v)] as a solvent, and further divided into 10 lower fractions (D ₃ -1 to D ₃ -10) by silica gel column chromatography. The separated _{D 3 -3 (33.5 mg) Phenomenex} Luna, phenyl-hexyl column (250 × 10.0 mm, 10 m m, flow rate: 2 mL / min) at 20% in MeOH (60 minutes using a 50% ) was purified by semi-preparative HPLC using a linear gradient solvent system, to give the compound _{8 (5.1 mg, t R =} 34.0 min) and compound _{9 (1.8 mg, t R =} 36.5 min).

Further, the above D ₃ -7 (1.1 g) was separated in RP-C ₁₈ column using 60% MeOH to obtain four lower fractions (D ₃ -71 to D ₃ -74). The resulting D ₃ -72 (506.7 mg) was dissolved in CH ₂ Cl ₂ -MeOH (10: 1, v / v) to CH ₂ Cl ₂ -MeOH-H ₂ O (1: 1: 0.25, v / Silica gel column chromatography was carried out to separate again into five lower fractions (D ₃ -721 to D ₃ -725). D ₃ -722 (316.4 mg) was again conducted to separate into 10 fractions to column chromatography on Sephadex LH-20 _{100% MeOH (D 3 -722A -} D 3 -722J), the separated D ₃ -722B ( 24.0 mg) a Phenomenex Luna, semi-preparative HPLC ( 19% MeCN, flow rate using a phenyl-hexyl column: to give 2 mL / min), compound 3 (6.3 mg, t _R = 29.0 min) And the compound _{4 (2.7 mg, t R =} 32.5 min) was obtained.

The above D ₃ -74 (127.6 mg) was applied to Sephadex LH-20 column chromatography with 100% MeOH to obtain nine lower fractions (D ₃ -741-D ₃ -749). The obtained D ₃ -746 (24.9 mg) a Phenomenex Luna, phenyl-hexyl column semi -preparative HPLC using a (flow rate: 2 mL / min ) to give compound 5 as a (3.5 mg, t _R = 30.5 min) and compound 6 was obtained (3.5 mg, t _R = 34.0 min). Further, to afford the D ₃ -10 (132.7 mg) of 9 sub-fraction (D ₃ to D -101 -109 ₃₎ applied to a Sephadex LH-20 column chromatography using 80% MeOH, D ₃ - 107 (16.6 mg) a Phenomenex Luna, phenyl-hexyl column semi-preparative HPLC using a (38% MeOH, flow rate: 2 mL / min) to give the compound _{1 (1.5 mg, t R =} 31.5 min) and compound 2 It was obtained (2.4 mg, t _R = 36.0 min). Finally, Compound 7 (2.2 mg, t _R = 27.0 min) Was obtained by semi-preparative HPLC (20% MeCN, flow rate: 2 mL / min) using a Phenomenex Luna, phenyl-hexyl column at D ₃ -109 (17.2 mg).

The obtained compounds 1 to 9 were dissolved in DMSO (dimethyl sulfoxide) at a concentration of 100 mM and stored at -20 DEG C. When necessary, a sample containing the compounds 1 to 9 was added to the cell culture medium at an appropriate concentration, The final DMSO concentration in the experiment was kept below 0.5%.

1-3. Cell culture

A2780 human ovarian carcinoma cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA), fetal bovine serum (FBS; Gibco BRL, Carlsbad, MD, USA), 100 units / mL penicillin penicillin, and 100 mg / mL streptomycin at 37 ° C in a humidified condition of 5% CO _{2 in} a Roswell Park Memorial Institute 1640 medium (Cellgro, Manassas, VA, USA).

1-4. MTT assay

A2780 cells were inoculated with 1 x 10 ⁴ cells / 100 μl in 96-well plate and cultured for 24 hours. Subsequently, the cells were further cultured in a cell culture medium containing a sample for 24 hours. Cell viability was determined using the Ez-Cytox cell viability assay kit (Dail Lab Service Co., Seoul, Korea) according to the manufacturer's instructions. More specifically, 10 占 퐇 of kit reagent was added to each well, the cells were incubated for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader (PowerWave XS, Bio-Tek Instruments, Winooski, Respectively.

1-5. Hoechst 33342 cell staining

Inoculated with A2780 cells to 4 x 10 ⁵ cells / mL 3 to 6-well plates and cultured for 24 hours. The cells were further cultured in a cell culture medium containing the samples for 24 hours, and 2 μl of Hoechst 33342 solution was added to each well and incubated for an additional 10 minutes. The stained cells were then observed with a fluorescence microscope.

1-6. Western blotting

Inoculated with A2780 cells to 4 x 10 ⁵ cells / mL 3 to 6-well plates and cultured for 24 hours. The cells were further cultured for 24 hours in a cell culture medium containing a sample, and the cells were harvested and cultured in a RIPA buffer (Cell Signaling Technology, Inc.) containing 1 × EDTA-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). , MA, USA). To enhance the detection of proteins isolated from precast 415% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, Calif., USA), a primary antibody and a secondary antibody specific for the epitope (Cell Signaling Technology, Inc., Danvers, MA, USA) and electrophoresed on PVDF membrane. The conjugated antibodies were detected using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK) and FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

1-7. Statistical analysis

All data were expressed as mean ± SD and statistical significance was determined using the Student's t-test. P values less than 0.05 were considered statistically significant.

Example 2. Identification of A2780 Ovarian Cancer Cell Lines Viability Reduction According to the Processing of Fractions of the Night School Door

2-1. Analysis of A2780 cell viability by treatment with the extracts and their fractions

In order to confirm the anticancer activity of the extract of the yam Kwan Kuan and its fractions prepared by the method of Example 1-1, the extract and the four fractions were respectively treated with A2780 cells, and the cell viability Respectively.

As a result, as shown in FIG. 1, methanol extract and BuOH fraction of the basement membrane had a small effect on the viability of ovarian cancer cells (see A and B), and the HX fraction and MC fraction But could be reduced (see C and E). EA fraction significantly inhibited the proliferation of ovarian cancer cells in a dose dependent manner (Fig. 1, IC ₅₀ : 77.25 ± 2.05 ㎍ / mL), confirming that EA fraction was the most active in inhibiting A2780 cell viability Reference).

2-2. Analysis of active compounds in the ethyl acetate fraction

The efficacy of A2780 cells in Example 2-1 was confirmed to be most effective in reducing the viability of the A2780 cells, and the effective compound of the EA fraction was isolated according to Example 1-2. The column chromatography and HPLC purification were repeated to separate four lignanosides (1-4), three flavonoid glycosides (5-7), and two phenolics (8-9), which are shown in FIG. The separated compounds, by comparing the spectroscopic data, physical data, including the LC / MS analysis and ^{1 H and 13 C NMR, (} -) - (8 S, 7 'R, 8' S) -isolariciresinol-9'- O -α-L-rhamnoside (1 ), aviculin (2), () -9'- O - (α-L-rhamnopyranosyl) lyoniresinol (3), (+) - 5'-methoxyisolariciresinol-9'- O - α-L-rhamnoside (4) , kaempferol-7- O -β-D-glucopyranoside (5), astragaline (6), orientin (7), vanilic acid (8), and syringic acid (9) it was confirmed.

2-3. Analysis of A2780 cell viability by treatment with active compound of ethyl acetate fraction

In order to evaluate the cytotoxic effect of the compound isolated from the EA fraction on the A2780 cells, it was confirmed that the EA fraction of the yamancom door was most effective in reducing the viability of the A2780 cell in Example 2-1. Compounds 1 to 9 were separated from the EA fraction by the method of Example 1, and each of them was treated with A2780 cells to perform the MTT assay according to Example 1-4.

As a result, as shown in Fig. 3, the compounds other than Compounds 3 and 9 did not significantly affect the viability of the cancer cells, but Compounds 3 and 9 showed that the viability of the cancer cells was reduced in a dose-dependent manner .

Example  3. A nightstand  Ethyl acetate Fraction  Identification of morphological changes of A2780 ovarian cancer cell line following treatment with Compound 3

In order to observe the morphological changes of A2780 cells according to the treatment of EA fractions, the compound 3 showing particularly cytotoxic activity on ovarian cancer cells was treated with a concentration of 50 or 100 μM to observe A2780 cells with a phase contrast microscope, 3, A2780 cells treated with Compound 3 at a concentration of 50 or 100 μM according to Example 1-5 were stained and observed under a light microscope.

As a result, as shown in Fig. 4, the control without any treatment had a normal form, but the cells treated with the compound 3 were separated from the culture dish to cause blebbing, shrinkage, And condensation (apoptotic cell) (A). In addition, while the control group showed slightly bright blue fluorescence by the glow-light microscope observation, the A2780 cell treated with Compound 3 showed bright blue fluorescence due to condensation of the chromatin of the dead cell. Morphological changes in these cells suggest that Compound 3 induces apoptosis of ovarian cancer cells.

Example 4. Confirmation of Increase of Expression of Apoptosis-Related Protein by Treatment of Compound 3 of the Ethyl Acetate Fraction of the School Bus

Western blotting was carried out in accordance with Examples 1-6 to confirm the mechanism of action of Compound 3.

As a result, as shown in FIG. 5, the expression of cleaved caspase-8, which is an apoptotic initiator caspase, was increased in the cells treated with Compound 3. The expression of cleaved caspase-3, an apoptotic effector caspase, It was confirmed that cleavage of the substrate PARP was increased. In addition, the pro-apoptotic factor BID was cleaved and the expression of Bax was increased and the expression of Bcl-2, an anti-apoptotic factor, was decreased. The results suggest that Compound 3 induces mitochondria-dependent cell death through activation of caspase-8 in ovarian cancer cells.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (12)

A pharmaceutical composition for preventing or treating cancer, which comprises a fraction of the extract of a camphor tree extract as an active ingredient.
The method according to claim 1,
Wherein the basilariae extract is a methanol extract.
The method according to claim 1,
Wherein said fraction is an ethyl acetate fraction of a yam door extract.
The method according to claim 1,
Wherein said fraction comprises a compound represented by the following Formula 1 at a concentration of 0.01 to 10 占 퐂 / mg.
[Chemical Formula 1]

The method according to claim 1,
Wherein said fraction comprises a compound represented by the following formula (2) in a concentration of 0.01 to 10 占 퐂 / mg.
(2)

The method according to claim 1,
Wherein the cancer is an ovarian cancer.
A health functional food composition for prevention or amelioration of cancer, comprising a fraction of the extract from a gazelle extract as an active ingredient.
8. The method of claim 7,
Wherein said basilariae extract is a methanol extract.
8. The method of claim 7,
Wherein the fraction is an ethyl acetate fraction of a yam cabbage extract.
8. The method of claim 7,
Wherein said fraction comprises a compound represented by the following formula (1) in a concentration of 0.01 to 10 占 퐂 / mg.
[Chemical Formula 1]

8. The method of claim 7,
Wherein said fraction comprises a compound represented by the following formula (2) in a concentration of 0.01 to 10 占 퐂 / mg.
(2)

8. The method of claim 7,
Wherein the cancer is ovarian cancer.

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