• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to a mixed herbal composition containing 5 to 90% by weight of soybeans, ganoderma lucidum, fish vinegar, licorice and licorice, respectively, and a method for preparing the same, more specifically 1) 5 to 90% by weight of soybeans and alcohol Preparing to extract soybean extract (first step); 2) preparing each extract by extracting 5 to 90% by weight of each of ganoderma lucidum, erukcho, jinjinmu and licorice with alcohol (second step); And 3) mixing and aging the soybean extract obtained in the first step and each extract obtained in the second step. Since the herbal composition of the present invention contains a natural herbal as an active ingredient, side effects and low toxicity are safe and can be usefully used for anticancer, immune enhancement, and atherosclerosis treatment.
    公开号:KR20030024739A
    申请号:KR1020030011164
    申请日:2003-02-22
    公开日:2003-03-26
    发明作者:정인택
    申请人:주식회사 보성；
    IPC主号:
    专利说明:

    The pharmaceutical composition and its preparation method of herb mixture for anticancer drug, immunological enhancement and curative for arteriosclerosis}
    [5] The present invention relates to a mixed herbal composition containing 5 to 90% by weight of soybeans, ganoderma lucidum, fish vinegar, licorice and licorice, respectively, and a method for preparing the same, more specifically 1) 5 to 90% by weight of soybeans and alcohol Preparing to extract soybean extract (first step); 2) preparing each extract by extracting 5 to 90% by weight of each of ganoderma lucidum, erukcho, jinjinmu and licorice with alcohol (second step); And 3) mixing and aging the soybean extract obtained in the first step and each extract obtained in the second step (third step), wherein the herbal composition is anti-cancer, immune enhancing And atherosclerosis treatment.
    [6] Cancer is an incurable disease of humans, so a specific anticancer drug is effective in some cancers, but not active in other cancers. Anticancer agent is a generic term for chemotherapeutic agents used for the treatment of malignant tumors. Most anticancer agents intervene in various metabolic pathways of cancer cells and mainly inhibit the synthesis of nucleic acids or exhibit anticancer activity. Anticancer drugs currently used in cancer treatment are classified into six categories of alkylating agents, metabolic antagonists, antibiotics, mitosis inhibitors, hormonal agents, and other agents according to biochemical mechanisms of action. Rather than damaging normal cells, especially tissue cells with active cell division, there have been problems involving various side effects such as bone marrow dysfunction, gastrointestinal disorders, and alopecia. Therefore, it is important to develop an anticancer agent that can increase the anticancer effect without the side effects of the human body.
    [7] The living body has a mechanism for excluding foreign substances out of the living body by identifying the self and the non-magnetic body by ingesting foreign substances invading the living body in order to protect self-homeostasis. What the living body should exclude includes not only foreign substances, such as bacteria and viruses, but also magnetic components such as waste tissue and cancerized cells. In living organisms, there are nonspecific defense mechanisms such as barriers such as skin and mucous membranes and phagocytosis by phagocytes, which are called natural immunity. On the other hand, a specific defense mechanism for the exclusion of newly acquired foreign substances by identifying invasive foreign substances as non-magnetic substances is called acquired immunity. Acquired immunity is divided into humoral immunity, which produces antibodies to produce an immune response, and lymphocytes directly into antigens and cellular immunity, which produces an immune response. The immune response is regulated by antigen recognition. Humoral immunity is that helper T cells recognize class II MHC-antigen complexes on the surface of antigen-presenting cells, such as macrophage, and helper T cells secrete lymphokines, and secreted lymphokines are B cells. It acts on B cells to differentiate and proliferate into plasma cells to make antibodies. On the other hand, cellular immunity is that lymphokines secreted from helper T cells activate killer T cells as described above, and the activated killer T cells bind to the class I MHC-antigen complex of cells infected with the virus to injure the cells. will be. Immune diseases with reduced immune function include rheumatoid arthritis, atopic dermatitis, AIDS, leukopenia, lupus erythematosus, myasthenia gravis, atrophic gastritis and autoimmune hemolytic anemia. Drugs marketed in Korea for the treatment of immune diseases can cause side effects such as allergic reactions, central nervous system side effects, indigestion, nausea, vomiting, anorexia, diarrhea. Therefore, it is important to develop an immune enhancer that can enhance the immune enhancing effect without the side effects of the human body.
    [8] Cholesterol is a major component of the cell membranes and myelin of cells in all parts of the body, including the brain, nerves, muscles, skin, liver, intestines and heart, and is a steroid hormone produced in the adrenal cortex, testes and ovaries. Precursor of vitamin D, which is produced in the skin by sunlight and sunlight, and bile acids, which are produced in the liver, are ingested through animal food or synthesized from acetyl-coenzyme A in vivo. Since cholesterol is hydrophobic, cholesterol itself cannot be present in the plasma, so when it is transported in the blood, it is combined with apoprotein and transported as a lipoprotein, which is high density lipoprotein (HDL). It is classified into Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) lipoprotein. Cholesterol is mainly contained in LDL and HDL, and triglyceride is mainly contained in VLDL. An amount of 20-30% of total cholesterol is carried in HDL form and the remainder is mostly in LDL form, where HDL is a factor that removes cholesterol from the vessel wall, while LDL is known as a risk factor for promoting atherosclerosis. Therefore, as the LDL increases, the walls of the blood vessels become thicker, and lime is deposited, causing the blood vessels to lose elasticity and narrowing, thereby causing arteriosclerosis, an adult disease in which blood vessels burst or interfere with blood circulation. Since atherosclerosis is a result of many years of lifestyle and physical condition, it is difficult to expect it to return to its original condition once occurring. Therefore, it was necessary to develop an effective atherosclerosis treatment because prevention was the only effective treatment until now.
    [9] Soybean is a seed of the plant family belonging to the legumes, containing 40% protein, 20% fat, 25% carbohydrate, and other biological activities such as vitamins, minerals, phospholipids, saponins, isoflavones and phytic acid. It also contains substances. Soybean is the most important source of protein in Korean diet, and it is known to prevent, dissolve, and have a formal effect.
    [10] Ganoderma lucidum is a kind of mushroom that grows on dead trees and stumps. It grows in strong wood such as oak and plum trees, and grows at right angles to the stump. Its shape is circular with horseshoe shape. Ganoderma Lucidum has no side effects and is said to be an excellent herb that can be taken for a long time.In herbal herbs, if you take Ganoderma lucidum for a long time, your body becomes light and not old, and it is long-lived and fresh. It is said that ganoderma has the effect of stroke, blood pressure, heart disease, liver, stomach, kidney, dementia, skin disease, etc.
    [11] Eoseongcho is a perennial herb belonging to more than three hundred, unusually fishy herb. Echo herb contains decanoyl acetaldehyde and flavonoids, which are fishy components, and have anti-inflammatory and blood purification effects, respectively. .
    [12] Injin mugwort is a perennial herb belonging to the Asteraceae family, and it is also called Sacheol mugwort because the stem survives in the winter. Injin mugwort contains coumarin, a useful ingredient for edema, and is used in herbal medicine for hepatitis, jaundice, stomach and cold. In Dongbobogam, Injin mugwort is very bitter, and it treats symptoms of yellow and yellow urine that are hard to pass through the jaundice caused by moist heat gathered in the body. It is recording.
    [13] Licorice is a perennial herb of legumes, also known as Gukro, meaning elder in medicine. Thus, licorice is used in combination with other drugs to harmonize the drug's effects and neutralize the poison. In addition, it is recorded that Dongbogam controls the disease of the five intestines, controls the physiology of eyes, nose, mouth, ears and faeces, communicates the blood vessels of the human body, and strengthens muscles and bones.
    [14] Recently, due to limitations of the development of disease treatments by synthetic compounds and problems related to side effects and toxicity during treatment, development of disease treatments based on herbal medicines has been actively conducted.
    [15] Therefore, the present inventors prepared a mixed herbal composition containing 5 to 90% by weight of soybean, ganoderma lucidum, eochochocho, jinjin wormwood and licorice in the course of examining the herbal medicine for anticancer, immune enhancement and atherosclerosis treatment, and the herbal composition The present invention has been found to have high safety and excellent anti-cancer, immune-enhancing, and atherosclerosis treatment effects because of lower side effects and toxicity than conventional anti-cancer, immuno-enhancing, or atherosclerosis treatments.
    [16] It is an object of the present invention to provide a mixed herbal composition containing 5 to 90% by weight of soybean, ganoderma lucidum, edible vinegar and licorice, respectively, and a method for preparing the same.
    [17] In addition, an object of the present invention is to provide a pharmaceutical composition comprising the herbal composition as an active ingredient.
    [1] 1 is a graph showing the effect on the TNF-α of the herbal composition of the present invention in the mouse,
    [2] Figure 2 is a graph showing the effect on lung metastasis of cancer cells in mice administered with the herbal composition of the present invention for 21 days,
    [3] 3 is a graph showing the antioxidant effect of the herbal composition of the present invention on cell-mediated LDL oxidation,
    [4] Figure 4 is a graph showing the antioxidant effect of the herbal composition of the present invention on conjugated double bonds formed during LDL oxidation during various times.
    [18] In order to achieve the above object, the present invention provides a mixed herbal composition containing 5 to 90% by weight of soybean, ganoderma lucidum, eojincho, phosphorus mugwort, and licorice, respectively, and a preparation method thereof.
    [19] More specifically, the herbal composition is 1) 5 to 90% by weight of soybeans boiled and cooled, then aged, the mature soybeans extracted with 10 to 90% C1 to C4 alcohol, filtered and concentrated in vacuo Preparing a soy extract (first step); 2) extracting 5 to 90% by weight of each of ganoderma lucidum, ergot vinegar, licorice and licorice using 10 to 90% C1 to C4 alcohol, followed by filtration and concentration in vacuo to prepare respective extracts (second step); And 3) mixing and aging the soybean extract obtained in the first step and each extract obtained in the second step, followed by filtration and vacuum concentration (third step).
    [20] In the first step, boil for 15 minutes at 121 ℃, it is preferable to aged for 3 days at 37 ℃, in the third step is preferably aged for 24 hours at 80 ℃. In addition, the alcohol is preferably 80% ethyl alcohol.
    [21] The present invention also provides an anticancer pharmaceutical composition containing the herbal composition as an active ingredient.
    [22] The herbal composition exhibits excellent anticancer activity such as solid cancer growth inhibition and lifespan extension in sarcoma cancer cells and excellent anticancer activity such as cancer cell proliferation inhibition in lung cancer cells.
    [23] In addition, the present invention provides a pharmaceutical composition for immuno-enhancing containing the herbal composition as an active ingredient.
    [24] The herbal composition exhibits immunopotentiating activities such as an increase in the number of leukocytes in blood, an increase in the total number of peritoneal cells, and an increase in the weight of immune-related organs in sarcoma cancer cells, an increase in nitric oxide (NO), and tumors in lung cancer cells. Necrosis factor-α (tumor necrosis factor-α, TNF-α) increase, such as immunosuppressive activity.
    [25] In addition, the present invention provides a pharmaceutical composition for treating arteriosclerosis containing the herbal composition as an active ingredient.
    [26] The herbal composition exhibits an arteriosclerosis therapeutic activity such as inhibiting oxidation of LDL, which is one of the causes of atherosclerosis, and inhibiting the formation of conjugated double bonds.
    [27] The composition may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical formulation.
    [28] That is, the composition of the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Is prepared using.
    [29] Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.
    [30] The effective dose of the herbal composition is 5 to 20 mg / kg, preferably 10 to 20 mg / kg, may be administered 1-6 times a day. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate, severity of the disease, and the like of the patient.
    [31] Soybean, ganoderma lucidum, fish vinegar, injin mugwort, and licorice, which are raw materials of the herbal composition of the present invention, have been secured as natural medicines. In particular, as a result of performing toxicity experiments when orally administering the herbal composition of the present invention to rats, Turned out to be a safe material.
    [32] In addition, the herbal composition can be widely used as a functional food or food additives for anticancer, immune enhancing or atherosclerosis prevention.
    [33] Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are presented to illustrate the present invention and are not intended to limit the scope of the present invention.
    [34] Example
    [35] Preparation of Mixed Herbal Compositions
    [36] 100 g of soybeans were boiled at 121 ° C. for 15 minutes and then cooled. Again aged for 3 days at 35 ℃. The aged soybeans were extracted with 80% ethanol at 80 ° C., filtered and concentrated in vacuo to obtain soy extract. On the other hand, 100g of each of Ganoderma lucidum, Eurseongcho, Injin mugwort and licorice were extracted using 80% ethanol, filtered and concentrated in vacuo to obtain respective extracts. 100 g of water was added to 25 g of the soybean extract, 20 g of ganoderma lucidum extract, 20 g of fish extract, 15 g of phosphorus mugwort extract, and 5 g of licorice extract, followed by aging at 80 ° C. for 24 hours, followed by filtration and vacuum concentration to obtain a mixed herbal composition 100 g. .
    [37] Experimental Example 1 Review of Anticancer Activity and Immune Activity Against Sarcoma Cancer
    [38] Experimental Animals and Tumor Cells
    [39] In this experiment, male ICR mice with a body weight of 18-22 g were used as experimental animals, and sarcoma-180 cells, a sarcoma carcinoma passaged at weekly intervals in the abdominal cavity of ICR mice, were used as tumor cells. .
    [40] 2. Solid Rock Growth Prevention Experiment
    [41] There are 7 experimental animals in each group, and each group is divided into medicinal herb composition administration group, which is dissolved in sterile saline solution, and administered at 50 mg and 100 mg / kg of mouse, and sterile physiological saline control group. It was.
    [42] First, subcutaneous transplantation of 0.1 mL (1.0 × 10 6 cell / mouse) of tumor cell suspension, which is passaged at a weekly interval in the laboratory, into the left groin of the experimental animal, and then the herbal composition and Physiological saline was administered intraperitoneally and killed on the 26th day of tumor cell transplantation. The resulting solid cancer was extracted and weighed.
    [43] As a result, as shown in Table 1, the inhibition rate of solid cancer growth was 68% at the concentration of 50 mg / Kg of the herbal composition of the present invention, and the blocking rate was rather low at 55% at 100 mg / kg.
    [44] Table 1
    [45] groupTumor weight (g) (mean ± standard deviation)Blockage rate (%) Control8.861 ± 0.5350 mg / kg2.83 ± 0.5268 100mg / kg3.86 ± 0.3255
    [46] 3. Life Extension Experiment
    [47] Seven experimental animals were used in each group, and each group was divided into a group administered with a herbal composition administered with 50 mg and 100 mg / kg of the mixed herbal composition prepared in Example in sterile saline solution and a control group administered with saline. .
    [48] First, subcutaneous transplantation of 0.1 mL (1.0 × 10 6 cell / mouse) of tumor cell suspension, which is passaged at a weekly interval in the laboratory, into the left groin of the experimental animal, and then the herbal composition and Physiological saline was administered intraperitoneally and survival was observed up to 35 days.
    [49] As a result, as shown in Table 2, the average lifespan of the control group is 18 days, whereas the herbal composition administration group of the present invention was shown as 20 days and 18 days, respectively.
    [50] TABLE 2
    [51] groupLife expectancy average (mean ± standard deviation)Blockage rate (%) Control18.00 ± 0.0650 mg / kg20.32 ± 0.2414 100mg / kg18.14 ± 0.635
    [52] 4. Blood White Blood Cell Count
    [53] The experimental animals were administered intraperitoneally for 10 consecutive days with 12 animals in each group, and the control group was administered sterile saline solution. On the 1st, 2nd, 4th and 7th day of the last day of each dose, 80 μL of blood was taken from the mouse's eye vein using heparinized capillary and mixed well with 320 μL of citric acid-physiological saline. , Tuk's solution was stained and leukocyte count was measured using a hemacytometer.
    [54] As a result, as shown in Table 3, it was confirmed that the blood leukocyte count was significantly increased in the herbal composition administration group compared to the control group. This result is considered to be a significant result considering that leukocytes are the primary effector cells of the body's immune mechanism.
    [55] TABLE 3
    [56] groupDay 1Day 2Day 47th day Control7,243 ± 7167,510 ± 2607,464 ± 1,0367,032 ± 629 50 mg / kg12,042 ± 46811,573 ± 2,58310,528 ± 4839,012 ± 653
    [57] 5. Effect on Total Peritoneal Cell Number
    [58] Experimental animals were divided into nine groups in each group, and then divided into the herbal composition administration group and the control group, and then the herbal composition and sterile saline solution were administered intraperitoneally for 3 consecutive days. On the 1st, 2nd, and 4th day after the last day of each administration, each mouse was lethal by cervical dislocation, washed well intraperitoneally with 5 mL of saline solution, and taken with ascites fluid to Tuk's solution. After staining, the total abdominal cell number was measured using a hemacytometer.
    [59] As a result, as shown in Table 4, the herbal composition-treated group showed a significant increase in the number of peritoneal cells on the first day of administration compared to the control group, but gradually decreased on the 2nd and 4th days. Because of the inclusion of nuclear and mononuclear cells, this increase in celiac cells is indicative of increased immune capacity.
    [60] Table 4
    [61] groupDay 1Day 2Day 4 Control2.84 ± 0.242.62 ± 0.262.89 ± 0.23 50 mg / kg3.40 ± 0.252.88 ± 0.122.24 ± 0.12
    [62] 6. Changes in Weight of Immune-Related Organs
    [63] Experimental animals were divided into 8 groups in each group, which was divided into the herbal composition administration group and the control group, and each herbal composition and sterile saline solution were administered intraperitoneally for 10 consecutive days, and the mouse was injected by cervical spinal bone decay method on the 8th day from the last day of administration. Liver, spleen and thymus were extracted and then organ weights were measured.
    [64] As a result, as shown in Table 5, it was confirmed that the weight of the liver, spleen and thymus of the herbal composition administration group is slightly increased compared to the control.
    [65] Table 5
    [66] groupWeight (g)Liver / body weight (%)Spleen / Weight (%)Thymus / Weight (%) Control23.68 ± 0.771.20 ± 0.04 * 4.10 ± 0.070.28 ± 0.01 50 mg / kg24.77 ± 0.841.32 ± 0.04 * 4.15 ± 0.120.35 ± 0.01
    [67] * p <0.05
    [68] Experimental Example 2 Investigation of Anticancer Activity and Immune Activity Against Lung Cancer
    [69] 1. Experimental Animal and Dosage
    [70] Males of 5 to 6 weeks of age, C57 BL / 6 mice were purchased from the Experimental Animal Center (negative Chungbuk) and used. The temperature was maintained at 20 ± 2 ℃ and the contrast period was 12 hours, and the solid feed and water were sufficiently supplied. Mice were reared under the same conditions before adaptation in an animal room environment. Feed was supplied with Samyang oil feed company and antibiotic-free mouse pellets. 10 ml of the herbal composition was diluted with 100 ml of tap water so that it could be freely eaten (5 per cage). The herbal composition was supplied for 21 days and the total amount was 3 g / 5 mice / 21 days.
    [71] 2. Test Cell
    [72] Isolation of lymphocytes from the spleen was performed as follows. Spleens obtained from mice were gently pressed into two sterile slide glasses in Hanks' balanced salt solution (HBSS, Sigma, St. Louis, Mo) to release lymphocytes from the spleen. Erythrocytes contained in this genus were removed by sterile lymphocyte distilled water with hypotonic shock, and the isolated lymphocytes were suspended in complete medium. Peritoneal macrophages were isolated according to Yoon's method. Three days after injection of 3 mL of Brewer's thioglycollate broth (Difco laboratory, detroit, MI) into the abdominal cavity of mice, 1% fetal calf serum (FCS), 10 mM HEPES, and penicillin (100 μg / mL) and Peritoneal effusion cells were collected using 10 mL HBSS (without calcium and magnesium) added with streptomycin (100 μg / mL). After resuspending in RPMI 1640 complete medium and incubating for 3 hours at 37 ° C, unattached cells were washed out with phosphate buffer, and the adherent cells were collected with 2.5 mM pyrophosphate and rubber policeman. It was used as celiac macrophages. The cancer cell lines were mouse mammary carcinoma cells, mouse leukemia cells, and human histocytic lymphoma.
    [73] 3. Measurement of the effect on the proliferation of cancer cells and normal immune cells
    [74] The cells prepared as above were dispensed into each well of a flat bottomed 96 well plate, and various concentrations of the herbal composition were added thereto to adjust the total amount to 0.2 mL, and put in a 37 ° C. 5% CO 2 incubator and incubated for 72 hours. At this time the culture cells 3 H- ssayimi Dean (3 H-thymidine, specific activity : 2.0 Ci / mmol, ICN Biomedicals, High Wycombe, Bucks, England) was incubated with 3 H- ssayimi Dean 0.5μCi per well end 18 After the addition of time, the measurement of 3 H-cymidine cohort was carried out using a β-counter after harvesting the cells on the glass fiber with a cell harvester (cell harvester).
    [75] Table 6 shows the results of measuring the proliferation of each cell after adding the herbal composition in the early stage of culture while culturing spleen cells and cancer cells. As the concentration of herbal composition increased, the proliferative capacity increased markedly in splenocytes, but it was found that the proliferative capacity was somewhat suppressed in cancer cell lines.
    [76] Table 6
    [77] Medicinal Herb Composition (㎍ / ml)CPM (count / min) SplenocytesFM3A / SP388 / SU937 / S 03001674866546802 250100,021512143214989 500101,119376839652234 10003900210021023990
    [78] 4. Measurement of cytotoxicity against cancer cells
    [79] The method using MTT ([3- (4,5-dimethylthizol-2yl) 2,5-diphenyltetrazolium bromide]) was modified. MTT was dissolved in a phosphate buffer (PBS) buffer solution at a concentration of 5 mg / mL, and then sterile filtered and stored in a dark place at 4 ° C., which was stored for 3 weeks or less. In each well of the microplate in culture, 160 μL of the upper layer was carefully added. At this time, MTT dilutions were added to wells containing no cells. After 4 hours of incubation at 37 ° C. and 5% CO 2 , about 40 μL was left to prevent cell loss by removing the upper medium. 100 μL of DMSO was added to each well, followed by shaking for about 20-30 minutes with a plate shaker at room temperature, and then absorbance was measured at 570 nm and 650 nm using an ELISA reader (Micro plate EL311). . In this case, the wells containing no cells were blanked and 650 nm was compared, and the value measured at 570 nm was subtracted from the measurement at 650 nm, and this value was used as a result. The cytotoxicity against was investigated.
    [80] The herbal composition was added at the beginning of the splenocyte culture and the cell viability was examined at 48 hours. As a result, direct cytotoxic activity was not observed as shown in Table 7.
    [81] TABLE 7
    [82] Medicinal Herb Composition (㎍ / mL)Survival rate (%) FM3A / SP388 / SU937 / S 045.945.246.5 5051.650.253.6 25049.449.851.8 50050.645.349.7 100044.741.946.1
    [83] 5. Changes in T Cell Subtypes
    [84] The T-cell subtype was composed of phycoerythrin (PE) -conjugated rat anti-mouse CD4 (L3T4) mAb and FITC conjugated rat anti-mouse CD8 (Ly-2) mAb, and IL-2 receptor was FITC-cojugated rat anti-mouse. CD25 mAb was used to analyze the manufacturer's instructions.
    [85] As a result, as shown in Table 8, the herbal composition-administered group increased the ratio of CD4 + and CD8 + T cells compared to the control group, but the CD4 + / CD8 + ratio did not differ between the two groups.
    [86] Table 8
    [87] Percent of single positive cells CD4 + CD8 - (L3T4 +) CD4 - CD8 + (Lyt2 + )CD4 + / CD8 + ratio Normal control20.35.14.0 50mg / kg (Medicinal composition treatment group)29.57.04.2
    [88] 6. Measurement of Nitric Oxide (NO) Production by Macrophages
    [89] Peritoneal macrophages are resuspended in cell culture plates to be 5 × 10 5 / mL cells and stimulated with interferon-gamma (IFN-γ, 10 U / mL) and lipopolysaccharide (LPS, 1 μg / mL). 48 hours of incubation and NO in the culture supernatant were measured by Griess reaction. That is, 100 µl of 1% sulfanylamide (30% acetic acid) and 100 µl of 0.1% N- (1-naphthyl) ethylenediamine dihydrochloride (60% acetic acid) were added to 100 µl of the culture supernatant and allowed to stand at room temperature. After 20 minutes the absorbance was measured at 570 nm using a ELISA reader (Bio Tek Instruments Inc, model EL311 SL) at a maintenance concentration of 800 μg / mL.
    [90] As a result of measuring the effect of the herbal composition on the production capacity of NO from the abdominal macrophages, the herbal composition slightly stimulated NO production as shown in Table 9. In addition, the results of examining the effects of the herbal composition on the production of NO are shown in Table 10.
    [91] Table 9
    [92] groupNitrite concentration (μM) Normal group (only administration) Herbal composition-administered group LPS + γ-IFN-administered group LPS + γ-IFN + herbal composition-administered group12 ± 3.428 ± 4.237 ± 3.236 ± 3.1
    [93] Table 10
    [94] Herbal Composition (㎍ / ㎖)Nitrite concentration ( M) 04.9 ± 1.3 258.2 ± 2.1 5012.2 ± 2.2 10015.9 ± 2.1 50021.8 ± 2.2 100016.7 ± 2.3
    [95] 7. Measurement of the titer of tumor necrosis factor (TNF-α)
    [96] To confirm the effect of herbal composition administration on TNF-α production in mice, the herbal composition was injected for 21 days (3 g / 200 mL / 5 mice). All groups of mice were sacrificed by cardiac puncture. Serum was prepared and assayed for TNF-α by ELISA reads. TNF-α was measured using an ELISA kit (ELISA kit, Genzyme, Boston, MA). That is, the anti-TNF antibody was attached to a 96well plate for 24 hours, washed, and 100 μl of the sample was added thereto and reacted in a 96 well plate for 1 hour. Tween 20 was reacted with anti-TNF monoclonal antibody for 90 minutes after washing with phosphate buffer (PBS) added to 10%. After washing, the reaction was performed for 60 minutes to react peroxidase-conjugated anti IgG antibody and washed again and then reacted with ο-phenylene diamine (orthophenylene diamine) solution for 15 minutes. After the reaction was stopped by adding 0.5N HCl solution, absorbance was measured at a wavelength of 450 nm. Measure the standard TNF solution at concentrations of 3200, 1600, 800, 400, 200, 100, and 50 pg / mL, measure the polynomial standard regression curve based on the absorbance measured in the standard solution, and use the sample The amount of TNF contained within was determined.
    [97] The effect of administration of the herbal composition on TNF-α production in mice is shown in FIG. 1. That is, the concentration of TNF-α was 262 pg / mL in the control group and 443 pg / mL in the herbal composition-administered group, and thus, it could be observed that TNF-α production could be somewhat enhanced.
    [98] 8. Lung metastasis experiment of melanoma cells (B16F10)
    [99] The cancer cell lines disclosed in this experiment were B16F10 mouse melanoma cells (Tumor Repoitory of the National Cancer Institute, Bethesda, MD), 10% fetal bovine serum, sodium pyruvate, nonnessential amino acids and L It was maintained in RPMI1640 medium containing L-glutamine. The preparation of the cancer cells was collected in 0.05% trypsin-0.02% EDTA solution of the logarithmic growth stage and washed twice to adjust the PBS roll concentration. B16F10 cells (2 × 10 5 ) were injected through the mouse microvein and lungs were removed on day 14 to determine the number of melanoma.
    [100] As shown in FIG. 2, the lung metastasis of B16F10 cells was reduced in number in the herbal composition-administered group compared to the control group, and the size of the metastasized cancer cell colonies was also significantly smaller in the herbal composition-administered group. It was also found that inhibiting the proliferation of.
    [101] Experimental Example 3 Investigation of the Activity Related to LDL Related to Arteriosclerosis
    [102] 1. Isolation of Human LDL
    [103] 50 mL of healthy male blood was placed in a plastic test tube containing 50 mL of EDTA (1 mg / H 2 O 50 mL), stirred, and left for 3 hours at 4 ° C. Plasma in blood was separated by centrifugation (2000 × g) for 20 minutes at room temperature, and then 1 mL of gentamicin sulfate (1 mg / 25 mL of water) was added thereto. LDL (d. 1.019 to 1.063 g / mL) was obtained by separating for 24 hours using an ultrafast centrifuge (46,000 × g). The isolated LDL was dialyzed for 16-20 hours as 200 mL of 0.01 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride, 0.01 M EDTA.
    [104] 2. Isolation and Culture of Macrophages
    [105] Female ICR mice were suffocated with CO 2 and washed with cold Dulbecco's phosphate buffered saline (Ca 2+ , without Mg 2+ ) collected in the abdomen, capturing macrophages, centrifuging and removing blood. Only cells were isolated. The cells 10% were inactivated fetal bovine serum and 2 mM / L, the L- glutamine, Dulbecco's Modified Eagle Medium (DMEM ) culture medium 1mL was added to 100 units / mL penicillin, and 0.17 mM / L streptomycin, 5% CO 2 Incubated in the presence of / air. After 24 hours of incubation at 37 ° C., the cells were replaced with fresh medium, and then LDL or oxidized LDL was added to DMEM medium containing 5% lipoprotein-deficient serum (LPDS) and L-glutamine at an appropriate concentration. .
    [106] 3. Oxidative Inhibitory Activity of LDL
    [107] 1) Cell Mediated LDL
    [108] 10 μl of LDL (100 μg protein / mL) and 8 μl of herbal composition (1 mg / ethanol mL) were added to macrophages and cultured cells of J774 (variant macrophage), respectively, and the total amount was added by adding phosphate buffer saline (PBS). After ul was incubated for 18 hours in the presence of 5% CO 2 to examine the degree of oxidation of LDL.
    [109] Oxidation of LDL was assessed as the formation of Thiobarbituric acid reacting substances (TBARS). 1.5 mL of 20% TCA (Trichloroaceticacid) was added to 100 μL of the culture mixture for each concentration containing 10 μL of LDL (100 μg protein / mL), followed by adding 0.67% TBA (Thiobarbituric acid) to 2 mL of 0.05M NaOH. The reaction mixture was then heated for 45 minutes on a 90 ° C water bath. After centrifugation (2,000 × g) for 10 minutes, the fluorescence of the supernatant was measured at 510 and 553 nm with a fluorescence photometer (Model 650-10S USA) manufactured by Perkin-Elmer. The number of TBARS in the sample is expressed as nmole of MDA from the standard curve of MDA made as malondialdehyde (MDA).
    [110] As a result, it is as shown in FIG. In other words, the oxidative control of macrophages had a TBARS of 29.60 ± 0.04 nmole MDA / mg LDL protein, and the TBARS of the standard control consisting of LDL only had a 2.42 ± 0.25 nmole MDA / mg LDL protein, whereas the herbal composition was 50 ㎍ / mL and 100 ㎍. The TBARS values of the groups administered with / mL were 4.20 ± 0.26 and 3.80 ± 0.17nmole MDA / mg LDL protein, respectively.
    [111] On the other hand, in J774, the control group had a TBARS of 33.87 ± 0.04 mnole MDA / mg LDL protein, whereas the TBARS of the group administered with 50 ㎍ / mL and 100 ㎍ / mL of the herbal composition was 4.62 ± 0.05 and 4.08 ± 0.04 nmole MDA / mg, respectively. LDL protein.
    [112] Therefore, the herbal compositions showed almost similar antioxidant activity of LDL in macrophages and J774 cells.
    [113] 2) Electrophoresis of LDL
    [114] Electrophoresis of LDL was carried out beforehand by evaporating 5 μl of Nile red (30 mg / acetone mL), 1) 10 μl of LDL (100 μg protein / mL), 2) 10 μl of LDL + 5 μl CuSO 4 , 3) LDL 10μl + 5μM CuSO 4 5μl + herbal composition (50μg / mL) 1mL, 4) LDL 10μl + 5μM CuSO 4 5μL + herbal composition (100μg / mL) 1mL each, mix, 0.1M barbital Agarose gel loaded with buffer (pH 8.6) was loaded (loaded) and developed for 20 minutes at 75V using 1M barbital buffer (pH 8.6) and confirmed by UV lamp.
    [115] On the other hand, in order to determine the electrophoretic mobility (LDL) of the LDL into the macrophages, 50 or 100 ㎍ / mL of the herbal composition of 1mL, respectively, developed in the same manner as described above, and then measured the movement of the LDL It was.
    [116] As a result, as shown in Table 11, it was confirmed that the herbal composition administered group is less LDL moving distance than the control group.
    [117] Table 11
    [118] groupRelative movementP LDL only1.0LDL + Macrophage1.89 ± 0.02<0.05 LDL + macrophage + composition 50 μg / mL1.38 ± 0.022<0.05 LDL + macrophage + composition 100 μg / mL1.23 ± 0.04<0.05
    [119] 4) Determination of conjugated conjugation
    [120] When LDL is oxidized, the fatty acid ester of LDL forms a double bond, which can be used to measure the degree of oxidation. That is, the formation of conjugated double bonds formed by LDL oxide LDL was measured at 234 nm with a fluorescence photometer (Model 650-10S USA) manufactured by Perkin-Elmer. That is, after mixing 10 μl of LDL (100 μg protein / mL), 5 μl of 5 μM CuSO 4 , and 160 μl of 50 and 100 μg / mL herbal composition (1 mg / mL), diluted to 2 mL with PBS (pH 7.4) (Final concentration 80 μg / mL) The solution was measured 7 times every 30 minutes while incubating at 37 ° C. for 210 minutes.
    [121] As a result, as shown in Figure 4, it can be seen that the formation of conjugated double bonds over time at 50 ㎍ / mL, 100 ㎍ / mL concentration of the herbal composition, respectively. This result is that the structural properties of the hydroxy group and the methoxy group in the structure of the herbal composition to suppress the formation of double bonds reduce the production of fatty acid esters.
    [122] Experimental Example 4 Acute Toxicity in Rats
    [123] Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Five animals per group were suspended in a 0.5% methylcellulose solution in a herbal composition prepared according to the examples and administered once orally at a dose of 1 g / kg, 5 g / kg and 10 g / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.
    [124] As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, the herbal composition of the present invention was determined to have no cytotoxicity and no cytotoxicity up to 50 g / kg in rats. Therefore, the oral administration intermediate dose (LD 50 ) was determined to be a safe substance of 100 g / kg or more of the herbal extract prepared according to the example.
    [125] Preparation Example 1 Preparation of Tablet
    [126] Wet granules by homogeneously mixing 100.0 mg herbal composition, corn starch 90.0 mg, lactose 175.0 mg, L-hydroxypropyl cellulose 15.0 mg, polyvinylpyrrolidone 90 5.0 mg and ethanol suitable raw materials prepared according to the embodiment Granules were added, and 1.8 mg of magnesium stearate was added and mixed, and the tablets were compressed to 400 mg.
    [127] Preparation Example 2 Preparation of Capsule
    [128] 100.0 mg of the herbal composition prepared according to the Example, 83.2 mg of corn starch, 175.0 mg of lactose and 1.8 mg of magnesium stearate were mixed homogeneously and filled to contain 360 mg in one capsule.
    [129] As described above, the present invention relates to a herbal composition containing soybean, ganoderma lucidum, fish vinegar, phosphorus mugwort and licorice and a method of manufacturing the same. Since the herbal composition exhibits excellent anticancer activity, immune enhancing activity and arteriosclerosis therapeutic activity, it can be usefully used as a medical anticancer agent, an immunostimulant and an arteriosclerosis therapeutic agent containing the herbal composition as an active ingredient, as well as a health food It can also be used as, especially because the extract of natural medicine, its stability is very high.
    权利要求:
    Claims (2)
    [1" claim-type="Currently amended] Mixed herbal composition consisting of soybean, ganoderma lucidum, eoseongcho, injin mugwort and licorice each 5 to 90% by weight.
    [2" claim-type="Currently amended] 1) 5 to 90% by weight of soybeans are boiled, cooled, and then aged, and the matured soybeans are extracted using 10 to 90% of C1 to C4 alcohol, filtered, and concentrated in vacuo to produce soy extract. Stage 1);
    2) extracting each of 5 to 90% by weight of each of Ganoderma lucidum, Echinacea, Injin mugwort and Licorice using 10-90% C1 to C4 alcohol, followed by filtration and concentration in vacuo to prepare respective extracts (second step);
    3) A method for preparing the herbal composition according to claim 1, which comprises mixing and aging the soybean extract obtained in the first step and the respective extract obtained in the second step, followed by filtration and vacuum concentration (third step).
    类似技术:
    公开号 | 公开日 | 专利标题
    KR20160036016A|2016-04-01|Composition comprising extracellular vesicles derived from fermented food, and use thereof
    Oršolić et al.2004|Immunomodulatory and antimetastatic action of propolis and related polyphenolic compounds
    Liu et al.2002|Antitumor activity of milk kefir and soy milk kefir in tumor-bearing mice
    US5270041A|1993-12-14|Sterols, their fatty acid esters and glucosides; processes for their preparation; spontaneously dispersible agents containing these compounds, and their use for treatment of tumors
    RU2383353C2|2010-03-10|Combined extracts from andrographis paniculata
    RU2341257C2|2008-12-20|Immunomodulatory agent, cancer agent and healthy food containing monoacetyldiacylglycerin derivatives
    US20110189220A1|2011-08-04|Mushroom compositions and methods for making and using
    KR100506384B1|2005-08-10|The pharmaceutical composition and its preparation method of herb mixture for cancer inhibition, recovery of immune system and protection of body from oxidative damage
    KR101740893B1|2017-06-13|COMPOSITION COMPRISING EXTRACELLULAR VESICLES DERIVED FROM Akkermansia muciniphila AS AN ACTIVE INGREDIENT FOR TREATING OR PREVENTING METABOLIC DISEASE
    KR20080081186A|2008-09-08|Novel lactobacillus strains and their use against helicobacter pylori
    JPWO2003039568A1|2005-02-24|Anticancer composition
    US7977379B2|2011-07-12|Method for angiogenesis inhibition or immunostimulation
    CN102791849B|2014-11-12|Lactic acid bacterium-containing preparation
    JP2012533290A|2012-12-27|Novel Lactobacillus plantarum and composition containing the same
    JP2005154432A|2005-06-16|Treated material of acerola containing polyphenol and/or vitamin c
    US20040138172A1|2004-07-15|Drug product for intestinal disease
    KR20040018118A|2004-03-02|Pharmaceutical composition comprising the extract of Actinidia arguta and related species for the prevention and treatment of allergic disease and non-allergic inflammatory disease
    Attia et al.2008|The anti-tumor effect of bee honey in Ehrlich ascite tumor model of mice is coincided with stimulation of the immune cells
    CN102470155B|2014-10-08|Method for producing novel gynostemma pentaphyllum extracts having increased amounts of damulin A and damulin B and pharmaceutical composition for treating metabolic diseases using the same
    EP1538198A2|2005-06-08|Lactic acid bacteria and their cellular components inducing immunoregulatory function, and method of obtaining the same
    Zhang et al.2016|Toxicology and immunology of Ganoderma lucidum polysaccharides in Kunming mice and Wistar rats
    US9056067B2|2015-06-16|Pharmaceutical composition for enhancing immunity, and extract of Poria
    US8822437B2|2014-09-02|Cerebral nerve cell neogenesis agent
    EP1154022B1|2010-10-13|Novel substance originating in basidiomycete culture, process for producing the same and use thereof
    Oršolić et al.2005|Synergystic antitumor effect of polyphenolic components of water soluble derivative of propolis against Ehrlich ascites tumour
    同族专利:
    公开号 | 公开日
    KR100521813B1|2005-10-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2003-02-22|Application filed by 주식회사 보성
    2003-02-22|Priority to KR10-2003-0011164A
    2003-03-26|Publication of KR20030024739A
    2005-10-14|Application granted
    2005-10-14|Publication of KR100521813B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2003-0011164A|KR100521813B1|2003-02-22|2003-02-22|The pharmaceutical composition and its preparation method of herb mixture for anticancer drug, immunological enhancement and curative for arteriosclerosis|
    [返回顶部]