KR101416191B1 - Pharmaceutical composition for preventing or treating cancer comprising extract of Jeju traditional citrus Palsac, Dangyusa or Iyegam as effective component - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating cancer and a composition for preventing or ameliorating cancer, which comprises as an active ingredient, Jeju native citrus fruits such as parachu, sugar beet or almond extract.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer, which contains, as an active ingredient, Jeju traditional citrus fruit extract,

The present invention relates to a pharmaceutical composition for preventing or treating cancer and a food for preventing or improving cancer, which contains Jeju native citrus fruits such as Echinosophora koreana, Echinochloa or Echinochloa crus-galli extracts, especially perianthracis extract as an active ingredient.

Citrus is an alkaline food with 87% moisture content. It is rich in any variety, essential oil or directional components. Citrus fruits are mainly composed of fructose, glucose and seodang, and contain citric acid, malic acid, . Citrus fruits from Jeju are good in taste and flavor and contain various vitamins, especially vitamins A and C. In addition, there are many pectin, vitamins, vitamins, bioflavonoids, limonoids, and hesperidin in the citrus fruits. It is one of the most commonly used fruits and has been widely used for medicinal purposes because of its effectiveness.

Currently, only citrus fruits are produced in Jeju and Namhae in Korea, and cultivated varieties are predominantly cold-tolerant orange (early-onset) citrus fruits introduced in Japan in 1911.

According to the folklore data and Dongwoo-bo-gyul, traditional citrus fruit which grows only in Jeju Island has the effect of increasing myocardial contractility and cardiac output and increasing cerebral blood flow. It is effective not only for anti-inflammatory action but also for suppression of skin allergic reaction, antimicrobial action, It has been reported that the herbal medicine effect is excellent and it contains a large amount of functional substance which is not found in other fruits. However, the traditional citrus fruits have no taste and no bitter taste for reproductive purposes and have been gradually upgraded to new varieties. Currently, about 22 varieties such as yuzu, palm, yi persimmon, mandarin orange, mandarin orange, Only a few native species remain.

Korean Patent Laid-Open Publication No. 2008-0088784 discloses an anticancer pharmaceutical composition containing an extract or an extract of a sugar alginate and a sugar alginate extract. Korean Patent Laid-Open Publication No. 2009-0120833 discloses a method of extracting citrus A method for extracting neohesperidin from a skin 'is disclosed. However, the method for extracting neohesperidin from the skin is different from the pharmaceutical composition for preventing or treating cancer, which contains, as an active ingredient, a saponin, sugar beet or almond extract of Jeju native citrus using supercritical extraction of the present invention Do.

The present invention has been made in view of the above needs, and the inventors of the present invention compared the activity of inhibiting cancer cell proliferation by extracts of each part and method in Jeju native citrus, By confirming the apoptosis of HeLa cells, a human gastric cancer cell, AGS and a human cervical cancer cell, in the treatment of supercritical extracts of parasite, sugar beet and aloe vera, The purpose of the present invention is to elucidate the therapeutic effect and various usefulness of the extract on cervical cancer or stomach cancer.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an extract of Echinosophora koreensis, a native Korean citrus fruit.

Further, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an extract of sugar alginate as an active ingredient of Jeju native citrus fruits.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which contains Jeju Native Tooth Extract as an active ingredient.

In addition, the present invention provides a food for preventing or improving cancer, which contains, as an active ingredient, an Echinosophora koreanum extract which is Jeju native citrus fruit.

Further, the present invention provides a food for preventing or improving cancer, which contains citrus peel extract of Jeju native citrus as an active ingredient.

In addition, the present invention provides a food for cancer prevention or improvement, which contains Jeju Native citrus extract as an active ingredient.

According to the present invention, since the Jeju native citrus extract is low in commerciality for reproductive purposes, the utility of the traditional citrus fruit gradually disappearing has been reexamined as a pharmaceutical composition and a food processing material. The anticancer pharmaceutical composition containing the Jeju native citrus extract as an active ingredient is a substance obtained from a natural material and has no cytotoxicity. Therefore, it can be used as a pharmaceutical composition for prevention and treatment of cancer or as a food for prevention and improvement of cancer have.

Figure 1 shows the effect of Jeju native citrus peel extract on inhibition of cell proliferation in AGS and HeLa cells. AGS cells were treated with Jeju native citrus skin extract (A: Hot water extract, B: 80% EtOH extract) at a concentration of 0-1000 μg / mL and cultured for 48 hours and 72 hours. HeLa cells were treated with Jeju traditional citrus peel extract (C: hot water extract, D: 80% EtOH extract) at the same concentration to measure cell activity. E: Fibroblasts were treated with Jeju native citrus peel extract (left: hot water extract, right: 80% EtOH extract) at a concentration of 0-1000 μg / mL for 48 hours and cell activity was measured. (●: party party, ○: parchment, ▼: Yi Yeon Sense)
Fig. 2 shows the effect of Jeju native citrus extract on inhibition of cell proliferation in AGS and HeLa cells. AGS and HeLa cells were treated with supercritical extract of Jeju native citrus at a concentration of 0-400 μg / mL and cultured for 48 hours. (A: pulp, B: peel), (●: parchment, ○: sugar, ▼:
Figure 3 shows the effect of the Jeju native citrus supercritical extract on HeLa cell morphology. Supercritical extracts were treated with HeLa cells at 0, 100, and 200 μg / mL for 24 hrs compared with control (A). Hoechst 33342 dye (left) and acridine orange dye (right) Cells were stained and observed under a microscope. (A: control, B: parchment crust extract, C: sugar crust extract, D:
Figure 4 shows the effect of Jeju native citrus supercritical extract on AGS cell morphology. Compared with control (A), supercritical extracts were treated with AGS cells at 0, 100, and 200 μg / mL for 24 hours and stained with Hoechst 33342 dye (left) and acridine orange dye And observed with a microscope. (A: control, B: parchment crust extract, C: sugar crust extract, D:

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

Further, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an extract of sugar alginate as an active ingredient of Jeju native citrus fruits.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which contains Jeju Native Tooth Extract as an active ingredient.

In the pharmaceutical composition according to an embodiment of the present invention, the extract of Paraxac, the sugar or the extract may be a freeze-dried parchment, a sugar or an extract, And may be, but not limited to, peel extracts of lyophilized, freeze-dried, sugar-free or yeast extract.

In the pharmaceutical composition according to an embodiment of the present invention, the extract of Paraxacum, Saccharomyces cerevisiae or Euglena persica may be, but is not limited to, a perianth supercritical extract of parachu, sugar cane or aloe vera.

In the pharmaceutical composition according to one embodiment of the present invention, the pericarp, supernatant or perilla supercritical extract is preferably prepared by lyophilization, pulverization, or pulverization of crushed parchment, sugar or peeled persimmon powder for 1 to 2 hours The supernatant may be prepared by supercritical extraction using a supercritical extractor at 280 to 320 bar and 45 to 55 ° C. More preferably, the supernatant is pulverized, And extraction with supercritical extraction at 50 < 0 > C and 300 bar with an extractor.

The supercritical extraction method of the present invention is a supercritical fluid extraction method and is an extraction system in which the principles of conventional distillation and extraction techniques are combined. A supercritical extraction system refers to the extraction of a substance using the solubility of a supercritical fluid.

"Supercritical fluid" refers to a substance that does not change its state even when heat and pressure are applied beyond the specified temperature (critical temperature) and pressure (critical pressure) when applying heat and pressure to the material. . Supercritical fluids are indistinguishable from liquid and gas states, and have inherent properties different from ordinary liquids and gases. Supercritical fluids are compressible fluids that have medium physical properties such as gas and liquid. They can diffuse like gases, dissolve other materials like liquids, and can change their properties to desired conditions by changing pressure and temperature.

Representative examples of supercritical fluids include, but are not limited to, carbon dioxide, water, methane, ethane, propane, ethylene, propylene, methanol, ethanol, The conditions of supercritical fluids for use in food and pharmaceuticals should be chemically safe, free from corrosion in the apparatus, and with low critical temperature and pressure, and good solubility.

In this regard, carbon dioxide is mainly used in the process. Supercritical carbon dioxide is relatively inexpensive and harmless to the human body, as well as non-flammable and chemically stable. Also, it is easy to change the physical properties at a low critical point (73.8 bar, 31.1 ° C) and can be used by recirculation. The physical characteristics of supercritical carbon dioxide are that they have medium physical properties between gas and liquid, and they can change physical properties such as density and solubility by controlling temperature and pressure. It has a high diffusion coefficient and a low surface tension, which allows penetration into a fine space, facilitates mass transfer, and has a specific gravity and density equivalent to that of a liquid. In particular, since supercritical carbon dioxide dissolves a nonpolar material having a low molecular weight well and is limitedly soluble for polar and high molecular materials, only one substance can be separated and extracted from the mixture. Since supercritical carbon dioxide has a lower critical point and a diffusion coefficient of 10 to 100 times higher than other solvents, it can be extracted without destroying the original value of the raw material to be extracted. Since the carbon dioxide is vaporized at the extraction, And can be used as an environmentally friendly solvent free from secondary pollution by < RTI ID = 0.0 >

In the pharmaceutical composition according to one embodiment of the present invention, the cancer may be gastric cancer, breast cancer, cervical cancer, lymphoma, hepatoblastoma, colon cancer or lung cancer, preferably gastric cancer or cervical cancer. .

The pharmaceutical composition for preventing or treating cancer of the present invention may contain 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the above extract, based on the total weight of the pharmaceutical composition.

The pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, 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 ), 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 administration 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 paper, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose does not in any way limit the scope of the invention.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides a food for preventing or improving cancer, which comprises the extract of Echinosophora koreana as an active ingredient.

Further, the present invention provides a food for preventing or improving cancer, which contains citrus peel extract of Jeju native citrus as an active ingredient.

In addition, the present invention provides a food for cancer prevention or improvement, which contains Jeju Native citrus extract as an active ingredient.

In the food according to an embodiment of the present invention, the extract of Echinosophora koreensis, Echinosophora koreensis or Echinochloa crus-galli can be preferably freeze-dried parchment, sugar or peel of persimmon, persimmon or passerine extract, Dried peach, sugar beet or peel extract, but is not limited thereto.

In the food according to one embodiment of the present invention, the extract of Echinosophora koreensis, Echinochloa or Echinochloa crus-galli is preferably a perianth supercritical extract of Echinosophora koreensis, Echinochloa spp.

In the food according to one embodiment of the present invention, the pericarp, supernatant or perilla supercritical extract of Eucalyptus persimilis is preferably freeze dried, and then pulverized parchment, sugar cane or pea powder is pulverized for 1 to 2 hours And may be prepared by supercritical extraction at 280 to 320 bar and 45 to 55 ° C with a critical extractor. More preferably, the freeze-dried, pulverized parchment, sugar cane or pea powder is pulverized for 2 hours in a supercritical extractor By extraction with supercritical extraction at 50 < 0 > C and 300 bar, but not limited thereto.

In the food according to an embodiment of the present invention, the cancer may be gastric cancer, breast cancer, cervical cancer, lymphoma, hepatoblastoma, colon cancer or lung cancer, preferably gastric cancer or cervical cancer.

The food is not particularly limited as long as it can be ingested to increase anticancer activity.

When the extract of the present invention is used as a food additive, the extract may be directly added, used in combination with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the extract of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, 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 extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention.

In addition to the above, the extract of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the extract of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. 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 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1. Traditional citrus Parch , Yuzuji  or Yi Yeon  Securing the extracts and testing the anticancer activity using them

In the case of hot water extraction, the peel, flesh, and pass of the freeze-dried Jeju native citrus fruits such as parmesan, sugar bean or almonds were crushed and put into distilled water at 1:30, Extracted from atmospheric pressure. After filtration, the mixture was concentrated, lyophilized, stored at room temperature in dark place, and dissolved in dimethyl sulfoxide (DMSO).

In the case of 80% ethanol extraction, the lyophilized Jeju native citrus fruits were pulverized, respectively, and each was subjected to ultrasonic extraction for 45 minutes at a ratio of 1:30 to 80% ethanol. The extract thus obtained was concentrated, lyophilized, dissolved in DMSO, and used in the experiment.

In the case of supercritical extraction, the lyophilized Jeju native citrus fruits were pulverized, respectively, and then extracted with CO ₂ supercritical extraction at 50 ° C and 300 bar with a 1 L supercritical extractor for 2 hours. Among them, the extracts were extracted for 1 hour and 30 minutes. The extracts were stored at room temperature under dark conditions and dissolved in DMSO.

Example  2. Traditional citrus in cancer cells Parch , Yuzuji  or Yea Heat number  And 80% Ethanol Extracts on Cell Growth

Through MTT analysis, the human gastric cancer cell (AGS), the human cervical cancer cell (HeLa cell), and the normal cell (human lung fibroblast), the Jeju native citrus extract Were measured. The cancerous activity was not shown to be high (Figure 1A-D) when 48 hours or 72 hours of hot water extract and 80% ethanol extract of Jeju native citrus were treated with each cancer cell. In order to confirm the effect of Jeju native citrus extract on normal cells, MTT analysis was performed on fibroblasts of normal cell line for 48 hours, and cytotoxicity was not observed (FIG. 1E). These results suggest that the extracts of hot water and 80% ethanol extract of Jeju Island have no anticancer effect against cancer cells.

Example  3. Traditional citrus in cancer cells Parch , Yuzuji  or Yea Supercritical  Detection of cell proliferation inhibitory effect of extracts

The antitumor activity of AGS and HeLa cells after 48 hours treatment of Jeju native citrus supercritical extract was investigated by MTT analysis. As a result, Was the best. (A) and perilla (B) extracts. The results showed that the extracts of perilla (B) showed better inhibitory effect on cell proliferation than those of flesh (A). In addition, when compared with the cell lines, it was confirmed that the perilla (B) extract showed cell-specific proliferation inhibitory activity in HeLa cells rather than AGS (FIG. 2).

Example  4. HeLa  In the cell, Parch , Yuzuji  or Yea Supercritical  Extract-induced cell death and Self-predation  ( autophagic ) In the form ( morphology ) Change observation

The shape of HeLa cells stained with Hoechst 33342 and Acridine Orange at a concentration of 100, 200 ug / ml was observed under a microscope. Apoptotic body formation was confirmed by Hoechst 33342 staining in the cells treated with Echinosophora koreana extract, and acidic vacuole organelle was stained with orange color even in acridine orange staining (FIG. 3B). In the case of Hoechst33342 staining of cells treated with saccharin extract, no apoptosis was observed, whereas in the case of staining with acridine orange, the orange point due to acidic vesicle staining, presumed to be autophagic morphorogy change dot) was increased (FIG. 3C). In addition, no death was observed in the Hoechst 33342 staining of the cells treated with the extract of Angelica keiskei koidz., And an increase in the orange point due to the acidic endoplasmic reticulum staining was confirmed in the acridine orange staining (Fig. 3D).

Example  5. AGS  In the cell, Parch , Yuzuji  or Yea Supercritical  Observation of morphological changes on cell death and autopatching by extracts

The shape of AGS cells stained with Hoechst 33342 and Acridine Orange at a concentration of 100, 200 ug / ml was observed under a microscope. Apoptotic body formation was confirmed by Hoechst 33342 staining in the cells treated with Echinosophora koreensis extract, and it was confirmed that acridine orange staining also formed an acidic endoplasmic reticulum and stained orange (FIG. 4B). In addition, apoptosis was observed in the Hoechst 33342 staining of the cells treated with the sugar extract and the extract of Echinosophora koreensis. In the case of staining with acridine orange, the orange spot estimated as autophagic morphorogy change . However, in the case of the cells treated with the extract of Ehrsia bark extract, an apoptosis was observed in Hoechst 33342 staining at 200 ug / ml (FIG. 4).

Example  6. HeLa  Cells and AGS  In a cell Parch Supercritical  Induction of cell death by extract

The cell cycle of HeLa cells and AGS cells was measured by FACS analysis after treatment of supercritical extracts of Jeju native citrus, Paracoccus, Saccharomyces cerevisiae, or Elianae (Table 1). The results of MTT analysis showed that subcutaneous extracts of subcutaneous ginseng extracts showed the best effect on the sub-G1 phase than the other extracts by FACS analysis. Unlike other Jeju native citrus fruits, it is believed to induce cell death through apoptosis.

Claims (14)

A pharmaceutical composition for preventing or treating gastric cancer, comprising 200 to 400 占 퐂 / ml of a supernatant extract of Echinochloa crus-galli which is native to Jeju Island as an active ingredient. delete delete delete delete The method of claim 1, wherein the supercritical extract of Echinochloa crus-galli is prepared by extracting the supernatant of Eustachia japonica crushed after lyophilization with a supercritical extractor at 280 to 320 bar and 45 to 55 ° C for 2 hours ≪ / RTI > delete Food for preventing or ameliorating gastric cancer, comprising 200 to 400 μg / ml of a supernatant extract of Echinochloa crus-galli which is native to Jeju Island as an active ingredient. delete delete delete delete 9. The method of claim 8, wherein the supercritical extract of Echinochloa crus-galli is prepared by extracting the Eustachian crust of pulverized Eustachian crust after lyophilization with a supercritical extractor at 280 to 320 bar and 45 to 55 ° C for 2 hours Food that features. delete

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