KR101791482B1 - The composition containing the extract sinsuk for cancer prevention or improvement - Google Patents
The composition containing the extract sinsuk for cancer prevention or improvement Download PDFInfo
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- KR101791482B1 KR101791482B1 KR1020150058662A KR20150058662A KR101791482B1 KR 101791482 B1 KR101791482 B1 KR 101791482B1 KR 1020150058662 A KR1020150058662 A KR 1020150058662A KR 20150058662 A KR20150058662 A KR 20150058662A KR 101791482 B1 KR101791482 B1 KR 101791482B1
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- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
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Abstract
The present invention relates to a composition for preventing or improving cancer, which comprises nephrolithic extract. More particularly, the present invention relates to a composition for prevention or amelioration of cancer, which comprises a nephrolithic extract and has a cytotoxic effect and a side effect, as well as effectively preventing cancer cell proliferation and preventing and improving cancer.
The composition for preventing or ameliorating cancer, which comprises the nephrite extract according to the present invention, is not cytotoxic and can be used without side effects, has an effect of inhibiting the proliferation of cancer cells, and can be most effective for inhibiting proliferation of gastric cancer cells.
In particular, the nephrite extract has excellent efficacy in suppressing cancer cells, and thus can be provided in various forms such as food or pharmaceutical composition utilizing the nephrite extract and easily ingested.
Description
The present invention relates to a composition for preventing or improving cancer, which comprises nephrolithic extract. More particularly, the present invention relates to a composition for prevention or amelioration of cancer, which comprises a nephrolithic extract and has a cytotoxic effect and a side effect, as well as effectively preventing cancer cell proliferation and preventing and improving cancer.
Cancer is a disease that is not normally regulated in the body and rapidly proliferates and causes infiltration and metastasis. It is also called malignant tumor, malignant neoplasia, carcinoma, (sarcoma), leukemia (leukemia), and lymphoma (lymphoma) are also included in the category of cancer.
Stomach cancer, especially, is the leading cause of cancer deaths. In particular, according to the data released by the Central Cancer Registry in 2011, 15.4% of the total cancer incidence in Korea is ranked second in stomach cancer in 2009, and it is known that cancer is the most prevalent cancer in men.
Stomach cancer is usually more than 55 years old, twice as common in men than women. These types of tumors are more common in the United States as well as in Japan, South Korea, Latin America, and Eastern Europe, where food is dried, sectioned, smoked and cut and preserved. Conversely, consumption of fresh fruits and vegetables may be against this disease.
Gastric cancer can develop in any part of the stomach and can spread to the stomach and / or other organs. These tumors can grow along the gastric wall cells and spread to the esophagus and intestines. If the tumor grows along the stomach wall, it can be transferred to the nearby lymph nodes and liver, and to the pancreas and the large intestine. It can even spread to the ovaries, lungs and distant lymph nodes. When gastric cancer tissue has metastasized to other parts of the body, these tumor cells have the same shape as the nature of the original tumor tissue. That is, the metastatic tumor cells are still stomach cancer cells. These tumor cells, which form one or more ovarian tumors and metastasize to the ovaries, are called Krukenberg tumors and are composed of transformed stomach cells that are not ovarian cells.
Since the symptoms of gastric cancer are nonspecific, it is not easy to detect at an early stage. These symptoms include indigestion, sore throat, abnormal pain, nausea, vomiting, diarrhea or constipation, loss of appetite, weakness, fatigue, and hemorrhage, such as blood that is detected in the stool and blood that comes with vomiting. Diagnosis usually takes place in the upper gastrointestinal tract and esophagus by X-ray and is measured after the patient has swallowed the soluble barium-labeled material. Endoscopy and gastroscopy of the stomach and esophagus can also be performed. If abnormal tissue is found, biopsy can be done through a cystoscopy. If this histological examination proves to be a tumor cell, the surrounding lymph nodes are also biopsied and the surrounding liver and pancreas tissues are also metastasized
To determine at which stage of the disease, a CT scan should be performed. The treatment of stomach cancer cells is a method of removing tumors from other metastatic tissues (partial or total gastrectomy) and chemotherapy, radiotherapy and immunotherapy of nearby lymph nodes ≪ / RTI > stimulation of the constituents).
The risk factors for gastric cancer are high salt diet, high fat diet, burnt food, tobacco, alcohol, Helicobacter pylori infection, genetic factors, and so on. It is known.
When people are exposed to these carcinogens during their normal lives, the carcinogenesis process is affected by the nature and exposure of the carcinogen itself.
Stomach cancer has various symptoms ranging from no symptoms to severe pain. In addition, the symptoms of gastric cancer do not have any characteristics but general digestive symptoms. In general, most of the stomach cancer in the early stages of the disease is mostly mild, even if a little digestive failure or upper abdominal discomfort is felt to the degree that most people are overlooked it can cause the death rate of stomach cancer is also increased.
Traditional methods of treating cancer classified as refractory disease include surgery, chemotherapy, and radiation therapy, but chemotherapy and radiation therapy are often used in combination with surgery. However, when chemotherapy and radiotherapy are given, it has been known to cause adverse effects on normal cells such as bone marrow cells and small epithelial cells (Hebert-Croteau et al., 2002; Guidry et al , 1996). Especially, various kinds of chemotherapeutic drugs used for chemotherapy have different pharmacological actions depending on individual patients, and toxic side effects are caused. Therefore, recently, substances having effective anticancer activity in natural products having little side effects have been discovered, Which is known to cause the selective death of cancer cells without affecting the normal cells.
Among the various methods used to treat cancer, effective treatment is to inhibit the division and growth of cancer cells and selectively remove cancer cells. In particular, apoptosis, which is known as apoptosis as a death of organized cells, is not only involved in normal development and differentiation in the developmental stage of the individual such as fetal morphogenesis, ovulation of oocytes, synaptic formation of neurons, (Han et al., 2008; Okada and Mak, 2004), which is a physiological process that removes abnormal cells in the body caused by infection and the like, as a defense mechanism for the survival of elaborate individuals under genetic control. . The induction of apoptosis involves interaction with various types of genes, such as increased or decreased expression of various genes, depending on the type of cell and the type of damage, and maintains biological phenomena and health such as morphogenesis, cell transformation and removal of damaged tissue or infected cells It is known to play an important role in However, apoptosis is an important target for the treatment of cancer at various stages of the process of cancer (Huerta et al., 2006; Jin Z and El-Deiry, 2006) 2005).
Therefore, there is a high demand for a composition for preventing or improving gastric cancer based on a composition derived from a natural extract or a natural extract. It can be processed and processed into food, health supplement, etc., without side effects, so that it can be effectively used for prevention or improvement of gastric cancer.
In this regard, prior art 1 (KR 10-2014-0005552 A, published on January 15, 2014) relates to a composition for preventing or treating cancer or aging which contains a camellia extract as an active ingredient will be. However, it is not specialized for gastric cancer, and it is necessary to develop a composition for prevention and improvement of gastric cancer using other natural substance extracts for the dissemination and utilization of various natural products.
It is an object of the present invention to provide a composition for preventing or ameliorating gastric cancer, which comprises a nephrolithic extract showing an effect of inhibiting proliferation of gastric cancer cells without cytotoxicity.
In particular, the nephrite extract can exert an excellent effect on the inhibition of gastric cancer cells, thereby providing a food or pharmaceutical composition utilizing the nephron extract.
Further, it is intended to provide a composition for preventing or improving gastric cancer, which is improved in the effect of inhibiting the proliferation of high gastric cancer cells by utilizing the nephritic extract and other naturally derived extract as a composite material.
Another object of the present invention is to provide a method for preparing a composition for preventing or improving gastric cancer, which comprises the nephron extract.
In order to achieve the above object, the composition for preventing or improving cancer comprising nephrite extract according to an embodiment of the present invention may include a nephrolithic extract and may have a preventive or ameliorative effect on gastric cancer.
The nephrite extract may be one in which water is used as a solvent.
The nephrite extract may comprise 100 parts by weight of nephrite and 10 to 180 parts by weight of sodium hydrogen carbonate with respect to the nephrite.
The composition for preventing or improving cancer, which comprises the nephrite extract, may contain 2000 to 6000 parts by weight of an extract of Trichoderma sp. For 100 parts by weight of the nephron extract.
The composition for preventing or improving cancer, which comprises the nephrite extract, may comprise 2000 to 6000 parts by weight of the extract of rhodochrous extract against 100 parts by weight of the nephron extract.
The cancer preventive or remedy composition containing the nephrite extract may contain 2000 to 6000 parts by weight of the extracts of Nunomari extract and 2000 to 6000 parts by weight of the extract of Neungyu for 100 parts by weight of the nephron extract.
The food composition for preventing or ameliorating gastric cancer according to another embodiment of the present invention may include a composition for preventing or ameliorating cancer including the nephrolithic extract.
The pharmaceutical composition for preventing or ameliorating gastric cancer according to another embodiment of the present invention may include a composition for preventing or improving cancer including the nephrolithic extract.
A method for preparing a composition for preventing or improving cancer, which comprises extracting nephrite according to another embodiment of the present invention, comprises the steps of mixing nephrite with water and adding sodium hydrogen carbonate And 180 parts by weight of an extract of the present invention, and heat-treating the mixture to prepare a nephrite extract; 100 parts by weight of pulverized pulverized product, 500 to 1400 parts by weight of water is mixed with the pulverized pulverized product and heat-treated at 70 to 160 ° C for 3 to 12 hours to prepare dried fruit extract; Drying the frozen lyophilized product to concentrate and dry the lyophilized lyophilized product; 100 parts by weight of rhizome crushed product and 800-2000 parts by weight of water are mixed with the crushed rhizome product and heat-treated at 70 to 160 ° C for 2 to 8 hours to prepare rhizome extract; Concentrating and drying the lyophilized lyophilized extract to prepare lyophilized lyophilized product; A lyophilized lyophilized product, a lyophilized lyophilized product, and water to prepare a mixture, and the mixture may have a preventive or ameliorative effect on gastric cancer.
The method for preparing a composition for preventing or improving cancer, which comprises the nephrite extract, may further comprise 0.01 to 10 parts by weight of willow to the nephrite.
Hereinafter, the present invention will be described in more detail.
The composition for preventing or improving gastric cancer according to one embodiment of the present invention includes nephrolithic extract.
Shinsuk (SS), an arsenic compound based on As 2 O 3 , is produced by burning ores containing arsenic oxide or arsenic containing minerals (arsenic containing minerals) (Lee, 1969) has been used in oriental medicine since 2000 for the purpose of curing illnesses.
The extract used throughout the specification of the present invention means a substance or an element extracted from an entire body and does not limit its property and is defined as including powder.
In addition, the term nephrite extract used throughout the specification of the present invention includes, in addition to the extract of the dictionary meaning, a septate, a mixture or a composition containing nephrite, which is an arsenic compound mainly composed of As 2 O 3 from mineral resources, And the stone itself.
The composition for preventing or improving gastric cancer may be one in which water is used as a solvent for the nephrolithic extract.
When the water extract is used, it is easy to disperse the nephrite, and it can be mixed with other natural extracts and can be provided in various formulations.
The nephrite extract may comprise 100 parts by weight of nephrite and 10 to 180 parts by weight of sodium hydrogen carbonate with respect to the nephrite.
When sodium hydrogencarbonate is contained, it is possible to lower the toxicity of the above-mentioned nephritis and to obtain the effective deliveries necessary for the improvement of the gastric cancer.
The composition for preventing or ameliorating gastric cancer may comprise 2000 to 6000 parts by weight of the extract for dryness, based on 100 parts by weight of the nephrite extract.
Gunchil (GC) is a medicinal herb that is used continuously from the records of "Divine Husbandman's Sacred Man", which is known as the oldest medicine book in oriental medicine to the " The liver is the small intestine (liver 脾 胃 large and small intestine) and acts on the blood to make blood 瘀 瘀 blood (瘀 瘀血) and traditionally 瘀 blood, 瘀 적 (聚血), (가 (가?), Cold and cold heartache (Song, et al., 2002).
When the above extract is further contained, the effect of inhibiting the proliferation of the gastric cancer cells of the nephritic extract is enhanced.
The composition for preventing or ameliorating gastric cancer may comprise 2000 to 6000 parts by weight of an extract of Guinea pea extract per 100 parts by weight of the nephron extract.
UGUNPI, UGUNPI, and UGP are dry skin and peeled cork layers of elm belonging to Ulmaceae. According to the records of the previous consent, It is mainly edematous, edematous, scabious, urine incontinence, incarcerated, and feverish, because it has the effect of gut dementia and detoxification of exanthema. It has been used to treat pathologies such as cough asthma, febrile fever, and antibiotic resistance (Shin, Min-kyo, 2000; Lee, 1984).
The present invention has an advantage that the effect of inhibiting the proliferation of gastric cancer cells of the nephritic extract is enhanced when the extract further contains the extract.
The composition for preventing or ameliorating gastric cancer may comprise 2000 to 6000 parts by weight of an extract of Guanyin and 2000 to 6000 parts by weight of Guanyinfuri extract, based on 100 parts by weight of the nephron extract.
The inhibitory effect of gastric cells on the above-mentioned nephritis may be best when the extract is mixed with the extract of Guanyin and Rootgrass. In particular, when the composition is used within the above-mentioned defined range, it has no cytotoxicity and thus has no side effects, and can effectively inhibit the proliferation of gastric cells.
Preferably, the composition for preventing or ameliorating gastric cancer may comprise 3000 to 4000 parts by weight of an extract of Rhododendron japonica extract and 3000 to 5000 parts by weight of Rhodiola extract in 100 parts by weight of the nephron extract.
In this range, the proliferation of gastric cancer cells can be minimized within a range of low cytotoxicity.
Preferably, the composition for prevention or improvement of gastric cancer may comprise 3500 to 5500 parts by weight of the extracts of Guanyin and 2000 to 4500 parts by weight of the extract of Guanyinfuri, for 100 parts by weight of the nephron extract.
In the above range, a unique flavor of the mixed extract is blended, and it is felt that the intrinsic bitterness of the kidney stones and the kidney stalks are uniformly mixed, thereby having a high preference with excellent flavor and texture.
Accordingly, most preferably, the composition for preventing or improving gastric cancer may comprise 3500-4000 parts by weight of the extracts of Guanyin and 3000-60000 parts by weight of the extract of Guanyinfuri, in 100 parts by weight of the nephron extract.
By the above range, effective inhibition of gastric cancer cell proliferation is possible, and since it can have a high flavor, it can be effective in providing a food composition or the like.
The food composition for preventing or ameliorating gastric cancer according to another embodiment of the present invention may include a composition for preventing or improving gastric cancer.
The food composition is considered to include all forms that can be used by those of ordinary skill in the art.
The pharmaceutical composition for preventing or ameliorating gastric cancer according to another embodiment of the present invention may include a composition for preventing or improving gastric cancer.
The pharmaceutical composition is considered to include all forms and forms that can be used by those skilled in the art. It may also be used within a pharmaceutically acceptable range.
According to another embodiment of the present invention, there is provided a method for preparing a composition for preventing or improving gastric cancer, which comprises mixing nephrite with water, mixing 10 to 180 parts by weight of sodium hydrogen carbonate with respect to 100 parts by weight of the nephrite A step of preparing a nephrite extract to produce a nephrite extract by heat treatment; 100 parts by weight of pulverized pulverized product, 500 to 1400 parts by weight of water is mixed with the pulverized pulverized product and heat-treated at 70 to 160 ° C for 3 to 12 hours to prepare dried fruit extract; Drying the frozen lyophilized product to concentrate and dry the lyophilized lyophilized product; 100 parts by weight of rhizome crushed product and 800-2000 parts by weight of water are mixed with the crushed rhizome product and heat-treated at 70 to 160 ° C for 2 to 8 hours to prepare rhizome extract; Concentrating and drying the lyophilized lyophilized extract to prepare lyophilized lyophilized product; Mixing the nephrite extract, the lyophilized lyophilized product, the lyophilized lyophilized product and the water, and a mixing step.
When the lyophilization process is performed, the toxicity contained in the dried crusts and rootstocks is reduced, so that it can be used without any side effects. In addition, the dried crusts and the crustaceans disappear, and the taste of the granite is covered, ≪ / RTI >
In addition, there is a problem in that when extracting from the above-mentioned ranges, the above-mentioned extracts of ganoderma extract and guinea fescue have an effect of obtaining an effective ingredient effective for suppressing gastric cancer cells, That is, when the temperature is lower than 70 ° C, the effect of obtaining the active ingredient is lowered. When the temperature exceeds 160 ° C, the other components are extracted together to lower the effect of inhibiting the proliferation of gastric cancer cells, And the bitter taste becomes stronger.
On the other hand, it may be more preferably extracted at 80 to 99 ° C. The above range is advantageous in that it is most effective in obtaining an active ingredient and does not cause problems such as increased specificity or intrinsic toxicity.
Therefore, it may be preferable to extract in the above temperature range within the above time range.
The method for preparing a composition for preventing or improving gastric cancer may further include 0.01 to 10 parts by weight of willow seeds to the nephrite.
When the willow is included in the heat treatment process in the step of preparing nephrite, the impurities may be removed to obtain an effective ingredient, and cytotoxicity due to impurities and other side effects may be improved.
Preferably the willow may be a willow branch. And the willow branches may be included in the thermal bath of the mixture containing the nephrite while removing bubbles generated during the heat treatment process using the branches.
The composition for preventing or ameliorating gastric cancer, which comprises the nephrite extract according to the present invention, has no cytotoxicity and can be used without side effects and has the effect of inhibiting proliferation of stomach cancer cells.
In particular, the nephrite extract has excellent efficacy in suppressing gastric cancer cells, and thus can be provided in various forms such as food or pharmaceutical compositions utilizing the nephrite extract and easily ingested.
Furthermore, compositions for preventing or improving stomach cancer, including nephrolithic extract, can be used as a composite material of the nephrite extract and other naturally-derived extracts to improve the effect of inhibiting the proliferation of high gastric cancer cells. In addition, the above-mentioned Shinseok and other natural materials can be combined to give a unique flavor, so that it is easy to supply it with a high-quality flavor and a good taste as a food of choice.
Another object of the present invention is to provide a method for preparing a composition for preventing or improving gastric cancer, which comprises the nephron extract.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the effect of a composition for preventing or improving cancer, including nephrolithic extract according to an embodiment of the present invention.
FIG. 2 shows the effect of a composition for preventing or ameliorating cancer, including nephrolithic extract according to an embodiment of the present invention.
FIG. 3 is a graph showing the effect of a composition for preventing or improving cancer, including nephrolithic extract according to an embodiment of the present invention.
FIG. 4 is a graph showing the effect of the composition for preventing or improving cancer, including nephrolithic extract according to an embodiment of the present invention.
FIG. 5 is a graph showing the effect of a composition for preventing or improving cancer, which comprises an extract of nephrolithiasis according to an embodiment of the present invention.
FIG. 6 is a graph showing the effect of a composition for preventing or improving cancer, which comprises an extract of nephrolithiasis according to an embodiment of the present invention.
FIG. 7 is a graph showing the effect of a composition for preventing or improving cancer, including nephrolithic extract according to an embodiment of the present invention.
Figure 8 relates to the effect of a composition for preventing or improving cancer comprising nephrolithic extract according to one embodiment of the present invention.
Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
[Preparation Example: Preparation of extract containing nephrite]
1. Sample Preparation
Dry crushed, yuukee, and nephrite used for this experiment were thoroughly washed with flowing water and then dried in a dry oven. After crushing roughly bark and woody parts, 800 ml of distilled water was added to 100 g of distilled water and the mixture was heated at 120 ° C for 6 hours under pressure. The extract was concentrated to 10 brix and lyophilized to obtain 36 g of solid component (GC) . To obtain wooly pea extract wool, 1 L of distilled water was added to 100 g of root weevil, and the extract was stirred at 120 ° C. for 4 hours under pressure and for 1 hour under pressure. The extract was concentrated to 15 brix and lyophilized. 30 g of solid component (UGP) was obtained.
In order to obtain Seongseok (SS) to be used in the experiment, Seongwanghwang was placed in pottery, sealed with mud made of wire and brine, heated for 3 hours with weak fire and 12 hours with strong fire, and white powder (nephrite) attached to the lid was collected. NaeHCO 3 (sodium hydrogen carbonate), 90% by weight of neodite, was added to distilled water, and the mixture was heated while mildly heated. Then, the mixture was continuously stirred until the bubbles disappeared. Then, 120% weight of nephrite was obtained. GC, UGP and SS obtained by the above method were finely pulverized with a mortar and sealed and stored at -70 ° C. In the experiment, the concentration was adjusted to 100 mg / ml using the third distilled water. Then, a 0.2 μm pore size syringe filter Was used to remove fine impurities and diluted in the medium at a proper concentration.
2. Experimental material
(TRAIL), death receptor 4 (DR4), DR5, Fas, Fas ligand (FasL), Bcl-2, and Bcl-2 were used for the analysis of protein expression changes by sample treatment. (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP-1), cIAP-2, survivin, caspase-3, caspase-8, caspase-9 (PLCP), and actin antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Inc.). Horseradish peroxidase (HRP) -conjugated anti-mouse and anti-rabbit antibodies used as secondary antibodies for immunoblotting were purchased from Amersham Life Science Corp. (Santa Cruz, CA, USA). (Arlington Heights, Ill., USA). Z-VAD-fmk, a pan caspase inhibitor used to inhibit the activity of caspases, was purchased from Calbiochem (San Diego, Calif., USA), and the colorimetric assay kit for in vitro assay of caspases was purchased from R & D Systems CA, USA).
[Experimental Method]
1. Cell culture
(RAW 264.7), myoblasts (C2C12) and human lung cancer cell lines (A549), colon cancer cell lines (HCT116), hepatocellular carcinoma cell lines (Hep3B), bladder cancer cell lines (T24) and gastric cancer cell lines (10% fetal bovine serum (FBS)) was added to the RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Grand Island, NY, USA) ml of streptomycin (Gibco BRL) at a concentration of 5
2. Cell proliferation inhibition assay by MTT assay
MTT assay was used to determine the extent of inhibition of GC, UGP, and SS by single or multiple treatment. In order to investigate the effect of tetrazolium bromide salt (MTT, Sigma-Aldrich) on the soluble substrate of yellow color, GC and UGP were treated with various concentrations of GC, UGP and SS in various kinds of normal cell lines and human cancer cell lines. Was diluted to a concentration of 0.5 mg / ml, and 2 ml of each was dispensed and reacted at 37 ° C for 3 hours. After the reaction was completed, the MTT reagent was removed and 1 ml of DMSO was added to each well. All of the insoluble purple MTT formazan produced in the wells was dissolved, and 200 μl of the MTT reagent was transferred to a 96-well plate. The resulting mixture was transferred to an ELISA reader (Molecular Devices, Sunnyvale, And the absorbance at 540 nm was measured. All three measurements were made, and mean values and standard errors were analyzed using the Microsoft EXCEL program.
3. Observation of cell morphology using an inverted microscope
The morphological changes of cancer cells by GC, UGP and SS alone or combined treatment were visually confirmed using an inverted microscope (Carl Zeiss, Germany). For this purpose, AGS cells were seeded at a density of 1 × 10 5 cells / ml in a 6-well plate for cell culture, and then cultured by single or combined treatment with GC, UGP and SS at a proper concentration. After 48 hours, the degree of visual inhibition and morphological change were observed at a magnification of 200 times and then photographed using the Axio Vision program.
4. Observation of nucleus morphology by DAPI staining
For confirmation of apoptotic body formation, the cells were collected and fixed in a 1: 9 ratio of 37% formaldehyde solution and phosphate-buffered saline (PBS) for 10 min at room temperature. After fixation, the fixing solution was removed and suspended in PBS. Cells were attached to the slide glass using cytospin (SHANDON, UK). Cells were washed with PBS and incubated at room temperature for 10 min with 0.2% Triton X-100 (Amresco, Solon, OH, USA). After incubation at room temperature with 2.5 μg / ml 4 ', 6-diamidino- 2-phenylindole (DAPI, Sigma-Aldrich) solution, and stained at room temperature for 15 minutes. After staining, morphological changes of cancer cell nuclei were observed at a magnification of 400 times using a fluorescence microscope (Carl Zeiss), and then photographs were taken using the Axio Vision program.
5. Measurement of apoptosis by Annexin V-Fluorescein Isothiocyanate (FITC) staining
To quantitatively analyze the degree of apoptosis induction, GC, UGP and SS were treated alone or combined. After 24 hours, the cells were collected and centrifuged at 2,000 rpm for 5 minutes to remove the supernatant. The cells were washed two or three times with PBS and suspended in annexin V binding buffer (Becton Dickinson, San Jose, CA, USA) containing 10 mM HEPES / NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2. V-FITC and propidium iodide (PI, Becton Dickinson) were treated for 15 min in the dark. After completion of the reaction, the cells were separated into single cells using a 35-mm mesh, and cells (V + / PI-) induced by apoptosis were measured by fluorescence reaction using FACS Calibur (Becton Dickinson). CellQuest software and ModiFit LT program Respectively.
6. DNA Fragmentation Analysis
The cells were collected and lysed at room temperature for 1 hour by adding lysis buffer [5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% Triton X-100] to the DNA fragments generated by apoptosis induction The supernatant was then recovered by centrifugation. The recovered supernatant was treated with proteinase K solution (Sigma-Aldrich) at a concentration of 0.5 mg / ml and reacted at 50 ° C for 3 hours. A phenol: chloroform: isoamyl alcohol mixture solution (25: 24: 1, Sigma-Aldrich) The mixture was stirred and then centrifuged to separate the supernatant. Isopropanol (Sigma-Aldrich) and 5 M NaCl were added to the separated supernatant, and reacted at 4 ° C for 24 hours. Then, the solidified DNA pellet was extracted by centrifugation. For RNA removal, the solidified DNA pellet was dissolved in an appropriate amount of TE buffer containing RNase A, and then added with 6X gel loading dye (Bioneer, Daejeon, Korea) and subjected to 1.6% agarose gel electrophoresis at 50 V Respectively. After electrophoresis, the agarose gel containing the separated DNA was stained with ethidium bromide (EtBr, Sigma-Aldrich) and DNA fragmentation was confirmed under UV.
7. Analysis of mitochondrial membrane potential (MMP, Δψm)
In order to measure the degree of change of MMP according to GC, UGP, and SS alone or in combination, the cells treated in the same manner as above were collected, and then 10 μM of 5,5 ', 6,6'-tetrachloro- The solution was treated with 3,3'-tetraethyl-imidacarbocyanine iodide (JC-1, Sigma-Aldrich) and reacted at room temperature for 20 minutes. After the reaction was completed, the supernatant was removed by centrifugation, cold PBS was added to suspend the cells, and the cells were separated into single cells using a 35 mm mesh. The prepared cells were applied to FACS Calibur to measure changes in MMP and analyzed using CellQuest software and ModiFit LT program.
8. Analysis of protein expression by Western blot analysis
To the cells was added an appropriate amount of lysis buffer [25 mM Tris-Cl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM phenymethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol After incubation for 1 hour, the total protein in the supernatant was separated by centrifugation. Protein concentration of the supernatant was quantified using Bio-Rad protein quantification reagent (Bio-Rad, Hercules, Calif., USA) and mixed with an equal volume of Laemmli sample buffer (Bio-Rad) ) -Polyacrylamide gel was used to separate proteins according to their molecular weights and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). Nitrocellulose membranes with transferred proteins were blocked with non-specific proteins using 5% skim milk, treated with primary antibody for at least 12 h at 4 ° C, washed with PBS-T, The secondary antibody for the secondary antibody was reacted at room temperature for 1 hour. After the reaction was completed, enhanced chemiluminoesence (ECL) slution (Amersham Life Science Corp.) was applied in the dark room and then exposed to X-ray film to analyze the amount of specific protein expressed.
9. Measurement of in vitro caspases activity
In order to confirm the activity of caspase enzymes, which are known to play important roles in inducing apoptosis, proteins were extracted and quantified in the same manner as described above. Then, 50 μl of a sample containing 150 μg of protein was added to 100 μM of a reaction 50 μl of buffer [40 mM HEPES (pH 7.4), 20% glycerol (v / v), 1 mM EDTA, 0.2% NP-40 and 10 mM DL-DTT) Respectively. Here, caspase-3-dependent substrates such as Asp-Glu-Val-Asp (DEVD) -p-nitroaniline (pNA); caspase-8, Ile-Glu-Thr-Asp (IETD) -pNA; (Leu-Glu-His-Asp (LEHD) -pNA] were added to each well at 37 ° C for 3 hours in a dark room, and the absorbance at 405 nm was measured using an ELISA reader Were measured. All three measurements were made, and mean values and standard errors were analyzed using the Microsoft EXCEL program.
10. Preference test
The GC, UGP and SS were blended at a constant ratio, and the degree of preference according to flavor and texture was indexed and evaluated. In the experiment, a total of 20 persons were repeatedly tasted with composition according to each blending ratio, and the palatability was evaluated with an index of 1 to 10, and the results were expressed as an average. The higher the number, the better the palatability.
11. Statistical Analysis
All of the above results are shown in SPSS ver. 18.0 Statistical significance was expressed as mean ± standard deviation (SD). The statistical significance of each item was analyzed at the p <0.05 level using Student t-test and Duncan's multiple range test after analysis of variance (ANOVA).
[Test Results and Analysis]
1. Effect of GC, UGP and SS on normal and cancer cell proliferation
MTT assay was performed to investigate the proliferation inhibition of GC, UGP and SS in various normal and cancer cells. As shown in FIG. 1, when GC and UGP were treated for 24 hours, no significant change was observed up to a maximum concentration of 40 g / ml. However, in the SS treatment group, concentration-dependent proliferation Inhibition was observed, indicating that the highest inhibitory effect of 1 g / ml was inhibited by about 66%.
Further, in order to confirm the inhibitory effect of the cancer cell proliferation by the combined treatment of GC, UGP and SS, the concentration of the SS was fixed and the effect was confirmed by changing the GC and UGP concentrations. In this case, the proliferation inhibition effect was remarkably increased by the combined treatment than the SS treatment group. That is, it was confirmed that GC and UGP induce a synergistic effect on the proliferation inhibition induced by SS. This synergistic effect was shown to be maximized in the complex treatment group in which GC and UGP were contained in 2000 to 4000 parts by weight based on 100 parts by weight of SS. On the other hand, when the GC and UGP are contained in an amount of more than 2000 to 4000 parts by weight based on 100 parts by weight of SS, it is confirmed that the effect of proliferation of cancer cells is lowered. This is analyzed as a result of relatively high GC and UGP contents.
Next, we investigated the proliferation inhibition and synergistic effect of GC and UGP and 0.6 μg / ml SS alone or in combination with 24 μg / ml of different types of cancer cells and normal cells. As shown in FIG. 2, in the case of RAW 264.7 macrophages and C2C12 myofibroblasts, when GC, UGP and SS were treated alone or in combination, proliferation inhibition and synergistic effect were not induced, It can be seen that In the case of cancer cells, no significant changes were observed in the lung cancer cell line A549 cells, and slight proliferation inhibitory and synergistic effects were observed in the colon cancer cells (HCT116), liver cancer cells (Hep3B) and bladder cancer cells (T24) It was found that the sensitivity was remarkably low as compared with the comparative example. Therefore, in the subsequent experiments, the anticancer efficacy of GC / UGP and 0.6 μg / ml SS alone or in combination with 24 μg / ml of AGS cells was investigated.
2. Preference survey according to combination of GC, UGP and SS
The concentrations of GC and UGP were varied while keeping the concentration of SS constant. The flavor was measured by the method of dispersing the compounded composition in hot water and evaluating palatability. The results are shown in Table 1 below.
The higher the number, the better the flavor and palatability.
(Unit: parts by weight)
Referring to the above Table 1, the palatability of the combination of the above extracts was confirmed in 2 to 5, and the fragrance of 1 and 6 was poor, and the palatability was reduced due to the problem of a strong smell.
Therefore, when it is in the above range, it can be used for food composition or food additive having high palatability.
3. Effect of GC, UGP and SS on the morphology of cancer cells
The morphological changes of AGS cells induced by GC, UGP and SS alone or in combination were observed using an inverted microscope. As shown in FIG. 3, large morphological changes were not observed in the GC and UGP alone treatment groups, but various morphological modifications were observed in the SS alone treatment group. In the GC, UGP and SS combined treatment groups, It was found that it was induced more strongly. In particular, the tendency of dendrite-like structure formation and adherence to the long and branched formation of dendritic-like structures was lost, and the tendency to float on the medium was remarkably increased with the decrease of cancer cell density. These results are consistent with the inhibition of proliferation by GC, UGP, and SS alone or in combination.
4. Effects of GC, UGP, and SS on morphological changes of nuclei
In order to investigate the effect of inhibition of proliferation and morphological changes on the morphology of nuclei by GC, UGP, and SS alone or in combination, DAPI staining, a fluorescent substance specifically binding to nucleic acid, was performed, Respectively. As shown in FIG. 4A, no change in the density and morphology of the whole nucleus was observed in the group treated with GC and UGP alone, but in the group treated with SS alone, the density of the nucleus was decreased and the chromatin condensation Apoptotic body was observed by GC, UGP and SS complex treatment group. This result shows that apoptosis is induced by SS single treatment and combined treatment of GC, UGP and SS, and especially that combination treatment of GC, UGP and SS induces apoptosis more strongly than SS single treatment.
5. Effect of GC, UGP and SS on Apoptosis Induction
Two additional experiments were conducted to provide direct evidence of apoptosis induction. As shown in FIGS. 4B and 4C, cancer cells cultured under the same conditions as above were treated with annexin V-FITC and PI, stained with DNA, and analyzed by DNA flow cytometry for quantitative evaluation of apoptosis induction. . The incidence of natural apoptosis in cultured cancer cells was very low, about 4.14%, and 5.6% and 5.36% in GC and UGP alone treatment groups, respectively, showing no significant difference from cancer cells grown in normal medium. However, apoptosis was induced in approximately 15.54% of cells in SS alone treatment group, and the frequency of apoptosis induced cells in GC, UGP and SS complex treatment group was drastically increased to 27.5%.
As a result of observation of DNA fragmentation phenomenon as another evidence of apoptosis induction, as shown in FIG. 4D, DNA fragmentation was not observed in GC and UGP alone treatment group but weak DNA fragmentation was observed in SS alone treatment group. GC and UGP And SS complex treatment group showed strong DNA fragmentation phenomenon. These results indicate that proliferation inhibition and morphological changes induced by SS alone treatment and GC, UGP and SS treatment are closely related to apoptosis induction in human gastric cancer cell line AGS cells, UGP and SS complex treatment groups, it is thought that GC and UGP are synergistic effects on apoptosis by SS.
6. Effect of GC, UGP and SS on MMP (Δψm)
The results of investigating the changes of MMP (Δψm) in order to confirm whether mitochondria are involved in the single treatment of SS and the induction of apoptosis by the combined treatment of GC, UGP and SS are shown in FIG. In the case of cancer cells grown in the normal medium, the normal MMP was 93.98% and the damaged MMP was 6.02%. When GC and UGP were treated alone, the normal MMP was 92.92% and 93.16% About 7.08% and 6.84%, respectively, indicating that mitochondrial damage was hardly induced. However, in the SS alone treatment group, the loss of MMP was increased to about 39.06%, and in the combined treatment group of GC, UGP and SS, the loss of MMP was found in about 71.2% of the cells. This suggests that induction of apoptosis of AGS cells by SS alone treatment and GC, UGP and SS combination treatments is associated with mitochondrial dysfunction through MMP disturbance, and this phenomenon is more strongly associated with GC, UGP and SS complex treatment .
7. Effect of GC, UGP and SS on expression of apoptosis inducing protein
Western blot analysis was used to investigate the changes in the expression of apoptosis - inducing proteins by GC, UGP, and SS alone or in combination with AGS cells. As shown in FIG. 6, the expression of all the proteins examined by GC and UGP alone was not significantly changed, but the expression of various proteins was changed by SS alone treatment and GC, UGP and SS combination treatments Respectively. First, the expression of various death receptors and death ligands involved in the extrinsic pathway was examined. As a result, expression of the death receptor and ligands Fas and FasL was increased, and the Bcl-2 family member, which is known to induce mitochondrial dysfunction in the intrinsic pathway In addition, the expression of Bcl-XL, which is an anti-apoptotic protein, is fragmented by activated caspase-8 by activation of death receptor. Therefore, Bid which is a pro-apoptotic gene causing malfunction of mitochondria is fragmented But the expression of total Bid protein was decreased. In addition, inhibition of caspase activity resulted in decreased expression of XIAP, cIAP-1 and survivin in the IAP family, which is known to inhibit apoptosis. Changes in the expression of these proteins were observed more strongly in GC, UGP and SS complexes than in SS alone, and several genes related to extrinsic and intrinsic pathways were involved in apoptosis induced by AGS cells.
8. Effects of GC, UGP and SS on Caspases activity and substrate protein expression
The effects of GC, UGP and SS on the expression and activity of caspase-3, -8 and -9, which are important regulators of apoptosis induction, were investigated among various caspases. As shown in the results of FIG. 7A, the expression of the active protein was increased with the SS alone treatment and the combined treatment of GC, UGP and SS, It was difficult to observe the difference of complex treatment accurately. As a result of the direct assay of the activity of these caspases through in vitro caspase activity assay, the degree of increase of caspase-3, -8 and -9 was increased in GC, UGP and SS complexes Treatment group. As shown in FIG. 7A, the target proteins PARP, β-catenin and PLC-γ1, which are specifically cleaved by the activated caspase-3 and are directly involved in the induction of apoptosis, also exhibit GC, UGP and SS And decreased expression and fragmentation in the combined treatment group.
9. Inhibition of apoptosis by inhibiting Caspases activity
These results indicate that caspase activation plays an important role in the apoptosis induced by GC, UGP, and SS complex treatment in AGS gastric cancer cells. Therefore, in order to confirm this, caspase inhibition inhibited apoptosis . As shown in FIGS. 8A and 8B, the increased apoptotic body by GC, UGP and SS complex treatment was significantly inhibited by z-VAD-fmk pretreatment as a pan-caspase inhibitor, and annexin V positive cells Of the patients were significantly reduced from about 27.28% to about 8.76%. As shown in FIG. 8C, AGS cell proliferation inhibition by GC, UGP and SS combined treatment was restored by z-VAD-fmk pre-treatment. In the DNA fragmentation phenomenon, z-VAD-fmk pretreatment. Therefore, it was found that the increase of caspase activity plays an important role in the apoptosis induced by GC, UGP and SS complex treatment.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.
Claims (10)
With respect to 100 parts by weight of the nephrite extract
Dried fruit extract 3500 to 4000 parts by weight
And 3000 to 4500 parts by weight of Rootgrass extract
The nephrite extract, ganoderma lucidum extract, and rhizome extracts were prepared by using water as a solvent
Which has a preventive or ameliorative effect on gastric cancer
A pharmaceutical composition for the prevention of gastric cancer including nephrolithic extract.
The nephrite extract
100 parts by weight of nephrite and 10 to 180 parts by weight of sodium hydrogen carbonate with respect to the nephrite
A composition for preventing cancer, comprising a nephrite extract.
100 parts by weight of pulverized pulverized product, 500 to 1400 parts by weight of water is mixed with the pulverized pulverized product and heat-treated at 70 to 160 ° C for 3 to 12 hours to prepare dried fruit extract;
100 parts by weight of rhizome crushed product and 800-2000 parts by weight of water are mixed with the crushed rhizome product and heat-treated at 70 to 160 ° C for 2 to 8 hours to prepare rhizome extract;
And a mixing step of preparing a mixture by mixing the nephrite extract, ganoderma lucidum extract, rhizome extract and water,
In the mixing step, the nephrite extract, the nephrite extract, and the rhizome extract are mixed with 100 parts by weight of the nephrite extract, 3500 to 4000 parts by weight of the extract of Rhododendron japonica, and 3000 to 4500 parts by weight of the extract of Rhodophylla
Wherein said mixture has a preventive or ameliorative effect on gastric cancer
A method for preparing a pharmaceutical composition for cancer prevention comprising a nephrite extract.
The step of preparing the nephrite extract comprises
0.01 to 10 parts by weight of willow is further included
A method for preparing a pharmaceutical composition for cancer prevention comprising a nephrite extract.
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| KR20240059915A (en) | 2022-10-28 | 2024-05-08 | 동의대학교 산학협력단 | Composition for preventing or improving cancer comprising root extract of adenophora triphylla var. japonica as an active ingredient |
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| KR101412057B1 (en) | 2012-07-04 | 2014-06-26 | 배용태 | Composition for preventing or treating aging or cancer comprising Camellia extract as an active ingredient |
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Non-Patent Citations (4)
| Title |
|---|
| Biosci. J., Uberlandia, 2010, Vol.26, No.2, pp.296-304* |
| European Journal of Cancer. 1999, Vol.35, No.8, pp.1258-1263* |
| Korean J. Biotechnol. Bioeng. 2001, Vol.16, No.6, pp.547-553* |
| Korean J. Oriental Physiology & Pathology. 2006, Vol.20, No.3, pp.701-709* |
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| KR20240059915A (en) | 2022-10-28 | 2024-05-08 | 동의대학교 산학협력단 | Composition for preventing or improving cancer comprising root extract of adenophora triphylla var. japonica as an active ingredient |
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