KR20080088784A - Pharmaceutical composition for anticancer comprising extracts of young dangyuja friut or leafs of dangyuja - Google Patents

Abstract

A pharmaceutical composition comprising the extract of young Dangyuja(Citrus tenuissima Tanaka) fruit or leaf is provided to induce apoptosis of cancer cells and inhibit toxicity by using natural food, so that the composition is useful to prevent and treat cancers. A pharmaceutical composition for prevention and treatment of cancer comprises the extract of young Dangyuja(Citrus tenuissima Tanaka) fruit or leaf which is prepared by (a) extracting young Dangyuja(Citrus tenuissima Tanaka) fruit or leaf powder with C1-C4 lower alcohol or its solution and filtering and separating the filtered solution, (b) separating solid from the filtered solution, and (c) extracting the separated solid with hexane or CHCl3, wherein the cancer is lymphoma, hepatoblastoma, uterine cancer, large intestine cancer, breast cancer, lung cancer or stomach cancer.

Description

Pharmaceutical Composition for Anticancer Comprising Extracts of Young Dangyuja Friut or Leafs of Dangyuja}

Figure 1 shows the survival rate of U937 cells treated with HFYD.

2 is a flow cytometry analysis to observe the number of apoptotic cells of U937 cells treated with HFYD.

Figure 3 shows the DNA fragmentation after electrophoresis of U937 cells treated with HFYD.

Figure 4 shows the expression changes of the Bcl-2 gene and Bax gene of U937 cells treated with HFYD.

Figure 5 shows the results of Western blot analysis of the activity of Bcl-2, Bax, caspase-3 and expression of cleaved PARP of U937 cells treated with HFYD.

Figure 6 is a graph showing the change in the concentration of pNA released by caspase-3 in HFYD treated U937 cells according to HFYD.

7 is a graph showing the survival rate of SNU-16 cells according to the concentration of the solvent fractions after SNU-16 cells were treated with various solvent fractions.

Figure 8 shows various cancer cell lines of CHCl ₃ After treatment with fractions, it is a graph showing the survival rate of cells according to the treated CFLD concentration.

Figure 9 shows the DNA fragmentation after agarose gel electrophoresis of SNU-16 cells treated with CFLD.

FIG. 10 shows the results of SNU-16 cells not treated with CFLD as a control and SNU-16 cells treated with CFLD, stained with fluorescent dyes, followed by fluorescence microscopy.

11 is CHCl ₃ of the sugar citron leaf The results of performing flow cytometry analysis by culturing the SNU-16 cells treated with the fractions.

12 is CHCl ₃ of the sugar citron leaf Bcl-2, Bax, caspase-3 activity and expression of cleaved PARP of SNU-16 cells treated with fractions were analyzed by Western blot.

13 is CHCl ₃ of the sugar citron leaf SNU-16 cells treated with the fractions are shown by the results of the gene comet analysis.

Field of invention

The present invention relates to an immature sugar extract and a sugar extract extract for the anti-cancer, more specifically, the immature sugar extract and sugar extract leaf extract and the immature sugar extract and sugar extracts that have the effect of cancer cell death It relates to an anticancer pharmaceutical composition containing as an active ingredient.

Background of the Invention

Citrus fruits refer to plants belonging to the family of citrus fruits and citrus fruits, citrus fruits include citrus, fortunella, poncirus and clymenia. The origin of citrus fruits is believed to be the southeastern and periphery of the Asian continent, from India to central and southern China, to the south via Indochina and the Malay peninsula, and to the east through China and to Korea and Japan. Jeju is the only mountainous area.

Citrus fruits are 87% water-based alkaline foods, rich in any variety, essential oils or aromatic ingredients, and citrus pulp is known to have a refreshing flavor with rich organic acids and sugars. In addition, the citrus peel contains a large amount of pectin, fiber, vitamins, various bioflavonoids and limonoids.

Ingesting a sufficient amount of food fiber, including pectin, generally reduces the absorption of fat, cholesterol and sugars in the intestine, which helps prevent atherosclerosis, hyperlipidemia and diabetes. In addition, pectin is a lipid associated with cardiovascular disease. It has been known to have various pharmacological activities of metabolic improvement effect, anticancer and antiviral action. Therefore, when ingesting citrus fruits containing a large amount of pectin as described above can be confirmed the same effect as described above.

Sugary milk contains ingredients beneficial to the human body, such as limonene, obacunone, nomiline and naringin. In particular, the sugar-containing sugar contains a multi-flavored limonoid component, has the effect of inhibiting and preventing cancer cell proliferation, and clinical experiments have been found to be excellent in inhibiting liver cancer, lung cancer, intestinal cancer and skin cancer (Kingsam, Citrus fruits industry. 104. Jeju Culture, Jeju, 2001).

On the other hand, the sugar bark is thick, sweet, non-toxic, removes the instrument in the stomach, loosen the poison and improves the taste. It has been reported to have antioxidant effects (Oh, HS et al . , Korean Journal of Culinary Research, 9:69, 2003; Mokbel, MS and Hashinaga, F., Food chemistry , 94: 529, 2006).

In particular, the immature fruit sugar is less mature than the mature sugar sugar family, but the content of citric acid (citric acid) is about 2 times higher to give a fresh taste, anti-allergic and antioxidant component flavonoids It is reported that naringin and hesperidin are 5 and 10 times as high, respectively. (Seunghwa Kim, Process Improvement Method using Citron Functional Substance, Jeju Agricultural Experiment Station, 2000).

 On the basis of the fact that the citrus immature fruit contains a large amount of beneficial ingredients, a cosmetic composition for whitening containing a citrus beverage (Korean Patent No. 344570) and an immature citrus extract using the citrus immature fruit as an active ingredient (Korea Patent Publication 2006-104054) has been published.

In addition, as a result of applying the components of the citrus extract in the pharmaceutical field, hesperidin or naringin separated and purified from the composition for improving lipid metabolism and lowering blood pressure (Korean Patent No. 314477), citrus rind extract, including citrus rind extract Cardiovascular disease prevention and treatment comprising a composition (Korean Patent No. 213895) and an anti-aging composition (Korean Patent No. 2006-13917) using a citrus-derived hesperetin (hesperetin) as an active ingredient.

As described above, various attempts have been made to explore the developmental potential of functional products using citrus extracts, but studies on sugar maturers, particularly sugar matured immature families, containing a large amount of beneficial ingredients as described above are insufficient.

Therefore, there is an urgent need in the art for the development of various functional products that can be utilized in the industry using sugar extract from citrus fruits.

Thus, the present inventors have made diligent efforts to improve the problems of the prior art, immature sugar extract and sugar extract extract obtained by using a variety of extraction solvents to induce apoptosis against cancer cells, showing an anticancer effect It confirmed and completed this invention.

It is an object of the present invention to provide an immature sugar extract extract exhibiting cancer cell death effect.

It is another object of the present invention to provide a sugar extract of the citrus leaves showing cancer cell death effect.

Still another object of the present invention is to provide an anticancer pharmaceutical composition containing immature sugar extract and sugar extract extract as an active ingredient.

In order to achieve the above object, the present invention provides an immature sugar yugwa extract exhibiting cancer cell death effect in one aspect.

In the present invention, the immature sugar extract can be characterized in that extracted with hexane or CHCl ₃ .

In the present invention, the immature sugar yugwa extract is (a) C ₁ ~ C ₄ Low alcohol or reflux extraction of the immature sugar yugwa powder in an aqueous solution thereof and then separating the filtrate; (b) separating solids from the separated filtrate; And (c) refluxing the separated solids in hexane or CHCl ₃ extraction solvent.

The present invention also provides an anticancer pharmaceutical composition containing the immature sugar extract.

In the present invention, the cancer may be selected from the group consisting of lymphoma, hepatoblastoma, uterine cancer, colon cancer, breast cancer, lung cancer and gastric cancer.

In another aspect, the present invention provides a sugar citron leaf extract exhibiting cancer cell death effect.

In the present invention, the immature sugar extract can be characterized in that it is extracted with any one extraction solvent selected from the group consisting of hexane, CHCl ₃ , ethyl acetate and butanol.

In the present invention, the sugar citrus leaf extract is (a) C ₁ ~ C ₄ Lower alcohol or reflux extraction of sugar citrus leaf powder in an aqueous solution and then separating the filtrate; (b) separating solids from the separated filtrate; And (c) refluxing the separated solids in any one extraction solvent selected from the group consisting of hexane, CHCl ₃ , ethyl acetate, and butanol.

The present invention also provides a pharmaceutical composition for anticancer containing the sugar extract of the yuja leaf as an active ingredient.

In the present invention, the cancer may be selected from the group consisting of gastric cancer, lymphoma, colon cancer, hepatoblastoma and lung cancer.

Hereinafter, the present invention will be described in detail.

The present invention utilizes the hexane or CHCl ₃ solvent extracts of immature sugar extracts and the hexane, CHCl ₃ , ethyl acetate and butanol solvent extracts of sugar extracts to induce apoptosis of cancer cells. It is characterized in that it provides an anticancer pharmaceutical composition containing the extract or sugar yuja leaf extract and the immature sugar yuja extract or sugar yuja leaf extract.

In the present invention, the effects of various solvent fractions of immature sugar extract and sugar extract extracts on cancer cells were investigated. It was confirmed that a large amount of cell death was induced against leukemia cells U937 and gastric cancer cells SNU-16.

Therefore, in the present invention, as an extract to be used as an anticancer composition, CHCl ₃ of immature sugar citrus and hexane fractions (HFYD) and sugar citrus leaves, which are toxic to cancer cells, are toxic to cancer cells. It is preferable to use the fraction (CFLD: Chloroform Fraction of Leaf of Dangyuja).

In the present invention, the immature sugar yugwa extract is preferably treated with hexane fractionation of the immature sugar yugwa extract to obtain an immature sugar yugwa hexane fraction.

In addition, in the present invention, it is preferable that the sugar citron leaf extract is subjected to CHCl ₃ fractionation on the sugar citron leaf extract to obtain CFLD.

In the present invention, HFYD and CFLD induce apoptosis in the manner of forming apopotic body, DNA fragmentation and apoptosis cells, thereby inhibiting the growth of cancer cells, thereby exhibiting anticancer efficacy.

In order to confirm the anticancer efficacy of HFYD and CFLD, HFYD and CFLD were treated in cancer cell lines, followed by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] assay. When viability was measured, it was confirmed that HFYD reduced the cell viability of U937 cells, lymphoma cells, and CFLD decreased the cell viability of SNU-16 cells, gastric cancer cell lines.

Therefore, U937 was treated with HFYD and SNU-16 was treated with CFLD to culture the cells, thereby observing apoptosis efficacy of HFYD and CFLD.

In SNU-16 cultured by treatment with U937 and CFLD treated with HFYD, DNA fragmentation, apoptotic body formation and an increase in the number of apoptosis were confirmed, and Bcl-2 expression, which is an anti-apoptotic gene, was inhibited and cysteine was suppressed. Caspase, a system protease, is activated, and the phenomenon of PARP cleavage required for DNA repair damaged by the enzyme was confirmed.

As a result, the immature sugar extract and sugar extract leaves extract according to the present invention exhibits anti-cancer efficacy in a manner of inducing apoptosis of cancer cells, can be used as a pharmaceutical composition for the treatment of various cancers in the future.

On the other hand, the composition for treating cancer containing the immature sugar yugwa extract and sugar yuja leaf extract may be administered in various formulations of oral and non-parenteral at the time of clinical administration, when formulated, commonly used fillers, binders, wetting agents, It is prepared using diluents or excipients such as disintegrants and surfactants. Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and include at least one excipient such as starch, calcium carbonate, water It is prepared by mixing cross, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents, water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, Tokyo dry preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In addition, the dosage of the immature sugar-oil extract and the composition for treating cancer containing the sugar-oil extract extract to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, Based on an adult patient weighing 70 kg, it is generally 100-1000 mg / day, preferably 100-500 mg / day, and also divided once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. It may also be administered.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1: immature Sugar Citron Division  Preparation of Extract

The immature sugar citron department was obtained from the National Institute of Subtropical Agriculture Jeju Province. The immature sugar cane was harvested about 30 days after flowering.

Air-dried immature sugar-oil pulp is ground using a mill, 84.98 g of the immature sugar-sugar fruit powder is added to 2 L of 80% methanol, stirred at room temperature for 3 days, then extracted, and extracted with immature sugar-oil and methanol Obtained.

Example  2: immature Sugar cane  Preparation of Various Solvent Fractions

The immature sugar-oil and methanol extract fractions obtained in Example 1 were filtered off, and evaporated to vaccum rotary evaporator under reduced pressure and lyophilized. The lyophilized extract was suspended in 1 L of an aqueous solution, extracted with n-hexane, CHCl ₃ , ethyl acetate, and n-butanol solvent, respectively, and then sequentially fractionated. N-hexane, CHCl ₃ , Ethyl acetate and n-butanol solvent fractions were prepared.

Further, n-hexane, CHCl ₃ , ethyl acetate and n-butanol solvent fractions of the immature sugar-oil extract were extracted three times at room temperature, and the solvent fractions obtained for each extraction were subjected to rotary evaporation, followed by freeze-drying. Finally, the extracted powder can be dissolved in DMSO and diluted in PBS (pH 7.4) to prepare a solvent fraction with immature sugar yugwa at various concentrations.

Experimental Example  1: immature Sugar cane  Cancer cell death efficacy test

end. Cell culture

Histocytic lymphoma cell line U937 (CRL-1593.2), colon cancer cell line HCT-15 (CCL-225), hepatoblastoma cell line Hep-G2 (HB-8065), breast cancer cell line MCF-7 (HTB-22), The lung cancer cell line NCI-H460 (HTB-177) and gastric cancer cell line SNU-16 (CLR-5974) cell lines were obtained from the American Type Culture Collection.

The cancer cell lines were in a 10% heat-inactivated fetal bovine serum, penicillin (100 units /) in RPMI 1640 or DMEM (Invitrogen Gibco BRL, USA) medium in a humidified incubator at 37 ° C. (5% CO ₂ /95% air). mL) and streptomycin (10 μg / mL) were added to the culture solution.

I. Immature Sugar cane  According to solvent fraction U937  Cellular Survival rate  analysis - MTT  analysis

To the medium containing MeOH, hexane fraction, CHCl ₃ fraction, ethanol acetate fraction, butanol fraction and H ₂ 0 fraction of immature sugar-oil family were added, respectively, and the concentration of each solvent fraction was 25, 50, 100 and 200 µg / At mL, U937 cells, which were histiocyte lymphoma cells, were incubated for 4 days, and then 5 mg / ml of MTT (Sigma, USA) was added to each well of 20 µl and further incubated at 37 ° C for 4 hours.

After incubation, the wells were centrifuged at 2500 rpm for 20 minutes at room temperature, the cultures were removed, the formed formazan crystals were dissolved in 150 mL of DMSO, and then 540 nm wavelength using a Micro reader (Sunrise, Australia). The absorbance was measured by.

As a result of MTT analysis, U937 cell viability cultured in medium containing hexane fraction of immature sugar ducts and CHCl ₃ was drastically reduced, resulting in the death of U937 cells from hexane fractions (HFYD) and CHCl ₃ fractions of sugar ducts. It can be seen that (Fig. 1).

All. HFYD Cells by Survival rate  analysis- MTT  analysis

The effect of HFYD on the viability of U937, HCT-15, Hep-G2, MCF-7, NCI-H460 and SNU-16 cells was investigated by MTT analysis.

To collect the tumor cells in log phase (exponential phase), respectively, captured transferred to each well (well) (approximately 10 ⁴ -10 ⁵ cells / well 180μL). HFYD was added at 25, 50, 100 and 200 μg / mL for each cancer cell, followed by incubation for 4 days, and then 0.1 mg of MTT (Sigma, USA) was added to each well and added at 37 ° C. for 4 hours. Incubated with

After incubation of the cancer cells, the wells were centrifuged at 2500 rpm for 20 minutes at room temperature, the culture solution was removed, the formed formazan crystals were dissolved in 150 mL of DMSO, and then 540 nm using a Micro reader (Sunrise, Australia). Absorbance was measured by wavelength.

As shown in Table 1, the MTT assay showed that the viability of cells cultured in the medium to which HFYD was added differed depending on the cell type. Accordingly, the IC ₅₀ (concentration of HFYD at 50% cell survival rate) was also clearly shown. In particular, HFYD was found to be the most toxic to U937.

Cell name Characteristic IC ₅₀ (μg / mL) U937 Histocyte Lymphoma Cells 59.57 HepG2 Hepatoblastoma Cells 131.45 HeLa Cervical cancer cells 286.52 HCT-15 Colorectal cancer cells 86.55 MCF-7 Breast cancer cells 143.76 NCI-H460 Lung cancer cells 72.67 SNU-16 Stomach cancer cells 90

la. Apoptosis bodies observed

U937 cells were transferred to 24 well plates at a concentration of 1.0 × 10 ⁵ cells / ml. After 16 hours of plating, the cells were treated with HFYD for 3 hours, and after 16 hours, 1.5 μl of the DNA-specific fluorescent dye 10 mg / ml Hoechst 33342 was added to 1.5 mL of the solution of each well, and then Well plates were incubated at 37 ° C. for 10 minutes. Cells stained with the fluorescent dye were observed using a fluorescence microscope.

As a result, it was confirmed that apoptosis bodies were formed in U937 cells cultured by HFYD treatment, and it was confirmed that HFYD induced death of U937 cells (FIG. 2B).

hemp. DNA  Segmentation observation

U937 (1 × 10 ⁵ cells / ml) was treated with 0, 25, 50, 100, and 200 μg / ml of HFYD for 24 hours, and then genomic DNA was obtained using the Wizard Genomic DNA purification kit (Promega, USA). Extracted by. 4 mg of extracted DNA was confirmed by electrophoresis.

As a result, a typical ladder pattern due to DNA destruction in nucleosomes, which is characteristic of apoptosis, could be identified, indicating that HFYD induces apoptosis of U937 cells (FIG. 3).

bar. Phosphatidylserine  Externalization ( Phosphatidylserine Externalization ) analysis

U937 cells (2 × 10 ⁵ cells / mL) were treated with 100 μg / mL of HFYD for 3 hours and in 100 μl of Annexin V-binding buffer (10 mM HEPES / NaOH, pH 7.4, 140 mM NaCl and 5 mM CaCl ₂ ). After suspension, Annexin V-FITC (BD Bioscience, NJ, USA) and the phosphor propidium iodide (PI) were added to a final concentration of 5 μg / mL. The cells were then incubated for 15 minutes at room temperature in the dark, diluted with 300 μl binding buffer, and flow cytometry analysis was performed using EPICS-XL FACScan flow cytometer (Coulter, Miami, FL, USA). Was performed.

At this time, the cells that are positive for Annexin V but negative for PI fluorescence are apoptotic cells (Zhong WB et al . , Anticancer Drugs 14 , 211: 217, 2003).

As a result of flow cytometry, the percentage of apoptosis cells in U937 cells treated with HFYD was increased compared to the control group not treated with HFYD (FIG. 2A), indicating that HFYD is involved in apoptosis of U937 cells. there was.

four. HFYD  By treatment Bcl -2 and Bax Analysis of Changes in Expression

U937 cells treated with HFYD were harvested, and expression of apoptosis related genes Bcl-2 and Bax was measured by RT-PCR.

RNA was extracted from the cells using TRI-zol (Invitrogen, USA). The absorbance of the RNA was determined by Sambrook et al. (Sambrook JE et. al . , In Molecular Cloning Laboratory Manual (2 nd ed .), Cold Spring Harbor Laboratory Press. United States of America, 7: 8, 1989).

PCR reactions were carried out using the following primers. It was performed using primers of Bcl-2 (SEQ ID NO: 1 and SEQ ID NO: 2) and primers of Bax (SEQ ID NO: 3 and SEQ ID NO: 4), and for amplification of the control GAPDH (primers of SEQ ID NO: 5 and SEQ ID NO: 6). ) Was used.

Bcl-2 sense: 5'-TGCACCTGACGCCCTTCAC-3 '(SEQ ID NO: 1)

Bcl-2 antisense: 5'-AGACAGCCAGGAGAAATCAAACAG-3 '(SEQ ID NO: 2)

Bax sense: 5'-ACCAAGAAGCTGAGCGAGTGT-3 '(SEQ ID NO: 3)

Bax antisense: 5'-ACAAAGATGGTCACGGTCTGCC-3 '(SEQ ID NO: 4)

GAPDH sense: 5'-GAAGGTGA AGGTCGGAGTC-3 '(SEQ ID NO: 5)

GAPDH antisense: 5'-GAAGATGGTGATGGGA TTTC-3 '(SEQ ID NO: 6)

Fragments of the amplified fractions of Bcl-2, Bax and GAPDH were observed by electrophoresis and the size of the fragments was 292 bp (Bcl-2), 364 bp (Bax) and 226 bp (GAPDH), respectively.

The expression of Bcl-2, an antiapoptotic protein, gradually decreased over time, and the expression of Bax, a pro-apoptotic protein, was not changed (FIG. 4). .

In conclusion, it was found that HFYD induces apoptosis by inhibiting the expression of the anti-apoptotic protein Bcl-2.

hemp. Weston Blot ( western blot Using analytics HFYD  Processed Bcl -2 and Bax Observation of Expression

HFYD treated cells were collected and washed twice with cold PBS, followed by lysis buffer [50 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO 3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg / ml aprotinin, 25 μg / ml leupeptin] and left to cool for 30 minutes. The cell lysate was then centrifuged at 12,000 RPM for 20 minutes at 4 ° C. and the supernatant of the cell lysate (protein 30-50 μg) was separated on 8-12% SDS-polyacrylamide gel. After electrophoresis, the protein on the gel was transferred to a polyvinylidene fluoride (PVDF) membrane (BIO-RAD, HC, USA). After blocking the non-specific site with 5% nonfat dried milk, the membrane was subjected to primary antihuman caspase 3 antibody, poly ADP Ribosyl Polymerase (PARP). ), Bcl-2 antibody and Bax antibody (Cell signaling, MA, USA), and further reacted with a secondary antibody (1: 5000, Vector, CA, USA) conjugated with peroxidase. The PVDF membrane was exposed to X-ray film and the protein bands were observed.

As a result, the expression of Bcl-2, an antiapoptotic protein, gradually decreased over time, and the expression of Bax, a pro-apoptotic protein, was not changed. 5). In conclusion, it was found that HFYD induces apoptosis by inhibiting the expression of the anti-apoptotic protein Bcl-2.

bar. Caspase -3 ( caspase -3) activity change analysis

U937 cells were treated with 25, 50, 100, and 200 μg / mL of HFYD for 24 hours and then lysed in cytolysis buffer to generate a mixture, wherein the substrate used DEAD- p NA ( p- nitroanyline). . The mixture was incubated overnight at 37 ° C. in wet conditions, and then the concentration of released p NA was measured.

As a result, as the concentration of HFYD increased, the concentration of released p NA increased, indicating that the activity of caspase-3, an enzyme that degrades p NA, increased (FIG. 6).

As the concentration of HFYD increases, the activity of caspase-3 also increases. At this time, since caspase-3 is an enzyme that promotes cell death through proteolysis, HFYD increases apoptosis by increasing the activity of caspase-3. It was found that to induce.

Example  3: Sugar leaf  Preparation of Extract

Air-dried sugar-oil leaves were ground using a mill, and 50 g of the powdered sugar-oil leaves were added to 2 L of 80% methanol, stirred at room temperature for 3 days, and then extracted to obtain a sugar-oil leaf methanol extract fraction. It was.

Example  4: Sugar leaf  Preparation of Various Solvent Fractions of Extracts

The methanol extract fraction of sugar palm leaves obtained in Example 1 was filtered, evaporated by vacuum vaccum evaporator (vaccum rotary evaporator) under reduced pressure and freeze-dried. The lyophilized extract was suspended in 1 L of an aqueous solution, and extracted with n-hexane, CHCl ₃ , ethyl acetate and n-butanol solvent, respectively, and then sequentially fractionated to extract n-hexane, CHCl ₃ , Ethyl acetate and n-butanol solvent fractions were prepared.

Each solvent was extracted three times at room temperature, and the solvent fractions obtained at each extraction were removed by rotary evaporation and then lyophilized. Finally, the extracted powder was dissolved in DMSO and diluted in PBS (pH 7.4) to obtain a final concentration.

Experimental Example  2: Sugar leaf  Experiment of cancer cell death efficacy of extract

end. Cell culture

Gastric cancer cell line SNU-16, histoblast lymphoma cell line U937, colon cancer cell line HCT-15, hepatoblastoma cell line Hep-G2 and lung cancer cell line NCI-H460 were incubated at 37 ° C (5% CO ₂ /95% air). ) Were cultured in RPMI 1640 or Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% FBS and 1% antibiotics.

I. Sugar leaf  According to solvent fraction SNU -16 cells Survival rate  analysis - MTT  analysis

To the medium containing MeOH, the hexane fraction, CHCl ₃ fraction, ethanol acetate fraction, butanol fraction and H ₂ 0 fraction of sugar palm leaves were added, respectively, and the concentration of each solvent fraction was 25, 50, 100 and 200 µg / In mL, SNU-16, a gastric cancer cell, was cultured, and then 0.1 mg of MTT (Sigma, USA) was added to each well and further incubated at 37 ° C for 4 hours.

After incubation, the wells were centrifuged at 2000 rpm for 10 minutes, the culture solution was removed, and 150 mL of DMSO was added to each well to dissolve formazan, and the absorbance was measured at 540 nm using a micro reader (Sunrise, Australia). It was.

As a result of the MTT analysis, the SNU-16 cell viability cultured in the medium containing hexane, CHCl ₃ , ethyl acetate and butanol fractions of sugar palm leaves dramatically decreased, which resulted in hexane, CHCl ₃ , ethyl acetate and butanol of sugar palm leaves. It can be seen that the fraction induces death of SNU-16 cells (FIG. 7).

All. Cells by CFLD Survival rate  analysis - MTT  analysis

HCT-15, SNU-16, HepG2, NCl-H460 and SK-N-SH were seeded in a 96 well plate at 1.0x10 ⁵ cells / mL, and the concentration of CFLD was determined after 4 hours. 25, 50, 100 and 200 μg / mL were added and incubated for 4 days, then 10 μl of MTT (Sigma, MO, USA) solution was added to each well and incubated for an additional 4 hours.

After incubation, the wells were centrifuged at 2000 rpm for 10 minutes, the culture solution was removed, and 150 mL of DMSO was added to each well to dissolve formazan, and the absorbance was measured at 540 nm using a micro reader (Sunrise, Australia). It was.

As a result of MTT analysis, the survival rate of cells after culture by CFLD was different according to the cell type, and in particular, CFLD was found to be the most toxic to SNU-16 (FIG. 8).

la. Apoptosis bodies observed

SNU-16 cells were treated with 50 µg / ml CFLD, and after 24 hours, Hoechst 33342, a fluorescent dye, was added to a final concentration of 10 µM and allowed to react at 37 ° C for 10 minutes. After the reaction, the SNU-16 cells were observed using a fluorescence microscope to confirm that apoptosis bodies were formed (FIG. 10). As a result, it was found that CFLD is involved in inducing apoptosis of SNU-16 cells.

hemp. DNA  Segmentation observation

SNU-16 (1x10 ⁵ cells / ml) was treated with 0, 25, 50, 100 and 200 μg / ml of CFLD for 24 hours, and then genomic DNA was purified using a DNA Genomic DNA purification kit (Promega, USA). Extracted using, and extracted DNA electrophoresis.

As a result, a typical ladder pattern due to DNA destruction in nucleosomes, which is characteristic of apoptosis, could be identified, and the results showed that CFLD induced apoptosis of SNU-16 (FIG. 9). ).

bar. Phosphatidylserine  Externalization ( Phosphatidylserine Externalization ) analysis

SNU-16 cells were treated with 50 μg / ml CFLD, centrifuged after 24 hours, suspended with the addition of 1 mL of PBS, and then centrifuged again. The supernatant was removed, fixed in 500 μL of 70% ethanol and washed twice with cold PBS (2 mM EDTA). Then, 5 μL of a PI solution containing 6 μL of PBS (2 mM EDTA), 6 μL of PI stock (2 mg / mL), and 6 μL of RNase was added, and then 30 minutes later using an EPICS-XL FACScan flow cytometer (Coulter, USA) at 37 ° C. Flow cytometry was performed.

Flow cytometry analysis showed that the percentage of Sub-G1 cells inducing apoptosis in CFLD-treated SNU-16 cells was higher than that in the control without CFLD (FIG. 11). Eventually, it was found that CFLD induces death of SNU-16 cells.

four. Weston Blot  analysis

SNU-16 cells treated with glucose-rich CHCl ₃ fractions were lysis buffer (50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO 3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg / ml aprotinin, 25 μg / ml leupeptin) and dissolved for 30 minutes at 4 ° C. and left to cool for 30 minutes. Cell lysates were then centrifuged at 13000 RPM for 20 minutes and cell lysates (protein 30-50 μg) of the supernatant were separated by SDS-PAGE. Then transferred to 5% NFDM and blocked overnight to inhibit nonspecific antibodies. Primary antibody nuclei were isolated on 8-12% SDS-polyacrylamide gel and glycine transfer buffer [192 mM glysin, 25 mM Tris-HCl (pH 8.8), 20% MeOH (v / v)]. Transfer to PVDF membrane (BIO-RAD, USA). After blocking the non-specific site with 5% nonfat dried milk, the membrane was the primary antibody, cleaved-caspase 3 antibody, cleaved-PARP antibody, Bcl -2 antibody and Bax antibody were cultured and further cultured using a secondary antibody conjugated with peroxidase (1: 5000, Vector, CA, USA). The PVDF membrane was exposed to X-ray film and the protein bands were observed.

As a result, over time, PARP of 116kDa decreased and PARP of 89kDa increased, confirming that PARP was cleaved (FIG. 12).

Ah. gene Comet  analysis( Comet assay )

950 μl of SNU-16 cells were seeded in a 24 well plate, treated with CHCl ₃ fraction to a concentration of ⁴ × 10 ⁴ cells / ml, and after 2 hours, the SNU-16 cells were collected for 3 minutes. Centrifuged at 2000 RPM. After centrifugation, the cells were washed with 1 ml of PBS and centrifuged again. After removal of the supernatant, 50 µl aliquots were applied to a slide coated with 1% agarose. Stored for minutes. Thereafter, the cover glass was removed and 0.7% low melting agarose was applied, and then again covered with the cover glass and stored at 4 ° C. for 20 minutes.

Fill the bottle with silver foil with 35 ml of lysis buffer, add 350 μl of Triton X-100 surfactant, and dissolve it in the bottle wrapped with silver paper for 90 minutes to dissolve the unwindng buffer. Prepared.

After adjusting the amount of the buffer so that the prepared cold unwinding buffer to the voltage 25V and the current 300mA, the cell was dispensed slide was placed upside down, and then electrophoresed in the dark for 20 minutes. After electrophoresis, the slides were washed three times with 0.4M Tris buffer (pH7.5) in the dark for 10 minutes, fixed with 70% EtOH, and the slides were dried in the dark for 24 hours to 1 week. Next, it was stained with EtBr and observed with a fluorescence microscope. As a result, it was found that the amount of DNA tail was increased in SNP-16 cells treated with CFLD (FIG. 13).

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described above, the present invention has an effect of providing an anticancer drug composition containing immature sugar extract and sugar extract extract. By using the anticancer composition containing the immature sugar milk fruit extract or sugar milk leaf extract of the present invention, by using a natural food extract as an anticancer composition, it can be used as a safe anticancer agent without toxicity, and the extract is treated with various solvent fractions. Depending on the type of anticancer drugs can be developed.

Claims (10)

Immature sugar extract extract showing cancer cell death effect. The immature sugar-oil extract of claim 1, wherein the extract is extracted with hexane or CHCl ₃ . The immature sugar cane extract of claim 1, which is prepared by the following method: (a) refluxing the immature sugar yugwa powder in C ₁ -C ₄ lower alcohol or an aqueous solution thereof, and then separating the filtrate; (b) separating solids from the separated filtrate; And (c) refluxing the separated solids in hexane or CHCl ₃ extraction solvent. A pharmaceutical composition for anticancer, comprising the immature sugar extract of any one of claims 1 to 3 as an active ingredient. The pharmaceutical composition according to claim 4, wherein the cancer is selected from the group consisting of lymphoma, hepatoblastoma, uterine cancer, colon cancer, breast cancer, lung cancer and gastric cancer. Sugar yuja leaf extract showing cancer cell death effect. The sugar citron leaf extract according to claim 6, wherein the extract is extracted with any one extraction solvent selected from the group consisting of hexane, CHCl ₃ , ethyl acetate and butanol. The sugar extract of claim 6, which is prepared by the following method: (a) refluxing the sugar citron leaf powder from C ₁ -C ₄ lower alcohol or an aqueous solution, and then separating the filtrate; (b) separating solids from the separated filtrate; And (c) extracting the separated solids under reflux in any one extraction solvent selected from the group consisting of hexane, CHCl ₃ , ethyl acetate and butanol. An anticancer pharmaceutical composition comprising the extract of any one of claims 6 to 8 as an active ingredient. 10. The pharmaceutical composition according to claim 9, wherein the cancer is selected from the group consisting of gastric cancer, lymphoma, colon cancer, hepatoblastoma and lung cancer.

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