KR20050030981A - Pharmaceutical composition comprising the extract of trichosanthes kirilowii var. japonica for treating or preventing cancer and leukemia disease - Google Patents

Abstract

A pharmaceutical composition comprising the extract of Trichosanthes kirilowii var. japonica is provided, which Trichosanthes kirilowii var. japonica extract has improved anticancer and leukemia cell apoptosis inducing effects, so that the extract is useful for prevention and treatment of cancer and leukemia. The pharmaceutical composition for prevention and treatment of cancer and leukemia comprises 0.1 to 50 wt.% of the crude extract of Trichosanthes kirilowii var. japonica, wherein the crude extract of Trichosanthes kirilowii var. japonica is produced with water, C1-C4 lower alcohol and mixed solvents thereof. The pharmaceutical composition for prevention and treatment of cancer and leukemia comprises 0.1 to 50 wt.% of the nonpolar solvent soluble extract of Trichosanthes kirilowii var. japonica, wherein the nonpolar solvent soluble extract of Trichosanthes kirilowii var. japonica is produced with hexane, chloroform, methylene chloride or ethylacetate.

Description

Pharmaceutical composition comprising the extract of Trichosanthes kirilowii var. japonica for treating or preventing cancer and leukemia disease}

The present invention relates to a composition for the prevention and treatment of cancer diseases containing a yellow skyweed extract, specifically, cancer or leukemia containing a yellow skyweed crude extract and a nonpolar solvent soluble extract having anti-cancer and leukemia cell apoptosis-inducing activity. It relates to a pharmaceutical composition for the prevention and treatment of diseases.

Leukemia refers to a disease in which leukocytes produced in the bone marrow develop malignant tumors. Because of the increase in leukocytes, blood appears whiter than normal, which is called leukemia. However, the peripheral blood of all leukemia patients does not show an increase in leukocytes. The increase in leukemia cells, which are cancer cells in the bone marrow, reduces the production of normal white blood cells, erythrocytes and platelets, thereby making them easier to infect, developing anemia and hemorrhagic tendencies (easily nosebleed or gum bleeding or bruising on the skin). If not, you will die from infection or bleeding.

Leukemia is classified according to the type of leukocytes with malignant proliferation. The leukemia is divided into myeloid and lymphocytic tissues. The leukemia is divided into acute and chronic according to the long and short course of natural history. Thus, there are four types of leukemias: acute lymphocytic leukemia in children, acute myeloid leukemia in adults, chronic myelogenous leukemia in Korea, and chronic lymphocytic leukemia, which is extremely rare in Korea. There are leukemias that are classified separately. In acute cases, leukemia cells are mostly immature, and in chronic cases, many mature cells are seen.

New therapies for leukemia include the use of multi-drug resistant conversion agents, which include the use of drugs that reduce the mechanism of becoming tumor cells to avoid damaging effects of chemotherapeutic agents (sometimes cause irresponsiveness or recurrence). ), The use of substances known as biological response modifiers (BRMS) as biological therapies. These substances are normally produced in small amounts as part of the body's natural response to cancer and other diseases. BRMS forms include monoclonal antibodies, and toxins are attached to antibodies that react with supplemental antibodies derived from tumor cells. Cytokines (eg, interferon, interleukin, and colony stimulating factor (CSFS)) are naturally occurring chemicals that stimulate blood cell production and help restore blood cell counts very quickly after treatment. Examples of these drugs include PSC 833, a multi-drug resistant converting agent, Rituxan, a monoclonal antibody, and the following cytokines: erythropoietin and Ipoietin, which stimulate the production of red cells; G-CSF, GM-CSF, filgrastim, and Sargramostim, which stimulates white cell production; And thrombopoietin that stimulates the production of platelets.

The treatment of leukemia is very complex and dependent on leukemia form. In addition, although significant clinical diversity is observed in patients with leukemia, these diversity occurs after one course of treatment. Patients who are resistant to treatment have very short survival and are not interested when resistance occurs.

Although widespread treatment programs show improvement in outcomes, there is a continuing need for the development of new therapies for the treatment of all types of leukemia.

The yellow fly ( Trichosanthes kirilowii var. Japonica ) used in the present invention is a perennial vine and grows on the hills or forest edges of southern Korea (Jeju-do, Heuksan-do, and southern tea islands) and the central region. The fruit of the yellow skyworms contains triterpenoid saponins, organic substances, fructose, pigments, and the like, and the seeds contain fatty oils. It also contains arginine, lysine, aniline, alanine, valine, glycine, and alkaloid complexes, and the protein in the fruit is different from the protein in the muscle. Shin Mingyo; Hyangjeomsa Dictionary, Yeonglimsa, pp960-961, 1998). In the oriental medicine, the roots of the yellow sky feathers are called King and Geun (王 瓜 根), the fruit is the fruit and vegetable (실 仁), and the seeds are called the fruit and fruit (土 瓜仁). The starch is extracted from the root and used for food. Fruits and roots are known to be used as medicinal herbs such as bruises, fish blood, jaundice, skin diseases, and seeds are used for expectoration, Jinhae, pain, or as anti-inflammatory agents (Kim, Tae-Jung; Korean Resource Plant, Seoul National University Press, p173, 1996). As mentioned above, the roots and fruits of the Yellow-Purple have been used in folklore for a long time, but the report on the composition and physiological activity of each site is still insufficient (Kondo, T., Inoue, M. et al., Biol. Pharm. Bull). ., 18 , p726, 1995), in particular, the report on the effect of inhibiting the tumor growth of the extract of each part of the yellow skyweed is still insufficient.

Therefore, the present inventors searched for anticancer effect of yellow native starfish, Jeju native plant, as part of efforts to develop anticancer drugs using natural-derived substances, and as a result, the anticancer effect of yellow starfish extract and apoptosis of HL-60 leukemia cells (apoptosis) The present invention was completed by confirming the efficacy of inducing.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of cancer and leukemia disease, which contains a yellow starch crude extract and a non-polar solvent soluble extract as an active ingredient having anticancer efficacy and apoptosis inducing effect of HL-60 leukemia cells. It is.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer and leukemia diseases containing the yellow starch crude extract and the non-polar solvent soluble extract as an active ingredient.

The crude extract includes water and extracts soluble in C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like, and mixed solvents thereof. The non-polar solvent soluble extract is soluble in hexane, chloroform, methylene chloride or ethyl acetate. One extract.

Hereinafter, the present invention will be described in detail.

The yellow star crude crude extract and the non-polar solvent soluble extract of the present invention can be obtained as follows.

The leaves, stems, roots, rinds and seeds of the yellow starling of the present invention are dried and pulverized, respectively, and then pulverized, to about 2 to 10 times, preferably about 3 to 5 times the weight of the sample for each site. A volume solvent of water and a polar solvent of C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like, or a mixed solvent having a mixing ratio thereof of about 1: 0.1 to 1:10, preferably methanol at about 5 at room temperature. The supernatant is recovered by vacuum filtration using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction for a period of time to 1 day, preferably 24 hours, and then Repeating the process 2 to 5 times, preferably 3 times to collect the supernatant and concentrated under reduced pressure to obtain a crude crude methanol extract per yellow sky.

In addition, the non-polar solvent soluble extract of the present invention is suspended in distilled water after the crude extract, the suspension is added to a nonpolar solvent such as about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, ethyl acetate, chloroform It can be obtained by extracting and separating the nonpolar solvent soluble layer 1 to 10 times, preferably 2 to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed . , Pp 6-7, 1998).

Preferably, the crude yellow crude extract of the yellow starch fraction obtained by the above process, preferably the organic crude solvent such as butanol, hexane, ethyl acetate and the like in the methanol crude extract of the yellow algae leaf from low solvent to high polar solvent By, preferably, hexane, ethyl acetate, butanol, water, and then sequentially fractionated solvent, concentrated under reduced pressure to obtain a solvent fraction of the yellow leaf.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer and leukemia disease containing the yellow starch crude extract and the non-polar soluble extract as an active ingredient.

In addition, the yellow starling has been used as a herbal medicine and edible for a long time, the extracts of the present invention extracted from them also have no problems such as toxicity and side effects.

The antitumor effect of the methanol extracts and the solvent fractions of the leaves of the yellow algae obtained by the above method was tested. The ethyl acetate and butanol fractions of the yellow algae leaf showed the highest growth inhibition effect. Growth inhibitory effect by the fraction of the sky leaf was confirmed to increase depending on the concentration. In addition, the apoptotic (apoptotic) cells were observed to increase in time-dependent manner by treatment with ethyl acetate fraction of yellow leaf. From these results, it was confirmed that the inhibitory effect of HL-60 leukemia cell proliferation of the yellow native bird, Jeju native plant, was due to apoptosis induction of HL-60 cells.

The pharmaceutical composition for preventing and treating cancer or leukemia disease of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

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

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, 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, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may 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 at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a dietary supplement comprising the extract and a food-acceptable food supplement additive that exhibits a prophylactic effect of a disease caused by cancer and leukemia. Examples of the foods to which the yellow starch extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing the disease caused by cancer and leukemia. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1.Preparation of Methanol Crude Extract of Yellow Leaf

After collecting yellow leaves of the wild sky native to Jeju Island in Wolpyeong, Jeju Island, and drying them, grind them with a grinding machine, and then finely powder 80% methanol, which is 3 times the weight of 312 g of yellow sky leaf fine powder sample, and deposit it at room temperature. After stirring for hours, the supernatant was recovered by vacuum filtration. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to obtain 44.89 g of a yellowish-brown methanol crude extract.

Example 2. Preparation of Methanol Crude Extracts for Different Yellow Spots

The stems, roots, rinds, and seeds of the yellow-sprayed native to Jeju Island are collected and dried in the Wolpyeong area of Jeju Island, and then finely ground by grinding with a crusher, and then finely divided by 45g, roots 42g, 31g and 35g seeds, respectively. 80% methanol corresponding to three times the weight of the sample was deposited and carried out in the same manner as in Example 1, yellow sky stalk (yield 1.3068 g), root (yield 3.3224 g), and rind (yield 5.2515 g). And crude crude extract of each part of the seed (yield 3.0081 g) was obtained.

Example 3. Preparation of Yellow Locust Fraction by Solvent

After 44.89 g of the yellow leaf methanol coarse extract obtained in Example 1 was suspended in 1 L of water, the solvents were sequentially separated from the solvent having the lowest polarity to the solvent having the highest polarity in the order of hexane, ethyl acetate, butanol and water. First, 1 liter of hexane was added to and dissolved in a crude yellow methanol extract, suspended in 1 liter of water, and then vacuum-dried by separating only the components dissolved in the hexane layer in a fractionated filter. This process was repeated three times to obtain a hexane fraction (yield 1.0863 g). 1 L of ethyl acetate was added to the remaining aqueous layer, and only the components dissolved in the ethyl acetate layer were separated and dried under vacuum. This process was repeated three times to obtain an ethyl acetate fraction (a yield of 2.2157 g), and 1 liter of butanol was added to the remaining aqueous layer to obtain a butanol fraction (a yield of 6.779 g). Finally, the remaining aqueous layer was concentrated under reduced pressure to obtain an aqueous layer fraction (1 g of yield).

Experimental Example 1. Cell Culture

HL-60 cell line (promyelocytic leukemia) derived from patients with acute promyelocytic leukemia was distributed from Korea Cell Line Bank (KCLB) at 100 units / ml penicillin-streptomycin (GIBCO) and 10% fetal bovine RPMI 1640 medium containing serum, GIBCO) was used and cultured in a 37%, 5% carbon dioxide incubator. Subcultures were performed once every 3 to 4 days.

Experimental Example 2. Measurement of metabolic activity of cells

2-1. Measurement of Cell Metabolic Activity of Leaf Extracts

Inhibition of HL-60 cell proliferation by extract treatment of yellow-flying star was determined by measuring the metabolic activity of the cells (Carmichael, J., DeGraff, WG et al., Cancer Research, 47 , p936, 1987). HL-60 cells (2.5 × 10 ⁵ / ml) were placed in each well of a 96 well plate, and the samples obtained in Examples 1 and 3 were collected at different concentrations (concentration interval 10 μg / ml-100 μg / Ml). After incubating for 4 days, 100 μg of MTT (3- (4,5-dimehtylthiazol) -2,5-diphenyltetrazolium bromide, Sigma) reagent was added thereto, followed by further incubation for 4 hours. The plate was centrifuged at 1000 rpm for 10 minutes, the medium was carefully removed, and then 150 μl of DMSO (dimethylsulfoxide, Sigma) was added to dissolve the formazan precipitate produced by the reduction of MTT, followed by a microplate reader. , BIO-TEK INSTRUMENIS. INC) was used to measure absorbance at 540 nm. The average absorbance value for each sample group was obtained, and growth inhibition was measured by comparing with the absorbance value of the control group.

As a result of the experiment, the 80% methanol extract of yellow leaf was shown to inhibit cell proliferation by about 43% when treated with 100µg / ml, 19.2% by hexane fraction, 73.2% by ethyl acetate fraction, butanol fraction By 47.6% of the HL-60 cell proliferation inhibitory effect was confirmed (see Fig. 1a). Ethyl acetate fractions with the highest inhibitory effects were treated with various concentrations (10, 25, 50, 100 μg / ml), 8.6% at 10 μg / ml, 24.3% at 25 μg / ml, and 50 μg. Cell growth inhibition was about 70% or more at the concentrations of / ml and 100 µg / ml (see FIG. 1B).

2-2. Determination of Cell Metabolism Activity of Extracts from Yellow Spotted Fields

80% methanol extract of the root, stem, seed, and rind of the yellow starling in Example 2 was carried out in the same manner as in Experimental Example 2-1, and various concentrations of 10, 25, 50, and 100 µg / ml were obtained. Cell proliferation inhibitory effect was measured when treated with.

As a result, it was confirmed that cell growth was significantly inhibited at the root concentration of 10 µg / ml, the stem of 50 µg / ml, the seed of 10 µg / ml and the rind of 100 µg / ml (see Table 1). This concentration was used to study the mechanism of tumor growth inhibition of the extract by site.

extract Cell growth inhibition degree (%) 10 μg / ml 25 μg / ml 50 μg / ml 100 μg / ml Root 62.9 ± 4.48 ^* 81.7 ± 1.02 ^* 80.5 ± 1.09 ^* 79.7 ± 2.98 ^* stem 2.78 ± 0.70 ^* 8.05 ± 3.07 ^* 88.2 ± 1.18 ^* 88.5 ± 0.58 ^* Seed 88.4 ± 0.5 ^* 87.7 ± 0.69 ^* 88.1 ± 0.41 ^* 89.2 ± 0.42 ^* pericarp 4.98 ± 0.66 11.46 ± 1.42 ^* 14.59 ± 1.41 ^* 45.7 ± 3.70 ^*

Experimental Example 3. Determination of HL-60 cell apoptosis inducing effect

3-1. DNA fragmentation analysis

The HL-60 cells (2.5 × 10 ⁵ / ml) were treated with the extracts of the respective parts of the yellow skyhopper obtained in Examples 1 to 3 and the solvent fractions of the leaves, and then cultured for 24 hours. After collecting the cells, the DNA was isolated using a Wizard genomic DNA purification kit ^{(Wizard ?? Genomic DNA Purification Kit,} Promega). The separated DNA was electrophoresed for 30 minutes (100V) on a 1.2% agarose gel, then stained with ethidium bromide and observed for DNA fragmentation under UV transilluminator (UV transilluminator). Purohit, A. et al., Int. J. Cancer, 85 , p584, 2000).

As a result, DNA fragmentation was best observed in HL-60 cells treated with ethyl acetate fraction of yellow leaf (see FIG. 2A; A line-100bp DNA chain size marker, B line-control, C-line-methanol extract, D-line-n-hexane fraction, E-line-ethyl acetate fraction, F-line-n-butanol fraction, and G-line-water fraction) DNA fragmentation was best observed when the extracts were treated (see Figure 2b; A line-1 kb DNA chain size marker, B line-control, C line-10 μg / ml root extract, D line-10 μg / ml Seed extract, E-line-50 µg / ml stem extract).

3-2. Cell cycle analysis

In order to observe the increase of sub-G1 hypodiploid cells by induction of apoptosis by the yellow-flying extract, the following experiment was performed. HL-60 cells (2.5 × 10 ⁵ / mL) were treated with the solvent fractions of the extracts and leaves of each part of the yellow sky, and then cultured for 24 hours, and then HL-60 cells were harvested and washed with PBS. HL-60 cells were fixed in 70% ethanol at −20 ° C. for 30 minutes, then washed with PBS, treated with RNase A (1 mg / ml) followed by propidium iodide (PI, Sigma) stained and analyzed for cell cycle using a flow cytometer (EPICS ^{?? ??} COULTER XL ^TM Flow Cytometer, BECKMAN COULTER) (Nicoletti, I., et al., J. Immunol. Methods, 139, p271, 1991) .

The ethyl acetate fraction of the yellow leaf was measured at 100 ㎍ / ml, and the results showed that the sub-G1 hypodiploid cells were 13.6% at 3 hours, 55.9% at 4 hours, 70.5% at 5 hours, and 6 hours. Apoptotic cells increased with time to 71.3% at 7 hours, 94.8% at 8 hours, and 96.9% at 8 hours (see Table 2). In addition, as shown in Figures 3a to 3d sub-G1 low diploid cells 37.7% by the stem extract 50 ㎍ / ㎖ treatment, 12% by root extract 10 ㎍ / ㎖, seed treatment for the yellow spotted extract treatment It was observed at 22.7% by treatment of the extract 10 ug / ml.

   time cell % Apoptosis cells G1 G2 / M 0 hr 0.46 55.3 18.3 3 hr 13.6 49.2 18.6 4 hr 55.9 27.2 5.86 5 hr 70.5 18.1 3.99 6 hr 71.3 20.2 2.41 7 hr 94.8 4.13 0.85 8 hr 96.9 4.60 0.09

3-3. Western blot analysis

The HL-60 cells (2.5 × 10 ⁵ / ml) were treated with the extract of each part of the yellow skyworm and the solvent fractions of the leaves by concentration (10, 50, 100 µg / ml), and then incubated for 24 hours. After washing the cells with PBS (Phosphate Buffered Saline) two or three times, 500 μl of lysis buffer was added for lysis, and then centrifuged at 14,000 rpm for 20 minutes to remove cell membrane components. Protein concentrations were quantified using the Bradford method (Bradford, MM, Anal. Biochem ., 72 , p248, 1976). 40 μg of lysate was denatured by 10% mini-gel SDS-PAGE (Polyacrylamide Gel Electrophoresis), which was applied to a polyvinylidene difluoride membrane (BIO-RAD) at 200 mA for 2 hours. Transferred. The membrane was reacted at room temperature for 2 hours using TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk. Antibodies to examine the expression level of PARP as anti-PARP (1: 1000) (Santa-Cruz) and Caspase-3 Ab (Cleaved) disconnected with antibodies to examine the expression levels of Caspase-3 caspase-3 Ab, 1: 1000) (Cell Signaling) was diluted in TTBS solution and used for 2 hours at room temperature. As a secondary antibody, anti-rabbit IgG (Herse Radish Peroxidase) conjugated anti-rabbit IgG (anti-rabbit IgG, Amersham Co.) was used diluted 1: 5000, the reaction proceeded for 30 minutes at room temperature. The membrane was washed three times with TTBS, reacted with ECL substrate (Amersham Co. RPN2106) for 1 to 3 minutes, and then exposed to X-ray film.

As a result of the experiment, as shown in Figure 4a, yellow leaf stem extract 50 ㎍ / ㎖ (A), root extract 50 ㎍ / ㎖ (B), seed extract 10 ㎍ / ㎖ (C), peel extract 100 ㎍ / ㎖ (D) And when treated with a concentration of 50 ㎍ / ㎖ (E) or more ethyl acetate solvent fractions of the leaves, the band of the active form of caspase-3 (19 kDa) was confirmed, which is a substrate by the activation of caspase-3 PARP (116 kDa) was delivered and closely matched the concentration at which the band of 85 kDa appeared (see FIG. 4B).

It describes an example of a pharmaceutical composition comprising a yellow sky feather extract of the present invention, the present invention is not intended to limit this, it is intended to explain in detail only.

Formulation Example 1 Preparation of Powder

Yellow Skyweed Extract Dry Powder 300 mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Yellow Skyweed Extract Dry Powder 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Yellow Skyweed Extract Dry Powder 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Yellow Skyweed Extract Dry Powder 50 mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Yellow Skyweed Extract Dry Powder 100 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Yellow Skyweed Extract Dry Powder 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Yellow Skyweed Extract Dry Powder 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the yellow star crude crude extract and the non-polar solvent soluble extract of the present invention exhibit anticancer efficacy and induction of apoptosis of HL-60 leukemia cells, and thus are useful for the prevention and treatment of cancer and leukemia diseases. It can be used as a composition.

Figures 1a and 1b shows the degree of growth inhibition of HL-60 cells of the yellow skyweed extract, Figure 1a is a diagram showing the degree of growth inhibition of HL-60 cells when treated with methanol extracts and solvent-specific fractions of yellow skyflower leaves, 1b is a diagram showing the degree of inhibition of HL-60 cell growth when the ethyl acetate fraction of the yellow leaf was treated at various concentrations.

Figures 2a and 2b shows the DNA fragmentation in HL-60 cells of the yellow starch extract, Figure 2a is a diagram showing the degree of DNA fragmentation when treated with methanol extracts and solvent-specific fractions of yellow skystar leaves, Figure 2b Figure showing the degree of DNA fragmentation when treated with extract for each site,

Figures 3a to 3d shows the effect of apoptosis induction measured by flow cytometry, Figure 3a is a diagram showing the degree of apoptosis when no treatment, Figure 3b is treated with a yellow gingival root extract of 10 ㎍ / ㎖ Figure 3c is a diagram showing the degree of apoptosis of the city, Figure 3c is a diagram showing the degree of apoptosis at the time of treatment with 10 ㎍ / ㎖ yellow celtic seed extract, Figure 3d is treated with 50 ㎍ / ㎖ yellow stalk stem extract Is a diagram showing the degree of apoptosis in the city,

Figure 4a is a diagram showing the degree of activation of caspase-3 (Caspase-3) when treated with the extract and the ethyl acetate fraction of the leaves of each part of the yellow sky, Figure 4b is the extract and leaves of each part of the yellow A diagram showing the segment of PARP when treated with an ethyl acetate fraction.

Claims (9)

A pharmaceutical composition for the prevention and treatment of cancer diseases, including crude extracts of Trichosanthes kirilowii var. Japonica . The pharmaceutical composition according to claim 1, wherein the crude extract is water, C ₁ to C ₄ lower alcohols, and extracts soluble in a mixed solvent thereof. A pharmaceutical composition for the prevention and treatment of cancer diseases comprising a non-polar solvent soluble extract of yellow sky. The pharmaceutical composition according to claim 3, wherein the nonpolar solvent soluble extract is an extract soluble in hexane, chloroform, methylene chloride or ethyl acetate. The pharmaceutical composition according to claim 4, wherein the non-polar solvent soluble extract is an extract soluble in ethyl acetate. The pharmaceutical composition of claim 1 or 3, wherein the cancer disease is leukemia. 4. A pharmaceutical composition according to claim 1 or 3 comprising 0.1 to 50% of said extract relative to the total weight of the composition. A dietary supplement comprising a yellow flyweed extract and a food-acceptable food supplement additive that has a preventive effect on cancer diseases. The health functional food according to claim 8, which is a health beverage.