KR20040091870A - Composition for inhibiting liver cancer containing an extract of the root bark of ulmus davidiana var - Google Patents

Abstract

PURPOSE: Provided are biofunctional material for rice and manufacturing method thereof based on oriental medicine, thereby inhibiting the loss of proteins, lipids and dietary fiber and preventing changes in the quality of rice and discoloration thereof. CONSTITUTION: Biofunctional material used for rice is manufactured by the steps of: removing impurities from charcoal with wind; pulverizing the charcoal to obtain 30-70 wt.% of powder thereof; mixing 2.3-4.2 wt.% of jade, 3-6 wt.% of ceramics powder and 70-90 wt.% of purified water then kneading the mixture thereof; forming the dough into a shape having a diameter of 6cm, a thickness of 1.1cm and an inner diameter of 1.5cm, and followed by drying and heating it at 500-1500 deg.C; washing bamboo, ginger, cinnamon, acorns, Quercus dentata leaves and pine needles with purified water then followed by drying and extracting them; concentrating the each extract thereof under reduced pressure; mixing the concentrate of each bamboo, ginger, cinnamon, acorns, Quercus dentata leaves and pine needles in a weight ratio of 3-5:1.25:2-3.5:2-3.3:1.5-4.5:1-3; adding silver powder having purity of 99.9% to the mixed concentrate and agitating them for 2 hours; fermenting the silver powder containing concentrate at 35-40 deg.C for 37-38days; adding 2-3 minerals extracted from loess to 96-98 wt.% of the fermentate thereof and fermenting them at 36-42 deg.C for 20 days; and adsorption the fermentate to the formed product above and drying it at 60-80 deg.C.

Description

Composition for inhibiting liver cancer comprising the extract of the roots of the roots {COMPOSITION FOR INHIBITING LIVER CANCER CONTAINING AN EXTRACT OF THE ROOT BARK OF ULMUS DAVIDIANA VAR}

The present invention relates to a hepatocarcinoma inhibitor comprising as an active ingredient a solvent extract of myocardium bark that can effectively inhibit the progression and metastasis of liver cancer by restoring intercellular signaling through gap junctions and inhibiting MMP activity without toxicity to liver cells. will be.

Recently, as the economic level of our country has risen, as the living environment has been improved and the diet has been enriched, western diets have been widely spread. As a result, chronic adult diseases such as cancer, arteriosclerosis, stroke, diabetes, and high blood pressure, which are presumed to be caused by excessive nutrition or an unbalanced diet, are increasing.

Many methods have been tried to treat cancer until now, and cancer treatment can be roughly classified into drug therapy, surgical therapy, radiation therapy, and the like. When oral or intravenous administration of anticancer drugs during drug treatment, the drug spreads throughout the bloodstream and reaches the lesion, so only trace amounts accumulate in the lesion. Therefore, in order to accumulate an appropriate amount in the lesion, a large amount of drugs must be administered, and the side effects thereof are a serious problem. In addition, there is a problem that a generally effective cancer inhibitor has a greater side effect such as pain or fever. In particular, radiation therapy is more problematic for its safety. For example, a substance such as Holmium, which is effective for liver cancer, has been produced with a complex with chitosan (Milican), and its effects have been proved, but the side effects are serious and still have problems (Watterson. JD et al., J. Urol . 168: 442-445, 2002; Peh, OH et al., Ann. Acad. Med. Singapore 30: 563-567, 2001).

In particular, in the search for cancer inhibitors, rather than a strategy for inhibiting cancer in the early stage of irreversible and short-term cancer, rather than looking for a substance or food that has an inhibitory effect on the long-term progression of cancer as a realistic and practical cancer prevention agent. Research is focused (Kang Jin-Seok et al., Chemical Cancer Prevention Korea Medicine, 2000; Surh, YJ, Mutat. Res. 428: 305-327, 1999; Sporn, MB, Lancet. 347: 1377-1381, 1996).

Inhibition of intercellular signaling through gap junction channels in the progression of liver cancer has been recognized as an important biochemical indicator, and deficiency of leucine 32 and 26 is known to be a major cause of liver cancer. On the other hand, substances that restore the inhibition of intercellular signaling have been recognized to inhibit the progression of liver cancer and have an inhibitory effect on metastasis of liver cancer (Eghbali B. et al., Proc. Natl. Acad. Sci. 87: 1328 -1331, 1990; Ara C et al., Cell Mol Life Sci. 59 (10): 1758-65, 2002; Evert M et al., Carcinogenesis 23 (5): 697-703, 2002; Muramatsu A et al. , Carcinogenesis 23 (2): 351-8, 2002).

In addition, matrix metalloproteinase (MMP) is an enzyme that is important for cancer migration and metastasis, and is a representative enzyme that dissolves extracellular matrix. To date, MMP expression is increased in several types of carcinoma, and gelatinase A (MMP-2, 72 kD) and gelatinase B (MMP-9, 92 kD) are the most directly related to cancer metastasis. Known. In addition, MMP-2 and MMP-9 play important roles in metastasis of liver cancer, and substances that inhibit MMPs have recently been spotlighted as novel cancer inhibitors (Kleiner DE et al., Analytical Biochemistry 218; 325-9, 1994; Stetler-Stevenson WG et al., Invasion Metastasis 14; 41-8, 1994; Lin LI et al., Oncology 55 (4); 349-53, 1998; Ming-Chung J et al., Biochemical and Biophysical Research Communication 282; 671-7, 2001; Lisa M et al., Science 295; 2387-92, 2002; Francesca T et al., The FASEB Journal 16, 2002).

On the other hand, the root of the root of the elm ( Ulmus davidiana var ) roots, roots of the roots have traditionally been widely used medicinal and health food. The root-derived oils are flavonoids, saponins, tannins, mucus (herbal ingredients: monolithic), resin, β-sitosterol, phytosterol, sigmasterol, tannin , Fatioil, etc., are known to contain medicinal effects and applications include sedimentation, species, single improvement (Chinese medicine standards: Ministry of Health and Welfare), cough, convergence, anti-inflammatory, hookworm, antibacterial (medicinal ingredients: It is written as a medicine such as eponymous note), diuresis, small poison, cure, embroidery supplement (herbology: changshu), and boiled by folk remedies Efficacy has been known for healing swelling, and it is known that it is effective in various ulcers such as gastric ulcer and duodenal ulcer in oriental medicine and folk medicine.

However, there have been no studies on liver cancer inhibitory effect of Rhizome extract.

The inventors of the present invention, while searching for liver cancer inhibitors derived from natural foods or plants that ensure human safety, gap junction cells that play an important role in maintaining homeostasis in vivo even at concentrations without cytotoxicity, and inhibited during liver cancer progression. The present invention has been completed by discovering the effect of restoring liver signaling and inhibiting the progression and metastasis of liver cancer by inhibiting the activity of MMPs that play an important role in metastasis of liver cancer.

Disclosure of Invention An object of the present invention is to provide a liver cancer inhibitor comprising the extract of E. coli as an active ingredient.

In addition, it is an object of the present invention to provide a food having an inhibitory effect of liver cancer using the root extract as an active ingredient.

1 is a result of measuring the cytotoxicity of the myocardium extract according to the present invention against human hepatoma cell line SK-Hep-1,

Figure 2 is a result of measuring the efficacy of restoring intracellular signal transduction through the gap junction suppressed in SK-Hep-1 cells according to the present invention,

Figure 3 is a result of measuring the efficacy of inhibiting the activity of MMP in SK-Hep-1 cells of the extract of Yugeun-pig.

In accordance with the above object, the present invention comprises an effective amount of the extract of the myrtle root as an active ingredient with a pharmaceutically acceptable carrier, to restore intercellular signaling through gap junctions and inhibit the activity of MMP to inhibit the progression and metastasis of liver cancer Provided are pharmaceutical compositions and foods that can be inhibited.

Hereinafter, the present invention will be described in detail.

The root extract of the roots of the present invention can be produced by solvent extraction of root roots or dried products thereof.

The extract of the roots of the present invention is added with an extractant of 2 to 20 times, preferably 3 to 10 times the volume of the dried root skin, and then 1 to 10 hours, preferably 4 to 6 hours at 25 to 100 ° C. After extraction, cooling and filtration, and then concentrated under reduced pressure to obtain a solvent extract from the root skin, wherein the filtration process may be repeated one or more times for the remaining residue after filtration. As the extraction solvent, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol, or a mixture thereof may be used, with 30 to 80% aqueous ethanol solution being most preferred.

Specifically, in a preferred embodiment of the present invention, after extracting refluxed root skin or dried product thereof under reflux for 5 hours using an aqueous 80% ethanol solution, centrifugation to obtain a supernatant, extract the remaining residue in the same manner, and then extract the obtained extract is filtered And freeze-dried to obtain a root extract.

The root extract of the present invention can be useful for restoring the progression and metastasis of liver cancer by restoring intercellular signaling through gap junction channels inhibited in liver cancer cells and inhibiting the activity of MMP.

In particular, compared to the conventional liver cancer preventive agent or therapeutic agent using a radioactive substance or a substance with strong toxicity even in normal cells, the root extract of the present invention can be safely ingested or administered in the body without special toxicity to obtain a liver cancer inhibitory effect. . In addition, the extract of Yugeun-derma according to the present invention can be prepared by separating the material showing a high liver cancer inhibitory effect only with a simple separation process is expected to lower the production cost compared to the various types of liver cancer inhibitors, including conventional liver cancer therapeutics. .

The root extract of the present invention may be mixed with a suitable pharmaceutically acceptable carrier or excipient or diluted with a diluent according to a conventional method to prepare a pharmaceutical composition having the above function. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulation may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the myofibril extract of the present invention range from 1 to 1,000 mg / kg body weight, preferably 10 to 100 mg / kg body weight, and may be administered once or in several divided doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage should in any way be regarded as the present invention. It does not limit the scope of.

In another aspect, the present invention provides a functional food or beverage composition comprising an effective amount of the extract of the myocardium extract, which can inhibit the progression and metastasis of liver cancer by restoring intercellular signaling through gap junctions and inhibiting the activity of MMP. Foods to which the extract of the present invention may be added to exhibit the above effects include, for example, various foods, meat, beverages, chocolate, snacks, confectionary, pizza, ramen, other noodles, gums, ice creams, alcoholic beverages, Vitamin complexes, alcoholic beverages and other health supplements, but are not limited thereto.

The root extract of the present invention may be added to the raw material during food preparation or properly mixed with the cooked food to prepare the above-mentioned health promoting food or beverage, and in this case, the content of the root root extract in the finally prepared food or beverage is In the range from 0.1 to 50% by weight.

Hereinafter, the liver cancer inhibitory effect of the extract of Yu-geun-pi will be described in more detail based on the examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

In addition, in the following examples solids and solid mixtures, liquids and liquids, and percentages for liquids and solids are based on weight / weight, volume / volume and weight / volume, respectively, and all reactions were carried out at room temperature unless otherwise noted. .

Example 1 Preparation of an Extract Having Hepatocellular Carcinoma Efficacy from Root Skin

After drying the dried root skin, 5 parts by weight of an aqueous solution of 50% ethanol (Duksan, Korea) at 80 to 90 ℃ to 1 part by weight of the dry weight was extracted by stirring under reflux for 5 hours. The extract was centrifuged at 8,000 rpm for 30 minutes at 4 ° C. (Vision, Korea) to collect the supernatant, and the remaining residue was extracted once again in the same manner. The extract thus obtained was filtered through filter paper (Whatmann 41), and then freeze-dried to obtain a myodermal extract.

In order to evaluate the hepatocarcinogenesis inhibitory effect of the extract of the roots, the cytotoxicity against hepatocarcinoma cells, the recovery efficacy of intercellular signal transduction through gap junctions, and the inhibition of MMP activity were measured as follows.

<Example 2> Cytotoxicity test of extract

MTT assay (JA Radosevich et al., Virchows Arch. B. Cell Pathol. Incl, Mol. Pathol . 63: 345-350, 1993) was performed to determine the cytotoxicity of the extracts.

Human lung cancer cell line, SK-Hep-1 cells 10% (Korea Cell Line Bank) FBS, penicillin (penicillin) 100 IU / ㎖ and streptomycin (streptomycin) 100 ㎍ / ㎖ is added by using the DMEM culture medium 5% CO ₂ Incubated in a 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA). The medium compositions used for cell culture were all used by GIBCO BRL (Grand Island, NY, USA).

In order to investigate the effect of inhibiting the growth of cancer cell lines by the addition of E. coli extract, the cultured liver cancer cells SK-Hep-1 were dispensed in a 96 well plate at a concentration of 1 × 10 ⁴ / well. Extracts were added at various concentrations and then incubated for 72 hours. Four hours before 72 hours, 20 μl / well of MTT reagent (Sigma Aldrich) was added to each well and incubated. After incubation, all of the supernatant was removed, leaving only the remaining cells. The remaining stained cells were lysed with 200 μl of DMSO (Dimethyl sulfuroxide) and absorbance was measured at 570 nm. This experiment was repeated three times.

Figure 1 and Table 1 shows the results of measuring the cytotoxicity of the extract of the roots.

Extract Treatment Concentration (㎍ / ml) Cytotoxicity (%) 0 0 10 -1.403 ± 9.935 20 -12.318 ± 10.327 50 -11.366 ± 9.096 100 15.407 ± 2.067

As shown in Figure 1 and Table 1 , the extract of the root of mycelium according to the present invention showed little cytotoxicity at a concentration of 50 ㎍ / ㎖ or less.

Example 3 Hepatocarcinoma Progression Inhibitory Effect of Rhizome Cortex Extract

Inhibition activity of hepatic carcinoma extracts in liver cancer progression was measured by an experiment of gap junctional intercellular communication assay through gap junction in liver cancer cells. This was measured using the SL / DT assay (Scrape Loading / Dye Transfer assay; Upham, BL, et al., Carcinogenesis 18: 37-42, 1997).

Specifically, DMEM medium containing 10% FBS, penicillin 100 IU / ml and streptomycin 100 μg / ml was added to SK-Hep-1 cells, a human liver cancer cell line, to measure intercellular signaling through gap junctions of liver cancer cells. Were incubated in a 5% CO ₂ , 37 ° C. incubator (Forma Scientific Co., Marjetta, OH, USA). The medium compositions used for cell culture were all used by GIBCO BRL (Grand Island, NY, USA). The liver cancer cells thus cultured were dispensed in a 2 ml plastic dish at a concentration of 1 × 10 ⁵ / ml and incubated for 24 hours. Thereafter, the culture solution of the cells cultured in each dish was changed to the same culture solution in which the extracts of the roots were mixed at various concentrations (experimental group). At this time, the control group was changed only to the culture medium containing no root extract. After 48 hours of further incubation, if cells grew more than 90% of the bottom of the dish, Lucifer yellow staining was used to determine whether intracellular signaling was restored through gap junctions inhibited in liver cancer cells. BioRad, Hercules, CA, USA).

Figure 2 and Table 2 shows the effect of the recovery of intercellular signaling through the gap junction of the root extract.

Control Root skin extract concentration (㎍ / ㎖) 12.5 25 50 Number of cells with intercellular signaling (dogs) 58.00 ± 7.937 80.41 ± 7.276 92.12 ± 9.052 99.67 ± 9.712

As shown in Figure 2 and Table 2 showed the effect of restoring the inhibition of intercellular signaling through gap junctions, which is an important phenomenon of liver cancer progression in proportion to the concentration.

<Example 4> MMP activity inhibitory effect of the extract

After culturing SK-Hep-1 cells, which are human liver cancer cells, for 24 hours, washed with PBS, incubated for 2 hours with serum-free media, and then treated with the extracts of the myocardium extract for 48 hours. Incubated. Supernatants were collected, centrifuged (vision, DN-5500), quantified (Beckman, DU650), and the same amount of total protein was electrophoresed on 10% SDS-PAGE gel containing 0.1% gelatin. After electrophoresis, the gel was washed three times with 2.5% Triton X-100. The gel was added to 40 mM Tris, 200 mM NaCl, and 10 mM CaCl ₂ mixed solution, treated at 37 ° C. for 18 hours, and then stained with 0.1% Coomassie brilliant blue. At this time, the whole background is dyed blue, and the gelatin decomposed portion appears as a white band.

Figure 3 shows the inhibitory effect of M. root extract of M. root, lane 1 is the untreated control; Lane 2 was treated with 5 μg / ml of the root extract; Lane 3 was treated with 10 μg / ml of the root extract; Lane 4 was treated with 20 μg / ml of the root bark extract; Lane 5 was treated with 40 μg / ml of E. coli extract; The first band shows the activity of MMP-9 and the second shows the activity of MMP-2.

As shown in FIG. 3 , it was confirmed that the myofibril extract inhibited the activity of MMP-9 at concentrations of 20 μg / ml and 40 μg / ml. In addition, MMP-2 showed a complete inhibitory effect even at 5 ㎍ / ㎖.

Therefore, the extract of the myodermis extract according to the present invention restores the inhibition of intercellular signaling through gap junction, which is the main phenomenon of liver cancer progression at a concentration that does not exhibit cytotoxicity, and effectively inhibits the progression and metastasis of liver cancer by inhibiting the activity of MMP. It was confirmed.

The root extract of the present invention may be used in the form of a powder, tablets, capsules, injections, solutions, and the like according to the methods commonly used pharmaceutically or in combination with excipients used alone or pharmaceutically.

Formulation examples are illustrated below.

Production Example 1 Powder

2 g of dried root extract

1 g lactose

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Tablet

100 mg of dried extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to the conventional tablet manufacturing method to produce a tablet.

Preparation Example 3 Capsule

100 mg of dried extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components to fill a gelatin capsule in accordance with the conventional capsule preparation method to prepare a capsule.

Production Example 4 Injection

100 mg of dried extract

Suitable amount of distilled water for injection

pH adjuster

According to a conventional injection preparation method, the active ingredient is dissolved in distilled water for injection, the pH is adjusted to about 7.5, and the whole is filled with 2 ml of ampoule with injection distilled water and sterilized to prepare an injection.

In addition, health foods and alcoholic beverages are prepared in the following manner.

<Manufacture example 5> Wire type

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to make a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then ground to a powder having a particle size of 60 mesh.

The root extract of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh using a grinder to obtain an extract dry powder.

Granules were prepared by combining the dry powders of the grains, seeds and root extracts prepared above in the following ratios.

Cereals: Brown rice 30% by weight, barley 15% by weight, barley 20% by weight, glutinous rice 9% by weight,

Seeds: perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight,

3% by weight of dried root powder, 0.5% by weight of ganoderma lucidum, 0.5% by weight of turmeric

Preparation Example 6 Chewing Gum

Chewing gum was prepared by a conventional method by combining 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 0.1% by weight of the root extract of the present invention.

Production Example 7 Candy

Candy was prepared by the conventional method by combining 60% by weight of sugar, 39.8% by weight of starch syrup, and 0.1% by weight of perfume and 0.1% by weight of the extract of Yukje skin.

Production Example 8 Biscuits

Force 1st class 25.59 wt%, 1st class gravity 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.78 wt%, ammonium 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt %, Rice flour 1.45 wt%, Vitamin B₁0.0001 wt%, Vitamin B20.0001 wt%, Milk flavor 0.04 wt%, Water 20.6998 wt%, Whole milk powder 1.16 wt%, Substitute milk powder 0.29 wt%, Calcium phosphate 0.03 wt% , Biscuits were prepared in a conventional manner by combining 0.29% by weight of spraying salt and 7.27% by weight of spray oil with 1% by weight of the root extract of the present invention.

<Manufacture example 9> Healthy drink

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of sodium riboflavinate, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 98.7362% by weight of water 1% by weight of the extract of the roots of the extract was prepared in a conventional manner for a healthy drink.

<Manufacture example 10> Sausage

Pork 65.18%, chicken 25%, starch 3.5%, soy protein 1.7%, salt 1.62%, glucose 0.5%, glycerin 1.5% by weight and 1% by weight of the extract of the present invention Sausage was prepared.

Production Example 11 Health Supplement

55% by weight of spirulina, 10% by weight of guar gum enzyme digestion, 0.01% by weight of vitamin B₁ hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, 22.2% by weight of lactose and 1.85% by weight of corn starch And 10% by weight of the root extract of the present invention was prepared tablet-type health supplement food in a conventional manner.

<Manufacture example 12> Alcohol

Soju extract containing 0.15 ~ 0.7% with soju, beer, liquor or fruit liquor is made into an emulsion state, and separated at 7,000 rpm with a centrifuge for 15 minutes in vacuum or at 9,000 rpm with a high speed mixer. Manufacture.

As described above, the root extract of the present invention can effectively inhibit the progression and metastasis of liver cancer by restoring intercellular signaling through gap junction channels of liver cancer cells and inhibiting MMP activity.

Claims (5)

A pharmaceutical composition for inhibiting liver cancer comprising the extract of Root-root skin as an active ingredient with a pharmaceutically acceptable carrier. The method of claim 1, A pharmaceutical composition, characterized by inhibiting the progression and metastasis of liver cancer through the recovery of intercellular signaling inhibition through gap junctions and inhibition of the activity of matrix metalloproteinase (MMP). The method of claim 1, Pharmaceutical extract, characterized in that the extract is obtained by solvent extraction of the roots of the roots or dried products thereof for 1 to 10 hours at 25 to 100 ℃. The method of claim 3, wherein Pharmaceutical composition, characterized in that the solvent is selected from the group consisting of methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol and mixtures thereof. A health-promoting food containing the extract of the myrtle root and having the effect of inhibiting the progression and metastasis of liver cancer.