KR101826177B1

KR101826177B1 - Composition for preventing, improving or treating hepatocellular carcinoma comprising purified extract of Citrus spp. fruit peels

Info

Publication number: KR101826177B1
Application number: KR1020150108242A
Authority: KR
Inventors: 김곤섭; 허정두; 이상준
Original assignee: 경상대학교산학협력단; 한국화학연구원
Priority date: 2015-07-30
Filing date: 2015-07-30
Publication date: 2018-02-06
Also published as: KR20170014595A

Abstract

The present invention relates to a composition for preventing, improving or treating hepatocellular carcinoma which contains citrus dermabase extracted tablets as an active ingredient. More specifically, the present invention relates to a composition for preventing, improving or treating hepatocellular carcinoma cells, Inhibiting, affecting the cell cycle, and inducing apoptosis, thereby effectively preventing, ameliorating or treating liver cancer.
In particular, the composition of the present invention is useful as an agent for increasing the expression of BaK protein, suppressing expression of Bcl-xL protein, inhibiting expression of caspase family protein, inhibiting expression of PARP protein, inhibiting phosphorylation of Akt protein, increasing phosphorylation of P38 MAPK protein, Increased phosphorylation of proteins and increased phosphorylation of ERK proteins can lead to apoptosis of the cells.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating hepatocellular carcinoma, which comprises purified citrus dermis extract as an active ingredient. fruit peels}

The present invention relates to a composition for preventing, improving or treating hepatocellular carcinoma which contains citrus dermabase extracted tablets as an active ingredient. More specifically, the present invention relates to a composition for preventing, improving or treating hepatocellular carcinoma cells, Inhibiting, affecting the cell cycle, and inducing apoptosis, thereby effectively preventing, ameliorating or treating liver cancer.

Dermis is a mature rind of citrus ( Citrus spp.) And has been known to exhibit digestive hyperactivity, diuretic action, antibacterial action, and antiallergic action from ancient times. The dermis is an area that is discarded after using the pulp, and there is an advantage that the amount of material that can be secured every year is unlimited. Therefore, by using these dermis well, very useful materials can be obtained from cheap materials.

If dermis can be extracted from dermis, it can be used for various purposes such as medicines, food or cosmetics. Since the dermis has not caused any adverse effects on human body, extracted substances have less side effects than other conventional synthetic medicines will be. Due to these various possibilities and advantages, various studies related to dermis have been conducted, and dermis application techniques such as Korean Patent No. 10-1187310 have been known.

According to the 2009 statistics released by the Ministry of Health and Welfare and the National Cancer Registration Center in 2011, there are about 16,000 cases of hepatocellular carcinoma in Korea, ranking 5th in terms of cancer incidence. Liver cancer in males, especially in males, is the fourth most common cause of cancer, with a higher incidence than females. The characteristics of hepatocellular carcinoma are high probability of liver metastasis and even if cancer is removed through surgical removal, the probability of recurrence is high and the survival rate of patients is very low for 5 years. Therefore, it is necessary to develop medicines which can effectively inhibit proliferation and death of liver cancer cells.

Accordingly, the present inventors have conducted various studies to develop a method that can be more usefully utilized based on the potential and advantage of the citrus dermis. Particularly, as a result of intensive study on the effect on cancer, it was found that the citrus fruit extract extract inhibits the cell viability of liver cancer cells, affects the cell cycle, induces apoptosis The inventors have confirmed that hepatocellular carcinoma can be prevented, ameliorated or treated effectively, and the present invention has been completed.

Korean Patent No. 10-1187310

Therefore, a main object of the present invention is to provide a composition which can effectively prevent, ameliorate or treat liver cancer.

According to one aspect of the present invention, there is provided a composition for preventing, ameliorating or treating liver cancer containing citrus dermabase extracted tablets as an active ingredient.

In the composition of the present invention, the citrus dermabase extracted tablets may be prepared by (a) lyophilizing and pulverizing citrus dermis; (b) extracting 50 to 90% (v / v) methanol by adding methanol to the citrus dandruff powder obtained in (a); (c) filtering the extract obtained in (b) above; (d) removing the solvent of the filtrate obtained in (c); (e) dissolving the residue obtained in (d) in methanol; (f) passing the solution obtained in (e) through a silica column; (g) washing the column with hexane after step (f); (h) eluting the column with ethyl acetate after the step (g); (i) removing the solvent of the eluate obtained in (h) above.

In the composition of the present invention, in step (b), it is preferable to add 200 to 1000 parts by weight of 50 to 90% (v / v) methanol to 100 parts by weight of the dicalcium phosphate powder and extract using ultrasonic extraction.

In the composition of the present invention, the citrus is preferably Citrus natsudaidai Hayata or Citrus platymamma Hort. Et Tanaka.

When the extract of tablets is used to produce an extract of tablets according to the above method, naringin, hesperidin, poncirin, isomeranzin, sinensetin, tetramethyl Preparation of extracted tablets comprising -O-tetramethyl-O-isoscutellarein, nobiletin, heptamethoxyflavone, trimethoxyflavone and tangeretin. can do. Particularly, on the basis of 1 kg of the citrus dermis used in this experiment, it was found that there were no significant differences between the two groups. Preparation of extracted tablets containing 0.9 ± 0.1 mg of O-isocetracaraine, 3.9 ± 0.1 mg of Novartin, 0.9 ± 0.1 mg of heptamethoxy flavone, 2.3 ± 0.1 mg of trimethoxy flavone and 5.1 ± 0.1 mg of marzeletin can do.

The purified extract prepared by using the above-described dicalcium salt reduces the cell viability of liver cancer cells, decreases the G1 phase in the cell cycle, induces apoptosis, Thereby effectively preventing, ameliorating, or treating liver cancer. In particular, the expression of BaK protein, inhibition of Bcl-xL protein expression, inhibition of caspase family protein expression, inhibition of PARP protein expression, inhibition of phosphorylation of Akt protein, or phosphorylation of P38 MAPK protein, Can be derived.

When the extract of tablets is prepared using the dermis according to the above method, it is possible to use neoeriocitrin, naringin, hesperidin, isosinensetin, sinensetin, But are not limited to, tetramethyl-O-isoscutellarein, nobiletin, tetramethoxyflavone, heptamethoxyflavone, tangeretin and hydroxypentamethoxy Extracted tablets containing hydroxypentamethoxyflavone can be prepared.

The purified extracts prepared using the ducks of the mandarin oranges reduce the cell viability of liver cancer cells, reduce the G1 phase in the cell cycle, induce apoptosis, Thereby effectively preventing, ameliorating, or treating liver cancer. In particular, it is possible to induce apoptosis through suppression of caspase family protein expression, inhibition of Bcl-xL protein expression, increase of phosphorylation of P38 MAPK protein, increase of phosphorylation of JNK protein or increase of phosphorylation of ERK protein .

In the present invention, (j) dissolving the residue obtained in (i) in methanol; And (k) performing a C ₁₈ column chromatography with the solution obtained in the above (j); may further include the C ₁₈ column chromatography to obtain 0.05 to 0.2% formic acid aqueous solution (A solution) and methanol and (B solution) in which acetonitrile is mixed at a ratio of 1: 0.5 to 1: 2 (volume basis) is used as a mobile phase, and the ratio of solution B is changed to 0% (v / v) for the first 5 to 15 minutes (V / v) (remaining A solution) from 20 to 30% (v / v), then the B solution is increased from 60 to 80% (V / v) (remaining A solution) over 3 to 7 minutes after the B solution is eluted to 40 to 60% (v / v) (remaining A solution) , And then eluting the solution with 40 to 60% (v / v) (remaining A solution) for 5 to 15 minutes is preferably used. The flow rate is preferably 0.3 to 0.7 ml / min, and the column temperature is preferably 30 to 40 ° C.

More preferably, a solution (solution B) in which 0.1% formic acid aqueous solution (solution A) and methanol and acetonitrile are mixed at a ratio of 1: 1 (volume basis) is used as a mobile phase. For the first 10 minutes, Is gradually increased from 0% (v / v) to 25% (v / v) (remaining A solution), then the solution is increased to 70% (v / v) (remaining A solution) over 10 minutes , Then eluting with isocratic for the next 30 minutes and then reducing the B solution to 25% (v / v) (remaining A solution) over 5 minutes and then eluting with isocratic for 10 minutes . The flow rate is 0.5 ml / min, and the column temperature is 35 ° C.

In the case of using a dicalcium phosphate berry, fractions containing a high concentration of each flavonoid compound can be obtained according to the retention time (retention time) under such C ₁₈ column chromatography conditions. The retention time is 16 to 17 minutes, 30 minutes to 31 minutes for pasonalin, 39 to 40 minutes for isomergene, 41.5 to 42.3 minutes for cinnensetine, 42.4 to 43.5 minutes for tetramethyl-O-isoscleralane, 46 to 47 minutes Novartin, 47.5 to 48.5 minutes is heptamethoxy flavone, 49.5 to 50.1 minutes is trimethoxy flavone, and 50.2 to 51 minutes is marjeretin.

In the case of using a bilberry skin, a fraction containing each flavonoid compound at a high concentration can be obtained according to the retention time (retention time) under such C ₁₈ column chromatography conditions. The retention time 13.5 to 14.5 minutes is equivalent to that of neoerythocyte, Isesinenetetin for 40.5 to 41.5 minutes, cinnexetin for 42.5 to 43.5 minutes, tetramethyl-O-isoscleralene for 44.5 to 45.5 minutes, 46 to 46.5 hours for 16 to 17 minutes, hesperidin for 18 to 19 minutes, Min for Novyletin, 46.6 to 47.1 minutes for tetramethoxy flavone, 47.2 to 48 minutes for hepatemethoxy flavone, 50 to 51 minutes for marzeletin and 51.1 to 52 minutes for hydroxypentamethoxy flavone.

The composition of the present invention can be used for medicines and foods.

At this time, the citrus dermabase extracted tablets according to the present invention can be formulated on the basis of a formulation standard of a conventional pharmaceutical agent of KFDA or a formulation standard of a health supplement.

The composition containing the citrus papaya extract of the present invention as an active ingredient can be prepared by a conventional method, by mixing the active ingredient with a pharmaceutically acceptable carrier according to the administration method, dosage form and therapeutic purpose, In the carrier.

When the carrier is used as a diluent, oral administration using at least one carrier selected from the group consisting of saline, buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose and ethanol For parenteral administration, formulations such as powders, granules, injections, syrups, solutions, tablets, suppositories, pessaries, ointments, creams or aerosols may be prepared. However, the carrier of the present invention is not limited to the above carrier. In this case, parenteral administration means administration of the active ingredient through rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, etc. in addition to orally.

The formulations may further comprise a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, an antiseptic, etc. to formulate the composition so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The dosage of the present invention can be adjusted according to the patient's condition, route of administration, and dosage form, and is not limited, and any person skilled in the art will be able to use the dosage within a wide range, Generally, in the present invention, it is judged that it is possible to continuously or intermittently administer 0.1 to 10 mg per 1 kg body weight of the citrus dermabase extracted purified product in an experimentally effective amount per day.

Based on the effective amount of the citrus fruit extract extract, the present invention provides a food containing the citrus fruit extract or its purified food or a pharmaceutically acceptable carrier as a basic dietary material, These foods include seasonings such as meat products, fish products, tofu, mushrooms, porridge, noodles such as noodles and noodles, seasonings such as soy sauce, miso, hot pepper paste, and mixed potatoes, sauces, confectionery products such as fermented milk and cheese, Can be used in foods of beverages such as pickles, fruits, vegetables, soybean milk, and fermented beverages. It will be apparent to those skilled in the art that the specific cooking methods and production methods are not described herein. The pharmaceutically acceptable carrier may also be the above-mentioned pharmaceutically acceptable carrier.

The composition of the present invention inhibits cell viability of liver cancer cells and affects the cell cycle. The composition of the present invention increases the expression of Bak protein, inhibits the expression of Bcl-xL protein, inhibits expression of Caspase family protein, It can effectively prevent, ameliorate, or treat liver cancer by inhibiting the expression of PARP protein, inhibiting phosphorylation of Akt protein, increasing phosphorylation of P38 MAPK protein, increasing phosphorylation of JNK protein, or inducing apoptosis of ERK protein have.

FIG. 1 shows the results of HPLC analysis of citrus fruit extract extracted and purified from dicalcium alginate.
FIG. 2 shows the structure of flavonoid compounds extracted from the dwarf bark.
Figure 3 shows the nomenclature of each part in the flavonoid glycoside.
FIG. 4 shows the results of HPLC analysis of citrus fruit extract extracted and purified from the dwarf calf.
FIG. 5 shows the results of an experiment on the effect of purified citrus dermis extract purified from Hepatica dermis on cell viability of HepG2 liver cancer cells.
FIG. 6 shows the results of experiments on the cell cycle of hepatocarcinoma HepG2 cells obtained from citrus fruit extract extracted and purified from dicalcium alginate.
FIG. 7 shows the results of an experiment on the effect of citrus dermabase extract purified and purified from dicalcium alginate on the apoptosis of HepG2 liver cancer cells.
FIG. 8 shows the results of the Tunel assay for examining the effect of citrus dermabase extracted and purified from the dicalcium alginate on apoptosis of HepG2 liver cancer cells.
FIG. 9 shows the results of an experiment on the effect of purified citrus dermis extract purified from dicalcium alfalfa on the expression of apoptosis-related proteins in HepG2 liver cancer cells.
FIG. 10 shows the results of an experiment on the effect of citrus dermabase extract purified and purified from dicalcium alginate on the phosphorylation (activation) of apoptosis-related proteins of HepG2 liver cancer cells.
FIG. 11 shows the results of an experiment on the effect of citrus dermabase extracted and purified from the ducks of the cabbage on the cell survival of HepG2 liver cancer cells.
FIG. 12 shows the results of an experiment on the effect of purified citrus dermis extract purified from the ducks of the Japanese cabbage on the cell cycle of HepG2 liver cancer cells.
FIG. 13 shows the results of an experiment on the effect of citrus dermabase extract purified and purified from the duck liver on apoptosis of HepG2 liver cancer cells.
FIG. 14 is a result of a double stain experiment for examining the effect of citrus dermabase extract purified and purified from duck liver on apoptosis of HepG2 liver cancer cells.
FIG. 15 shows the results of an experiment on the effect of purified citrus dermis extracts purified from the ducks of mackerel on the expression of apoptosis-related proteins (caspase 3 and caspase 9) in HepG2 liver cancer cells.
FIG. 16 shows the results of an experiment on the effect of citrus dermabase extracted and purified from the duck liver of Senegal on the expression of apoptosis-related proteins (Bcl-xL and Bax) in HepG2 liver cancer cells.
FIG. 17 shows the results of an experiment on the effect of citrus dermabase extracted and purified from the dwarf shark on phosphorylation (activation) of apoptosis-related proteins (p38, JNK and ERK) of HepG2 liver cancer cells.
FIG. 18 is a graphical representation of the effect of purified citrus extract obtained from citrus dermis on purified hepatocellular apoptosis-related factors.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Preparation of Extract Tablets Using Citrus Dermis

Citrus dermis and mandarin dermis were used to prepare each citrus fruit extract.

Tangerine dyestuff (100 g) was freeze-dried (lyophilizer, PVTED50A, Ilsin Bio Base Co. Ltd., Yangju, Republic of Korea) and ground into fine powder. 70% methanol (30 ml) was added to citrus fruit powder (5 g) and homogenized for 2 minutes (PT-MR 2100, Kinematica, Lucerne, Switzerland). The homogenized mixture was mixed with a vortex mixer (G560E, Scientific Industries Inc., Bohemia, NY, USA) for 2 minutes and then extracted with an ultrasonic bath (Bransonic 3510R-DTH, Branson Ultrasonics Co., Banbury, USA) for 10 minutes. After repeated extraction twice, the samples were mixed, filtered using a filter paper, and then centrifuged at 3200 x g for 10 minutes. The supernatant was placed in a 200 ml flask and the solvent was removed under reduced pressure using a rotary evaporator (Eyela NVC-2100, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The residue was dissolved in 1 ml of methanol and loaded into a silica cartridge (1.7 x 2 cm) produced in a laboratory. The cartridge was washed with 5 mL of hexane and then eluted with 5 mL of ethyl acetate. The solvent was removed under reduced pressure. The residue was redissolved in 1 ml of methanol, passed through a 0.45 μm cellulosic membrane, transferred to a silicon bottle and stored at -20 ° C. until analysis.

Experimental Example 1. Purification and structure confirmation of purified citrus dermis extract from citrus dermis

In the above Example 1, the components of the extract and purified product prepared using the dicalcium phosphate were confirmed.

Separated flavonoid components were identified by comparison with RP-HPLC with C ₁₈ column, ESI-MS / MS data in cation mode and reported data.

HPLC analysis was performed using an 1100 series chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with G1322A degasser, G1312A pump, G1313 autosampler and G1316A oven. Chromatographic separation was performed on a Zorbax Stable Bond Analytical SB-C18 column (4.6x250 mm, 5 [mu] m, Agilent Technologies, MD, USA). The mobile phase solvent system consisted of 0.1% formic acid aqueous solution (A) and methanol / acetonitrile (1: 1) (B), increasing from 0 to 25% B over the first 10 minutes and 70% over 10 minutes. Eluted with isocratic for 30 minutes, then B was reduced to 25% over 5 minutes and then eluted with isocratic for 10 minutes. The flow rate was 0.5 ml / min, the column temperature was 35 ° C, and the injection amount was 10 μl in each experiment. Chromatographic data were collected using the ChemStation, Rev.B.0301 program. In the whole experiment, data of 250 ~ 600nm were collected with resolution of 2nm and quantification of flavonoid was done by extracting chromatogram at 280nm. The analytical curves of hesperetin and polymethoxy flavone compounds were quantified using nobiletin calibration curves.

MS / MS experiments were performed using a 3200 QTRAP LC / MS / MS system (Applied Biosystems, Forster, CA, USA) equipped with a Turbo V ™ source and a Turbo Ion Spray probe. Mass spectra were performed in positive ion and SIM mode. A total of 14 MS / MS transitions of healthy citrus and 16 MS / MS transitions of diseased citrus were monitored. BioAnalyst ™ version 1.4.2 and software version 1.4.2 were used for instrument manipulation and data collection, respectively. Nitrogen was used as a fog and dryer. The pressure of the two gases was 45 psi. The electron spray voltage was 5.5 kV and the source temperature was 500 ° C. The resolution of the first quadrupole and the ion trap was between 0.6 and 0.8 (unit resolution). The mass spectrometry spectrum was recorded between m / z 50 and 800 at a step size of 0.05 amu.

Figure 1 shows partial chromatograms from 10 to 60 minutes of each extracted tablets recorded at a detection wavelength of 280 nm. The MS / MS data and structure of the identified components are summarized in Table 1 and FIG. Using MS / MS, you can get information on the aglycone skeleton, the form of carbohydrate or the presence of various substituents, the order of glycans, the linkage of glycosides, and the binding site of aglycons and substituents. The glycoconjugate nomenclature proposed by Domon and Costello (Domon & Costello, 1988) is used to illustrate the production of flavanone glucoside, and the ions produced from aglycon are used for the nomenclature proposed by Ma and Li (Ma et al ., 1997). These are shown in FIG. ^{k, l} X _j, Y _j and Z j is to represent the ion containing the aglycone, where j is the number of bond between the glycoside being broken when calculated from the aglycon the combined decomposition between k and l are carbohydrate ring . The glycoside bond between the glycan moiety and the aglycone is represented by zero. When the charge is in the hydrocarbon moiety, the resulting ions are ^{denoted k, l} A _i and B _i , where I (≥1) represents the number of glycoside bonds that are decomposed when calculated from the non-reducing end. ^{i, j} A ⁺ ₀ and ^{i, j} B ⁺ ₀ represent the product ions containing the intact A- and B-rings. Where the superscripts i and j represent the bond of the C-rings to be disassembled. The knee letter 0 to the right of A and B is used to avoid confusion with the A _i ⁺ and B _j ⁺ ( i ≥ 1) labels used to represent carbohydrate slices containing units per terminal.

NO. Compound r.t (min) [M + H] < + > Fragment (m / z) One Naringin 16.5 581 H + 2H2O] +, 545 [M + H-2H2O] +, 543 [M + Na] +, 563 [M + H- H] +, 401 [Z1 + H-H2O] +, 383 [Z1 + H-2H2O] +, 365 [Z1 + H-3H2O] +, 273 [Y0 + +, 147 [1,4B0 + H-H2] +, 121 [1,3B0 + H] + 2 Hesperidin 18.5 611 H + 2H2O] +, 575 [M + H-2H2O] +, 553 [M + H] H] +, 431 [Z1 + H-H2O] +, 413 [Z1 + H-2H2O] +, 395 [Z1 + H- +, 177 [1,4B0 + H-H2] +, 151 [1,3B0 + H] + 3 Poncirin 30.5 595 417 [Z1 + H? H2O] +, 397 [Z1 + H? 2H2O] +, 379 [Z1 + H] +, H [3H2O] +, 287 [Y0 + H] +, 179 [0,4B0 + H] +, 135 [ 4 Isomeranzin 39.6 261 243, 201, 189, 177, 161, 159, 145, 131, 128, 115, 103 5 Sinensetin 42.2 373 [M + H-CH3] +, 343 [M + H-2CH3] +, 340 [M + H- +, 162 [1,3B0] +, 147 [1,2B0-H2] + 6 Tetramethyl-O-isoscutellarein 42.6 343 H + 2 H-CH3] +, 313 [M + H-2CH3] +, 299 [M + H- +, 181 [1,3A0 + H-2CH3] +, 135 [0,2B0] +, 133 [1,3B0 + H] + 7 Nobiletin 46.4 403 M + H-CH4-2OCH3] +, 241 [1,3A0 + H], 388 [M + H-CH3] H] +, 211 [1,3A0 + H-OCH3] +, 181 [1,3A0 + H-2OCH3] 8 Heptamethoxyflavone 48.1 433 H] +, 211 [1,3A0 + H-2CH3] +, 193 [1,3B0 + H] +, H] +, 165 [0, 2B0] +, 149 [1,2B0] + 9 Trimethoxyflavone 49.8 313 298, 269, 255, 181 10 Tangeretin 50.7 373 M + H-OCH3-H2O] +, 297 [M + H-2CH3-H2O-CO] +, 241 [ 3A0 + H] +, 211 [1,3A0-2CH3] +, 135 [0,2B0] +

Experimental Example 2. Purification and structure confirmation of purified citrus extract from citrus fruit dwarf

Using the same method as in Experimental Example 1, the components of the extract and purified product prepared using the dicalcium alginate in Example 1 were confirmed.

The partial chromatograms for 10 to 60 minutes of each extracted tablets recorded at a detection wavelength of 280 nm are shown in FIG. The MS / MS data and structure of the identified components are summarized in Table 2 and FIG.

NO. Compound RT
(min) [M + H] < + > / [M-H] - MS / MS One Neoeriocitrin 13.85 - / 595 459, 329, 311, 287, 151, 135, 107 2 Naringin 16.78 - / 579 459, 313, 271, 193, 151 3 Hesperidin 18.43 - / 609 608, 325, 301 4 5 6 7 8 Isosinensetin 41.07 373 358, 357, 343, 329, 181, 165 9 Sinensetin 43.09 373 358, 357, 343, 340, 329, 312, 162 10 Tetramethyl-o-isoscutellarein 45.19 343 328, 313, 299, 285, 240, 152, 133 11 Nobiletin 46.30 403 388, 373, 355, 327, 241, 211, 165 12 Tetramethoxyflavone 46.95 343 327, 313, 282, 150 13 Heptamethoxyflavone 47.50 433 418, 403, 385, 211, 165 14 Tangeretin 50.43 373 358, 343, 325, 297 15 Hydroxypentamethoxyflavone 51.59 389 374, 359, 341, 165

Experimental Example 3: Determination of flavonoid compounds extracted from dicalcium alginate

Nine flavonoid components and one coumarin derivative were quantitated from the peak area on the HPLC-UV chromatogram recorded at 280 nm. They were validated on the basis of representative flavonoid standards of the same group. That is, flavanone, flavone and coumarin derivatives were validated using hesperetin, noviletin and coumarin, respectively. Calibration curves were prepared by periodic analysis of each reference material. The regression equation used y = ax + b, where y and x represent the peak area and concentration of each compound, respectively. Correlation coefficient ( r ² ) was high as 0.9994, 0.9970 and 0.9981 for hesperetin, nobiletin and coumarin, respectively. The detection limits (LOD) were 0.01, 0.10 and 0.02 mg / l for hesperetin, nobiletin and coumarin, respectively. The limits of quantitation (LOQ) were 0.05, 0.30 and 0.06 mg / l for hesperetin, nobiletin and coumarin, respectively.

The amount of each compound in the shell is summarized in Table 3. The total amount of flavonoid was 9229.7 ± 0.5 mg / kg. The total amounts of the compounds of flavanone, flavone and coumarin derivatives were 9213 ± 0.3, 13.7 ± 0.1 and 2.6 ± 0.1 mg / kg, respectively. The amount of naringin was the highest at 5010.0 ± 4.5 mg / kg and the amount of sinensetin was the lowest at 0.6 ± 0.1 mg / kg.

NO. Compound Mean ^a ± SD ^b One Naringin 5010.0 + - 4.5 2 Hesperidin 4121.2 ± 0.9 3 Poncirin 82.2 ± 0.2 4 Isomeranzin 2.6 ± 0.1 5 Sinensetin 0.6 ± 0.1 6 Tetramethyl- O- isoscutellarein 0.9 ± 0.1 7 Nobiletin 3.9 ± 0.1 8 Heptamethoxyflavone 0.9 ± 0.1 9 Trimethoxyflavone 2.3 ± 0.1 10 Tangeretin 5.1 ± 0.1 Total 9229.7 ± 0.5

* ^a mg / kg fresh peel weight.

* ^b Standard deviation.

Experimental Example 4: Activity of purified citrus dermis extract purified and purified from dicalcium dipalpase against cancer cells

4-1. Effect on viability of HepG2 cells

In order to investigate the effect of the extracted tablets prepared by using the dicalcium dipalpin in Example 1 on the viability of HepG2, the cell viability was drastically reduced as a result of MTT treatment at various concentrations for 24 hours and 48 hours . It was confirmed that the cell viability decreased more clearly in the 48 hour-treated experiment (see FIG. 5).

4-2. Effect on cell cycle of HepG2 cells

The cell cycle phase was analyzed by flow-cytometry in order to examine the effect of the purified extract prepared by using the dicalcium dipalpin in Example 1 on the cell cycle of HepG2. When the extracted extract was treated, a significant increase in subG1 phase was confirmed and a decrease in the G1 phase was confirmed in proportion thereto (see FIG. 6).

4-3. HepG2 Cell apoptosis Effect on

The cell stage was confirmed by flow cytometry in order to confirm the effect of the purified extract prepared using the dicalcium dipalpin in Example 1 on the apoptosis of HepG2 cells. Treatment of the 48 hour extract resulted in an increase in the subG1 phase. To prove this, nuclear staining with Hoechst stain methods was performed to confirm cleavage of the nucleus. In addition, PI / annexin V staining was performed to confirm the increase of subG1 phase, and the increase of apoptosis was confirmed (see FIG. 7).

4-4. Effect of HepG2 on cell death

In Example 1, nuclear changes were observed to examine the effect of the purified extract prepared using the dicalcium dipalpalis on HepG2 cell apoptosis. Tunel assay showed high levels of DNA damage when treated with the extract (see FIG. 8).

To identify apoptosis at the translation level, expression patterns of related proteins were analyzed by western blotting. As a result, it was confirmed that mitochondria-dependent pathway was formed through the increase of Bak protein and Bcl-xL protein, and apoptosis end product was confirmed by decreasing expression of caspase family and PARP protein. In addition, the mitochondrial membrane poteintial was measured and abnormal mitochondrial function was confirmed in the extract-purified water treatment group (see FIG. 9).

4-5. Effect of HepG2 cells on apoptosis-related protein expression

In order to confirm the high signal transduction mechanism causing apoptosis, the MAPK family protein P38 protein was identified as total form and phosphorylation form, respectively. The expression level of Akt / Pi3k protein was also confirmed. Akt was found to reduce activity by reducing the degree of phosphorylation, and it was found that the MAPKs family protein increased the degree of phosphorylation of P38 (see FIG. 10).

Experimental Example 5: Activity of purified citrus dermis extract purified from the duck bark of Cancer against cancer cells

5-1. Effect on viability of HepG2 cells

In order to investigate the effect of the extracted tablets prepared by using the ducklings in Example 1 on the viability of HepG2, the cell viability was drastically reduced as a result of MTT treatment for 24 hours and 48 hours. . And it was confirmed that the cell viability was further reduced in the 48 hour-treated experiment (see FIG. 11).

5-2. Effect on cell cycle of HepG2 cells

The cell cycle phase was analyzed by flow-cytometry in order to examine the effect of the purified extract prepared by using the dwarf beetle on the cell cycle of HepG2 in Example 1 above. When the extract was treated, it was confirmed that the subG1 phase was significantly increased and the G1 phase was decreased in proportion thereto (see FIG. 12).

5-3. Apoptosis of HepG2 cells

The cell stage was confirmed by flow cytometry in order to confirm the effect of the purified extract prepared by using the ducklings in the Example 1 on the apoptosis of HepG2 cells. Treatment of the extract with 24 hour extraction resulted in nuclear condensation and decomposition of the chromosome. To prove this, nuclear staining with Hoechst stain methods was performed to confirm cleavage of the nucleus. In addition, ladder assay was performed to confirm the degradation of chromosome, and the increase of apoptosis was confirmed (see FIG. 13).

5-4. Effect of HepG2 on cell death

In order to examine the effect of the purified extract prepared by using the dwarf beetle on HepG2 cell apoptosis in Example 1, nuclear changes were observed. Double stain was performed to confirm a high level of apoptosis when the extract was treated (see Fig. 14).

To identify apoptosis at the translation level, expression patterns of related proteins were analyzed by western blotting. As a result, it was confirmed that mitochondria-dependent pathway was formed by the decrease of Bcl-xL protein, and apoptosis end product was confirmed by decreasing expression of caspase family protein. In addition, mitochondrial membrane poteintial was measured and abnormal mitochondrial function was confirmed in the extract-purified water treatment group (see FIGS. 15 and 16).

5-5. Effect of HepG2 cells on apoptosis-related protein expression

In order to confirm the high signal transduction mechanism causing apoptosis, the related proteins P38, JNK and ERK proteins were identified as total form and phosphorylation form, respectively. As a result, it was confirmed that the degree of phosphorylation of P38, JNK and ERK proteins was increased (see FIG. 17).

Claims

(a) Citrus platymamma (Hort. et Tanaka); freezing and drying the dermis;
(b) adding 50 to 90% (v / v) methanol to the dicalcium alginate powder obtained in the step (a) and extracting it;
(c) filtering the extract obtained in (b) above;
(d) removing the solvent of the filtrate obtained in (c);
(e) dissolving the residue obtained in (d) in methanol;
(f) passing the solution obtained in (e) through a silica column;
(g) washing the column with hexane after step (f);
(h) eluting the column with ethyl acetate after the step (g); And
(i) removing the solvent of the eluate obtained in (h) above, as an active ingredient, a composition for preventing, improving or treating liver cancer.

delete

The method according to claim 1,
The citrus dermabase extracted tablets
Neoeriocitrin, naringin, hesperidin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein, novolac, Prevention, improvement or treatment of liver cancer, characterized in that it comprises nobiletin, tetramethoxyflavone, heptamethoxyflavone, tangeretin and hydroxypentamethoxyflavone. / RTI >

8. The method of claim 7,
The composition induces cell apoptosis through inhibition of Caspase family protein expression, inhibition of Bcl-xL protein expression, phosphorylation of P38 MAPK protein, phosphorylation of JNK protein, or phosphorylation of ERK protein Or a pharmaceutically acceptable salt thereof.