KR100950564B1 - Anti-Cancer Composition Containing an Extract of Chinese Herb as an Effective Ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for anticancer having an inhibitory effect of MMP-9, and more specifically, to the composition of peony, Angelica, Sesame, Cinnabar, Cultivator, Safflower, Licorice, Prickly pear, Brown root, Sansa, Dansam and persimmon soup broken the following medicinal herbs, C _{3 -6} of a lower alcohol, water, a lower organic acid, a lower alcohol ester, peony, extracted with a solvent selected from the group consisting of a lower ketone, and halogenated hydrocarbons, Angelica, Eucommia, locking, Cassia tora, safflower, licorice, The present invention relates to a pharmaceutical composition and health food for anticancer containing herbal extracts consisting of prickly pear, brown root, hawthorn, salvia and persimmon soup as active ingredients.

According to the present invention, the herbal extracts are useful as anticancer compositions because they inhibit the expression of MMP-9 in human breast cancer cell lines and inhibit the infiltration and metastasis of cancer.

Matrix metalloproteinase (MMP), phorbol 12-myristate 13-acetate (PMA), cancer, tumors, metastasis, invasion

Description

Anti-Cancer Composition Containing an Extract of Chinese Herb as an Effective Ingredient}

The present invention relates to a pharmaceutical composition for anticancer having an inhibitory effect of MMP-9, and more specifically, to the composition of peony, Angelica, Sesame, Cinnabar, Cultivator, Safflower, Licorice, Prickly pear, Brown root, Sansa, Dansam and persimmon soup It relates to an anticancer pharmaceutical composition and health food containing the herbal extract as an active ingredient.

The metastasis process of cancer is a series of processes in which metastatic cancer cells leave the site of origin and invasion through the blood vessels into the surrounding tissue to form secondary tumors by proliferation at other sites. In this process, matrix metalloproteinases (MMPs) play an important role in inducing and metastasis of cancer cells by breaking down the extracellular matrix (ECM) and basement membrane (BM). As proteolytic enzymes, more than 20 species have been isolated and confirmed so far. MMP is an endoproteinase that uses zinc as a coenzyme and is divided into collagenase, gelatinase, stromelysins, and Membrane-type MMP according to its structure and properties. In particular, MMP-2 (72 kDa type IV collagenase; gelatinase A) and MMP-9 (92 kDa type IV collagenase; gelatinase B), which are type IV collagenase, are enzymes that degrade type IV collagen, an important component of the basement membrane. , Most commonly associated with cancer migration and metastasis (Nabeshima, K. et. al ., Pathol. Int ., 52 , pp 255-64, 2002). In addition, MMP-9 is known to play an important role in invasion and metastasis of breast cancer (Scorilas A. et. al ., Br . J. Cancer , 84 , pp 1488-96, 2001). In addition, since the promoter region of MMP-9 has a transcription factor AP-1 and NF-kB binding region, cancer-causing stimulants such as cytokines and PMA (phorbol 12-myristate 13-acetate) are It is known to regulate the expression of MMP-9 by regulating the activation of transcription factors such as NF-kB (Sato H., et. al ., Oncogene , 19 , pp2904-2912, 2000; Lee SO, et al ., Biochem . Biophys . Res . Commun . , 354 , pp 165-171, 2007). As described above, substances that inhibit transcription factor activity to inhibit MMP-9 expression have been spotlighted as novel cancer inhibitors.

Peeonia lactiflora Pall . ) Is distributed in Korea, Mongolia and East Siberia, and is used for gardening because of its beautiful flowers. Roots are used for pain, abdominal pain, amenorrhea, dysmenorrhea, hemostasis, anemia, and bruises.

Angelica gigas Nakai ), also known as tongdang (土 當歸), Sunggeumcho, Chosun donkey, pharmacologically promotes the blood flow in the coronary artery, and stimulates the production of red blood cells.

Eucommiae Cortex ) is a Chinese specialty plant, which uses bark as a tonic tonic, strengthens the cerebral and lungs, and manages knee pain and nectarism. In the private sector, the leaves are decocted and used to treat neuralgia hypertension and are also taken as tea.

Cinnamomum cassia Blume is a medicinal herb made from broilers of broilers or other related plants. It has an unusual scent, its color is darker, and its weakness is hot and warm. Opening the pores of the skin during the early cold to sweat, relieve pain in the shoulders and back, pain in the extremities joints, promote circulation of blood and treat lack of yang. The pharmacological action is known to inhibit the development of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, pneumococcus pneumoniae, inhibition of influenza virus, and diuretic effect.

Cassia tora L. ) means seeds that brighten the eyes, and is also known as Yangming, Embossing, Horse Racing, Horse Crystal, Yannokdu, and Gandu. Hepatic (肝火) down the eyes, redness, swelling, pain, tears flowing symptoms to treat, night blindness is used to constipation caused by fever accumulated in the large intestine is effective. It is also used to lower blood pressure and prevent atherosclerosis. Pharmacological effects include diuresis, constipation, uterine contraction and skin fungal suppression, cholesterol lowering.

Safflower ( Carthamus tinctorius L. ) is called hongram (이), dill, safflower, safflower, and when it gets wet in the early morning dew, dried flowers are called safflower. In oriental medicine, it is used to treat women's diseases, pain, abdominal pain, etc. Seed oils contain a lot of linolic acid, which is good for preventing atherosclerosis from excess cholesterol.

Licorice ( Glycyrrhiza uralensis Fisch ) is a medicinal plant and is distributed in northeastern China, Siberia and Mongolia. The root is sweet and is used as a sweetener and herbal medicine.

Acanthopanax senticosus ) is also called thorny ogapi , and it is known that ogapi, which is peeled from the bark of ogopi tree, is highly effective in treating back pain, hand-to-hand pain and incompatibility . Roots, stems, leaves, berries and flowers can all be used for medicinal purposes.

Brown root ( Pueraria thunbergiana Benth ) has the tinnitus of gynecology, mung bean, dried dried walnut, dried dried persimmon, dried bran, and yellow root. Since it is one of the nine species of grass, it is also called Kekkukeun. Sweating, lowering your fever, treating high fever, headaches, and quenching thirst. Indigestion, headache, anemia, dysentery, abdominal pain, alcohol poisoning, colds, vomiting, women's bleeding is used to help digestion. The roots are crushed to drink juice.

Sansa ( Crataegus pinnatifida Bunge is Sanlihong, Red Fruit, Buksansa, Red Sweets, Sanjohong, Yangguja, and Billiards. , Woolen yarn, Sanriaceae, narrative, narrative, Yanggu, red tide room, Husa, Guja, falcon It is called 繫 梅), has a peculiar smell, tastes a little sour and sweet, and its properties are slightly warm. Warm the stomach to promote digestion, eat meat and pretend to be effective, and is used for the treatment of abdominal pain, vomiting, diarrhea, excessive stomach acid, chronic enteritis. In addition, it acts on blood to help blood flow and eliminates blood. Pharmacological action, cardiac action, blood circulation improvement, blood pressure lowering action have been reported.

Salviae Miltiorrhizae Radix is distributed in Korea, China, Japan, etc., and its root has a peculiar smell and a bitter taste. It is used to treat dysmenorrhea, dysmenorrhea and postpartum abdominal pain. It is used to treat hemolytic heart pain and bruises, and to treat insomnia skin rash.

Gamguk ( Chrysanthemum indicum ) is a plant of the Asteraceae, also known as Huangguk, and is distributed in Korea, Taiwan, China, and Japan. The flowers are dried and drunk, the young leaves are used as herbs, and the flowers have a dark scent, making them also ornamental. It is prescribed for the treatment of colds, pneumonia, bronchitis, headache, gastritis, enteritis, and boils. Folk remedies crush the entire grass and attach it to the affected area or wash the affected area with fresh herbs.

Prior art related to suppression of MMP-9 expression includes epigallocatechin gallate (Korean Patent Publication No. 10-2005-0045770), which inhibits the expression of matrix metalloproteinase-9 and osteoclast formation. Inhibitory Effect of Resveratrol, a Major Component, on MMP-9 Expression (Woo JH et al ., Oncogene , 23 , pp 1845-1853, 2004), Salvita miltiorrhia BGE inhibits MMP-9 activity (Jin UH et al ., Vascular Pharmacol ., 44 , pp345-353, 2006) and MMP-2 and MMP-9 Inhibitory Activities of Traditional Korean Herbal Medicines, Shihoga-Keol-Moyo-tang (Ha KT et. al ., Pharmacol. Res ., 50 , pp 279-285, 2004).

On the other hand, pedigree is a Chinese herbal extract consisting of Peony, Angelica, Tofu, Gyeji, Clan, Safflower, Licorice, Prickly Pear, Brown Root, Sansa, Salvia, and Ginseng, and has long been effective in treating diseases such as cardiovascular diseases, brain diseases, and inflammatory diseases in China. The drug has been proven and clinically used, but the activity of inhibiting expression of MMP-9 and cancer cell invasion and metastasis activity of the bloodline has not been reported at all.

Thus, the present inventors have made efforts to develop a natural extract with anti-cancer effect, as a result, Chinese herbal medicine extract consisting of peony, Angelica, tofu, gyeji, missing, safflower, licorice, spiny ginseng, brown root, hawthorn, sweet ginseng and gamguk in human breast cancer cell line Inhibiting the expression of MMP-9, effectively inhibiting cancer cell infiltration and metastasis, and thus effective in anticancer and completed the present invention.

It is a main object of the present invention to provide a pharmaceutical composition for anticancer, containing a Chinese herbal extract consisting of peony, Angelica, sessip, ginseng, ginseng, deficiency, safflower, licorice, shiitake, brown root, hawthorn, salvia, and persimmon soup as an active ingredient.

Another object of the present invention is to provide an anti-cancer health food comprising a Chinese medicine extract consisting of peony, Angelica, tofu, gyeji, gyeoljak, safflower, licorice, shiitake, brown root, hawthorn, red ginseng and persimmon soup and food acceptable additives To provide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for anticancer containing Chinese herbal medicine extract consisting of peony, Angelica, sesame, Gyeji, Cassia, safflower, licorice, thorny ginseng, brown root, hawthorn, salvia, and persimmon soup as an active ingredient. .

The present invention also provides an anti-cancer health food comprising a Chinese medicine extract consisting of peony, Angelica, sesame, Gyeji, gyeoljak, safflower, licorice, thorny red pepper, brown root, hawthorn, red ginseng and persimmon soup, and a food supplement acceptable additive. do.

The pharmaceutical composition according to the present invention can suppress the expression of MMP-9 in human breast cancer cell lines, and effectively inhibit cancer cell infiltration and metastasis, so can be used as an effective drug for anticancer.

The invention in one aspect, peony (Paeonia lactiflora Pall . ), Angelica gigas Nakai , Toothworm ( Eucommiae) Cortex ), Cinnamomum cassia Blume ), Cassia tora L. ), safflower ( Carthamus) tinctorius L. ), licorice ( Glycyrrhiza uralensis Fisch ), Acanthopanax senticosus ), root ( Pueraria thunbergiana Benth ), Crataegus pinnatifida Bunge , Salviae Miltiorrhizae Radix ) and Chrysanthemum indicum relates to a pharmaceutical composition for anticancer containing an herbal extract as an active ingredient.

In the present invention, the herbal extract may be characterized in that the extract with water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, with respect to 100 parts by weight of peony extract, 50 to 150 parts by weight, extract of Tofu extract 10 to 150 parts by weight, cinnamon extract 50 to 200 parts by weight, Clarifier extract 50 to 250 parts by weight, safflower extract 50 to 250 parts by weight, licorice extract 150 to 250 parts by weight, prickly pear extract 150 to 250 parts by weight brown root extract 150 to 300 It can be characterized in that it comprises a weight part, 150 to 300 parts by weight of the hawthorn extract, 150 to 300 parts by weight and Dankook extract 150 to 350 parts by weight.

The composition ratio of the herbal composition as described above is obtained on the basis of the results of many animal experiments and clinical experiments, when the composition ratio is out of the range, there is a problem that can not obtain a satisfactory anti-cancer effect.

Herbal medicine extract of the present invention, after lyophilizing and grinding the Peony, Angelica, Sesame, Cinnamon, Lactobacillus, safflower, licorice, spinach, brown root, Sansa, Dansam and persimmon soup, about 1 to 20 times the weight of the dried sample, preferably Preferably, about 5 to 15 times the amount of C ₁ to C ₄ lower alcohols such as ethanol, methanol, water or a mixed solvent thereof, about 50 to 100 hours at room temperature, preferably 60 To 85 It can be obtained by using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for a period of time, preferably cold needle extraction and then concentrated under reduced pressure.

In the present invention, the herbal extract may be characterized by having a function of inhibiting the expression of MMP-9, it may be characterized by having a function of inhibiting infiltration and metastasis of cancer cells.

In the present invention, the cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer Cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract Cancer, Pancreatic cancer, Small intestine cancer, Colorectal cancer, Anal cancer, Bladder cancer, Kidney cancer, Male genital tumor, Penile (urethral) cancer, Prostate cancer, Female genital tumor, Cervical cancer, Endometrial cancer, Ovarian cancer, Uterine sarcoma , Vaginal cancer, female genital external cancer and female urethral cancer may be selected from the group consisting of.

As a result of confirming MMP-9 expressed in cancer cells using zymograghy, western blot, and promoter assay, the herbal extract according to the present invention was expressed in MMP-9 in human breast cancer cell lines. And significantly inhibited the invasion and metastasis of cancer cells in the invasion experiments using Matrigel (Pribol 12-myristate 13-acetate) as in the control group not induced by PMA (phorbol 12-myristate 13-acetate). The composition containing the extract of the present invention can be used in combination or formulated with drugs such as antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics that are already in use.

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 compositions 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 any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterile injectable solutions 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 oils such as olive oil, injectable esters 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 10 mg / kg, preferably 0.001 to 10 mg / kg per day. 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.

Extracts of the invention can be administered to a mammal by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

In another aspect, the present invention ( Peonia) lactiflora Pall . ), Angelica gigas Nakai ), Eucommiae Cortex ), Cinnamomum cassia Blume ), Cassia tora L. ), safflower ( Carthamus) tinctorius L. ), licorice ( Glycyrrhiza uralensis Fisch ), Acanthopanax senticosus ), root ( Pueraria thunbergiana Benth , Crataegus pinnatifida Bunge ), Salviae Miltiorrhizae Radix and Chrysanthemum indicum are related to the anti-cancer health food comprising a medicinal herb extract and food acceptable food supplements.

Compounds containing the extracts of the present invention or pharmaceutically acceptable salts thereof can be used as the main ingredient or additives and auxiliaries of foods in the manufacture of various functional foods and health foods.

As used herein, the term "functional food" means a food product having improved functionality of a general food product by adding the extract of the present invention to a general food product. Functionality can be roughly divided into physical properties and physiological functions. When the extract of the present invention is added to general foods, the physical properties and physiological properties of general foods will be improved, and the present invention provides a comprehensive 'functional food product of such improved functions. 'Is defined.

For example, anti-cancer functional foods can be prepared using the anticancer effect of the extract of the present invention. In addition, it will be possible to produce a functional fortified food and the like using the anticancer effect.

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 extract of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is generally selected in the range of 0.01 to 20 parts by weight per 100 parts by weight of the extract of the present invention.

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

Example 1 Preparation of Herbal Medicine Extract

Commercially purchased Peony, Angelica, Sesame, Gyeji, Lamiaceae, Safflower, Licorice, Prickly Pear Root, Sansa, Salvia, and Persimmon Soup were mixed, ground and powdered in 10 parts by weight of powder. It was extracted for 3 days at room temperature. After the extract was filtered, the residue was repeatedly extracted with methanol, filtered through a filter paper, concentrated under reduced pressure with a rotary vacuum evaporator, and freeze-dried with a lyophilizer to obtain an herbal extract (hereinafter referred to as HM). .

Medicinal herbal name Dose amount (g) One Paeonia lactiflora Pall . (Peony) 1.0 2 Angelica gigas Nakai (Donkey) 1.0 3 Eucommiae Cortex 0.7 4 Cinnamomum cassia Blume 1.3 5 Cassia tora L. (deflector) 1.7 6 Carthamus tinctorius L. (Safflower) 1.7 7 Glycyrrhiza uralensis Fisch (Liquorice) 2.0 8 Acanthopanax senticosus ( Kashiogapi ) 2.0 9 Pueraria thunbergiana Benth 2.3 10 Crataegus pinnatifida Bunge (Sansa) 2.3 11 Salviae Miltiorrhizae Radix 2.3 12 Chrysanthemum indicum (immigration) 2.7 Total 21.0

Example 2: Confirmation of proliferation inhibitory activity of human breast cancer cell line (MCF-7)

In order to confirm the effect of HM treatment on the proliferation inhibition of cells in MCF-7 cells (ATCC, USA) was tested by the following method.

Formula of Dulbecco containing heat treated 10% fetal bovine serum (FBS, Gibco-BRL, USA), penicillin (100 units / ml), streptomycin (100 µg / ml) and sodium bicarbonate (2.2 g / ml) MCF-7 cells were added to a 96 well plate containing medium (DMEM, Gibco-BRL, USA), followed by 37 ° C. and 5% CO ₂ incubator to 1 × 10 ⁴ cells per 100 μl of DMEM culture medium. Incubated at. Cell proliferation assay was measured by purchasing the proliferation assay kit-II (XTT, Boehringer Mannheim, Germany).

Cultured MCF-7 cells were added to 100 μl fresh media with HM at 0, 10, 50, 100, 150, 200, 250 and 300 μg / ml concentrations, respectively, followed by 37 ° C., 5% CO ₂ hours incubator was incubated for 24 hours. After incubation, MCF-7 cells were washed with PBS saline, and then added to each well containing 50 µl of XTT reagent prepared by mixing 5 ml of XTT-labeled reagent and 100 µl of electron coupling reagent. After incubation for 4 hours at 37 ℃, 5% CO ₂ incubator, the absorbance was measured at 490nm with an ELISA meter (Bio-Rad, CA, USA). The data were performed three independent experiments each, and the results were expressed as mean ± SD.

As a result, as shown in Figure 1, it was confirmed that the proliferation of MCF-7 cells in proportion to the treatment concentration of HM.

Example 3: Confirmation of inhibition of MMP-9 enzyme activity

In order to determine the effect of HM treatment on MMP-9 enzymatic activity gelatin degrading activity in MCF-7 cells induced with PMA, gelatin zymography was performed. Add 1 × 10 ⁵ MCF-7 cells to a 6-well plate containing Dulbecco's modified medium (DMEM, Gibco-BRL, USA) containing 10% fetal bovine serum (FBS, Gibco-BRL, USA) and 37 After 12 hours of incubation in a 5% CO ₂ incubator, the cultured MCF-7 cells were added again to a 6-well plate containing serum-free DMEM medium, and 12 hours in a 37 ° C, 5% CO ₂ incubator. Incubated.

In order to induce expression of MMP-9, 100 nM PMA (phorbol 12-myristate 13-acetate) was treated to cultured MCF-7 cells, and treated with HM at concentrations of 0, 50, 150 and 200 μg / ml, respectively. Incubated for 24 hours at 37 ℃, 5% CO ₂ incubator. 20 μl of medium obtained from MCF-7 cell cultures contained 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS and 0.00625% (w / v) bromophenol blue After dilution in 5 μl of the prepared buffer, electrophoresis was performed without heating on a 10% polyacrylamide gel containing 0.1% (w / v) gelatin. After electrophoresis, the gel was washed with 2.5% (v / v) Triton X-100 for 1 hour to remove SDS, and warmed in reaction buffer for 24 hours to degrade the gelatin substrate. The data were performed three independent experiments each, and the results were expressed as mean ± SD.

As a result, as shown in Figure 2 (A), it was confirmed that when compared to the PMA induction control, MMP-9 activity is reduced in proportion to the treatment concentration of HM. In particular, it was found that the MMP-9 activity was inhibited at a concentration of 200 ㎍ / ml similar to the control group not induced with PMA.

Western blot was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control to measure the amount of MMP-9 expression. MCF-7 cells were treated with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% NaNO ₃ , 100 μg / ml PMSF (phenylmethanesulphonylfluoride, phenylmethylsulphonyl fluoride), 1 μg / ml aprotinin and 1% Triton X-100 Crushed with the buffer containing this. Protein quantitation was measured with a protein quantification kit (Bio-Rad, USA). SDS-PAGE electrophoresis of total cell extract protein (20 μg) samples was performed to determine the amount of MMP-9 expression. MMP-9 and GAPDH protein detection was performed by purchasing MMP-9 antibody (Cell Signaling, USA) and GAPDH antibody (Chemicon, USA), respectively. Antigen-antibody reaction with horseradish peroxidase-binding anti-mouse antibody followed by protein detection with an ECL chemiluminescence kit (Amersham, USA), with data of 3 independent in each The experiment was carried out and the results were expressed as mean ± SD.

As a result, as shown in FIG. 2 (B), it was found that the amount of protein decreased depending on the HM concentration. In particular, MMP-9 enzyme production disappeared at a concentration of 200 μg / ml. The same results were found in the enzyme zymography and Western blot analysis.

Example 4: Confirmation of MMP-9 expression inhibitory activity

4-1: MMP  Transcription Inhibition for Expression

RT-PCR was performed to confirm the effect of HM treatment on MMP-9 expression in PMA-induced MCF-7 cells. In this case, beta-actin was used as a control, and primers used for RT-PCR are shown in Table 2 below.

Target genes Primer sequence (sense / antisense) SEQ ID NO: Product size (bp) Beta-actin 5'-CAA GAG ATG GCC ACG GCT GCT-3 '5'-TCC TTC TGC ATC CTG TCG GCA-3' 1 2 247 MMP-9 ORF (open reading frame) 5'-GGA GCC GCT CTC CAA GAA GCT T-3 '5'-CTC CTC CCT TTC CTC CAG AAC AGA A-3' 3 4 520

PMA-induced MCF-7 cells were treated with HM at concentrations of 0, 50, 150 and 200 μg / ml, as in Example 3, and TRIzol reagent (Invitrogen, USA) was prepared from the cultured MCF-7 cells. Total RNA was isolated. CDNA was synthesized using 1 μg of each total RNA isolated (AMV RNA PCR kit; Takara, Japan), and then RT-PCR was performed using the primers (SEQ ID NOs: 3 and 4) of Table 2 (94 ° C). : 1 minute, 60 ° C .: 1 minute, 72 ° C .: 1 minute, 30 cycles). The resulting PCR product was analyzed by agarose gel electrophoresis and stained with ethidium bromide.

As a result, as shown in Figure 3 (A), the expected target size of MMP-9 was confirmed to be 520 bp, the transcription inhibitory effect of MMP-9 increased depending on the concentration of HM treatment, in particular, 200 ㎍ It was found that the transcription of MMP-9 was completely inhibited at the / ml concentration. Also, except that the primers of SEQ ID NO: 2 (SEQ ID NOs: 1 and 2) were used to synthesize cDNA of beta actin (control) and electrophoresis analysis in the same manner, HM treatment did not affect the expression of beta actin. It was confirmed that no effect.

4-2: MMP-9 Promoter Activity Inhibition

In order to confirm the effects of HM treatment on MMP-9 promoter activity in PMA-induced MCF-7 cells, the primers of the 5'-promoter region of human MMP-9 (Accession No. D10051) was cloned by performing RT-PCR. The resulting PCR product (MMP-9 promoter portion) was analyzed by electrophoresis, and the base sequence was confirmed.

Target genes Primer sequence (sense / antisense) SEQ ID NO: Product size (bp) MMP-9 promoter 5'-ACA TTT GCC CGA GCT CCT GAA G -3 '5'-AGG GGC TGC CAG AAG CTT ATG GT-3' 5 6 710

The gene of the cloned MMP-9 promoter portion was inserted into the pGL2-Basic vector containing the polyadenylation signal upstream region of the luciferase gene. After smearing MCF-7 cells in 6-well plates at 1 × 10 ⁵ cells / well density, 1 μg of the MMP-9 promoter-luciferase reporter construct was constructed using LipofectAMINE method (Invitrogen, USA). 1 μg of beta galactosidase reporter plasmid (β-galactosidase reporter plasmid) was cotransduced. MCF-7 cells transduced in Dulbecco's modified medium (DMEM, Gibco-BRL, USA) containing 10% fetal bovine serum (FBS, Gibco-BRL, USA) were treated at 37 ° C. with 5% CO _2. After incubating for 4 hours in the incubator, the medium was changed, and then treated with 100 nM PMA (phorbol 12-myristate 13-acetate) to cultured MCF-7 cells, and treated with 200 μg / ml concentration of HM, followed by 37 C, incubated for 24 hours in a 5% CO ₂ incubator. Luciferase activity and beta galactosidase activity were measured by luciferase and beta galactosidase assay kit (Promega, USA), and luciferase activity was corrected by beta galactosidase activity and independent for each cell extract. Was carried out. The data were performed three independent experiments each, and the results were expressed as mean ± SD.

PMA untreated group (pGL2 and MMP-9WT) and PMA treated group {WT (PMA) and WT (PMA) + HM (200 μg / ml)} was divided into HM (200 μg / ml) of the PMA treatment group WT (PMA) treated only with 100nM PMA (phorbol 12-myristate 13-acetate) and 100nM PMA (phorbol 12-myristate 13-acetate) were treated, and WT (treated with 200 μg / ml HM) was treated. Experiments were divided by PMA) + HM (200 μg / ml).

As a result, as shown in Figure 3 (B), the treatment of HM inhibits the expression of luciferase than the control group, it can be seen that the strong MMP-9 promoter inhibitory activity. In particular, in the case of the WT (PMA) + HM (200 μg / ml) group treated with 100 nM PMA (phorbol 12-myristate 13-acetate) and treated with 200 μg / ml HM, luciferase induced by PMA It was found that the activity was inhibited to a level similar to the control group not induced with PMA by treatment with HM.

Example 5: Confirmation of metastasis and infiltration inhibitory activity of human breast cancer cell line (MCF-7)

To determine the effect of HM treatment on cell metastasis and invasion inhibition in PMA-induced MCF-7 cells (8 μm, Becton Dickinson, USA) Matrigel analysis was performed. Smear MCF-7 cells (5 × 10 ⁴ cells / 200 μl) in the infiltration chamber (upper chamber) and 500 μl of serum-free DMEM medium in the lower chamber and incubate for 12 hours in a 37 ° C, 5% CO ₂ incubator. Then, 100 nM PMA (phorbol 12-myristate 13-acetate) was treated, HM at concentrations of 0, 100 and 200 μg / ml, respectively, and then incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. . MCF-7 cells were infiltrated from the upper chamber to the lower chamber while incubated for 24 hours, and the non-infiltrated cells remained on the surface of the upper chamber and removed using a cotton swab. Cells infiltrated into the lower chamber were stained with Hemotoxylin-Eosin reagent, the filter was removed, placed on a slide glass, fixed with Eikitt fixative, and the cells infiltrated into Matrigel under the microscope counted the total number of cells. The data were performed three independent experiments each, and the results were expressed as mean ± SD.

PMA non-treated group (a) and PMA treated group {(b), (c) and (d)} was divided into 100nM PMA (phorbol) according to the treatment of HM (100 and 200 ㎍ / ml) among the PMA treated group (B) treated only with 12-myristate 13-acetate (b), treated with 100nM PMA (phorbol 12-myristate 13-acetate), treated with 100 μg / ml HM (c) and 100nM PMA (phorbol 12-myristate) 13-acetate) and treated with 200 μg / ml HM (d).

As a result, as shown in Figure 4 (A), it was confirmed that the cell infiltration occurred in (b) and (c), it can be seen that the infiltration of MCF-7 cells is inhibited depending on the concentration of HM treatment. there was. In particular, it was confirmed that at 200 μg / ml concentration, infiltration of cells was inhibited to a level similar to that of the control (a) not induced by PMA by treatment (d) of HM. On the other hand, as shown in Figure 4 (B), it can be seen that the metastasis of MCF-7 cells is also inhibited in proportion to the treatment concentration of HM. In particular, it was confirmed that at 200 μg / ml concentration, the metastasis of cells was inhibited to a level similar to that of the control group not induced with PMA by HM treatment.

    One example of the formulation of the pharmaceutical composition comprising the extract of the herbal medicine of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation Example 1 Preparation of Powder

HM 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2: Preparation of Tablet

HM 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional manufacturing method of the tablet.

Formulation example  3: Manufacture of capsule

HM 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  4: Preparation of Injection

HM 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

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

Formulation example  5: Liquid  Produce

HM 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The solution was prepared by sterilization.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a graph confirming the change in cell activity by the treatment of HM in MCF-7 cells using XTT assay.

Figure 2 (A) is a result of measuring the MMP-9 enzyme inhibitory activity by treatment with HM in PMA-induced MCF-7 cells, using (Z), (B) is MCF-7 induced by PMA Western blotting to confirm the inhibitory effect of MMP-9 production by HM treatment in cells.

Figure 3 (A) is the result of confirming MMP-9 gene expression by HM treatment in PF-induced MCF-7 cells by performing RT-PCR, (B) in MCF-7 cells induced by PMA MMP-9 promoter inhibitory activity by treatment of HM is a graph confirming the use of luciferase activity.

Figure 4 (A) is a result of measuring the infiltration inhibition effect of MCF-7 cells by treatment with HM in PMA-induced MCF-7 cells using the Matrigel assay, (B) is MCF induced with PMA It is a graph showing the effect of inhibiting metastasis of MCF-7 cells by HM treatment in -7 cells.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Anti-Cancer Composition Containing an Extract of Chinese Herb as          an Effective Ingredient <130> P08-B042 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for beta-actin <400> 1 caagagatgg ccacggctgc t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for beta-actin <400> 2 tccttctgca tcctgtcggc a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for MMP-9 ORF <400> 3 ggagccgctc tccaagaagc tt 22 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for MMP-9 ORF <400> 4 ctcctccctt tcctccagaa cagaa 25 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for MMP-9 promoter <400> 5 acatttgccc gagctcctga ag 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for MMP-9 promoter <400> 6 aggggctgcc agaagcttat ggt 23  

Claims (14)

Peony ( Paeonia lactiflora Pall. ), Angelica gigas Nakai , Toothworm ( Eucommiae Cortex ), Cage ( Cinnamomum cassia Blume ), Cassia tora L. , Safflower ( Carthamus tinctorius L. ), Licorice ( Glycyrrhiza uralensis Fisch ) , Chinese herbal medicine consisting of Acanthopanax senticosus, Pueraria thunbergiana Benth , Sansa ( Crataegus pinnatifida Bunge ), Salviae Miltiorrhizae Radix and Chrysanthemum indicum An anticancer pharmaceutical composition containing an extract extracted using a solvent as an active ingredient. delete According to claim 1, The medicinal herb extract is based on 100 parts by weight of Peony extract, 50 to 150 parts by weight of Angelica extract, 10 to 150 parts by weight of Tobacco extract, 50 to 200 parts by weight of Gyeji extract, 50 to 250 parts by weight of extract Safflower extract 50 to 250 parts by weight, licorice extract 150 to 250 parts by weight, prickly pear extract 150 to 250 parts by weight brown root extract 150 to 300 parts by weight, hawthorn extract 150 to 300 parts by weight, salvia extract 150 to 300 parts by weight and persimmon extract 150 To pharmaceutical composition comprising 350 parts by weight. According to claim 1, wherein the herbal extract is a pharmaceutical composition, characterized in that it has a function of inhibiting the expression of MMP-9. The pharmaceutical composition of claim 1, wherein the herbal extract has a function of inhibiting infiltration and metastasis of cancer cells. The method of claim 1, wherein the cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer ( Sinus) cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, Biliary tract cancer, pancreatic cancer, small intestine cancer, colon (rectal) cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile (urethral) cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterus Pharmaceutical composition, characterized in that selected from the group consisting of sarcoma, vaginal cancer, female genital external cancer and female urethral cancer. The pharmaceutical composition of claim 6, wherein the cancer is breast cancer. Peony ( Paeonia lactiflora Pall. ), Angelica gigas Nakai , Toothworm ( Eucommiae Cortex ), Cage ( Cinnamomum cassia Blume ), Cassia tora L. , Safflower ( Carthamus tinctorius L. ), Licorice ( Glycyrrhiza uralensis Fisch ) , Chinese herbal medicine consisting of Acanthopanax senticosus, Pueraria thunbergiana Benth , Sansa ( Crataegus pinnatifida Bunge ), Salviae Miltiorrhizae Radix and Chrysanthemum indicum An anti-cancer health food comprising an extract extracted using a solvent and a food supplement acceptable food additive. delete According to claim 8, The medicinal herb extract is based on 100 parts by weight of Peony extract, 50 to 150 parts by weight of Angelica extract, 10 to 150 parts by weight of Tobacco extract, 50 to 200 parts by weight of gyeji extract, 50 to 250 parts by weight of the deficiency extract, Safflower extract 50 to 250 parts by weight, licorice extract 150 to 250 parts by weight, prickly pear extract 150 to 250 parts by weight brown root extract 150 to 300 parts by weight, hawthorn extract 150 to 300 parts by weight, salvia extract 150 to 300 parts by weight and persimmon extract 150 Health foods comprising from 350 parts by weight. The health food according to claim 8, wherein the herbal extract has a function of inhibiting the expression of MMP-9. The health food according to claim 8, wherein the herbal extract has a function of inhibiting infiltration and metastasis of cancer cells. The method of claim 8, wherein the cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer ( Sinus) cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, Biliary tract cancer, pancreatic cancer, small intestine cancer, colon (rectal) cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile (urethral) cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterus Health food, characterized in that selected from the group consisting of sarcoma, vaginal cancer, female genital external cancer and female urethral cancer. The health food of claim 13, wherein the cancer is breast cancer.

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