KR20000007345A - Medicine of extract from long aged platycodon grandiflorum(korean name "doraji") - Google Patents

Abstract

PURPOSE: A medicine prepared by using an extract from aged Doraji or selectively adding another herb medicine or extract thereof to the extract, is provided for supplemental health food to promote the use of Doraji. CONSTITUTION: The extract for the medicine is obtained by a method in that "Doraji" roots of over 20 years age are dried and crushed to powder, to be heated in water or organic solvent at the temperature of 90-100°C for 4-6 hours to extract the essence twice in the process for filtration, the filtered extract being added with another herb medicine or its extract selectively. The prepared medicine is used to treat bacterial disease, inflammation and rheumatism.

Description

Pharmaceuticals made with Jangsaeng Bellflower Extract

Field of invention

The present invention relates to various pharmaceuticals and health supplements based on the various pharmacological effects of Jangsaeng Doraji, produced over 20 years of age, according to the "Perennial Bellflower Cultivation Method" of Korean Patent No. 45791.

Background of the Invention

Bellflower (Gilkyung Platycodon grandiflorum A. DC) is a herbal medicine that has been used for respiratory diseases such as drainage, expectoration, bronchitis, asthma and anti-inflammation, and herbal medicines containing bellflower are 273 in consent and 49 in chemotherapy. It is known to have a variety of pharmacological action.

The major pharmacological component of bellflower is terpene-based saponins (prestigotin A, C, D, D ₆ , polygalacin D, D ₂ and 18 species), accounting for about 2% of the root. In animal experiments, terpene-based saponins are hemolytic, localized, antitussive, expectorant, central nervous inhibitory (sedative, analgesic, antipyretic), anti-inflammatory against acute and chronic inflammation, anti-allergic, anti-ulcer and gastric secretion , Anticholinergic to lower blood pressure by expanding blood vessels, lowering blood sugar, improving cholesterol metabolism [Igarashi, K., Sogoigaku, 3, 1 (1951) and Lee Eun-bang, Journal of Pharmacognosy, 5, 49 (1974 )]. In addition, steroidal compounds such as alpha-spinasterol, stigmas-7-enol, alpha-spinasterol glucoside and the like and polysaccharides such as inulin, platicodidine and betulin were also isolated.

In addition, the hot water and ethanol extracts of the bellflower inhibit the fungal apratoxin [Hitokoto, H., Morozumi, S., Wake, T., Sakai, S. and Ueno, I., Mycopathologia, 66, 161 (1979) ], The inulin fraction has phagocytosis [Nagao, T., Matsuda, H., Nambo, K. and Kubo, M., Shoyakugaku Zasshi, 40, 367 (1986)], the inulin, a fructose polymer, is a solid cancer It is known to have antitumor efficacy against ascites and ascites (Naga, T., Matsuda, H., Makata, K., Namba and Kubo, M., Shoyakugaku Zasshi, 40, 375 (1986)).

According to the invention utilizing the pharmacological properties of the bellflower, Japanese Patent Laid-Open No. 60-89427 discloses that the inulin fraction in 70% methanol extract of bellflower has 31-39% and 32-44, respectively, inhibitory effects against ascites and solid cancers in ICR mice. In combination with the chemotherapeutic drug mitomycin, it is shown that not only can suppress solid cancer by 61% but also suppress the side effects of mitomycin, such as weight loss and white blood cell count. Due to the excellent anti-tumor activity of the bellflower, Japanese Patent Application Laid-Open No. 2-172921 was administered to Enrlich ascites cancer cell lines using inulin obtained from tissue culture of bellflower to improve many difficulties in separating inulin from bellflower. Disclosed are the effects of the invention that result in inhibition rates and life-expansion rates of 140-145%. Japanese Patent Laid-Open No. 3-264534 discloses a 40% long-light extract as an alcohol absorption inhibitor.

Currently, about 30 kinds of herbal medicines containing bellflower are sold in Korea, China, Japan, etc., and five kinds of herbal medicines such as five-Yam Soon Ki-san, Tong-hwan, and Soon-gi-won are treatments for stroke, arthritis, etc. As a therapeutic agent for, Jamomile extract is used as a therapeutic agent for bronchitis, pulmonary tuberculosis, pleurisy and the like, and manokdan is used as a therapeutic agent for hypertension, arteriosclerosis and diabetes, respectively. Other herbal preparations containing bellflower are used for bronchial diseases such as asthma, expectoration, drainage, and Jinhae.

However, the bellflowers, which have been used in the herbal medicine and experiments until now, were grown for two to four years because the roots decayed when grown for more than four to five years. More than 20 years of perennial Jang Saeng Dora produced by the 'perennial bellflower cultivation method' recently patented No. 45791 is a patent protection herbal medicine is not used as a herbal medicine for sale at all.

Therefore, the present inventors have developed the use as a medicinal supplement such as health supplements and herbal medicines by utilizing the fact that Jangsaeng Bellflower is significantly superior in pharmacological action compared to the common bellflower of 2-4 years old.

An object of the present invention is to provide the use as a herbal medicine of Jang Saeng Doraji having excellent pharmacological action and physiological activity compared to the common bellflower of 2-4 years old.

Another object of the present invention is to provide an antimicrobial agent using Jangsaeng Bellflower Extract.

Still another object of the present invention is to provide an anti-inflammatory therapeutic agent using Jangsaeng Bellflower Extract.

It is another object of the present invention to provide an herbal preparation that is nontoxic in vivo and has no other side effects.

The above and other objects of the present invention can be achieved by the present invention described below.

Figures 1a-1g shows the binding and cell types of cancer cells to laminin when treated with B16-F1 mouse melanoma cell lines at concentrations of 0.031, 0.062, 0.125, 0.25, 0.5 and 1 mg / ml It is a photograph observed with a microscope.

The present invention relates to the use of the extract prepared by preparing Jangsaeng Dorage as a powder and then extracting it using hot water extraction or an organic solvent as an antimicrobial agent, hyperlipidemia treatment agent, diabetes treatment agent, cancer treatment agent or inflammatory treatment agent. In the following, the difference between the constituents and pharmacological action of the common bellflower and Jangsaeng bellflower will be examined in detail.

Common Bellflower and Jangsaeng Bellflower have no big difference in moisture, crude fat, mineral salt, etc. among common ingredients. The total amount of dietary fiber and the amount of irradiated phononine were detected as high as 3.1% and 0.4% in 22 years old, respectively. The total content of inulin in the constituent sugar components of the bellflower was almost unchanged from 70 to 73 mg / g, but there was a big difference in the degree of polymerization of fructose, the constituent sugar component of inulin. I.e. the higher the content of the Jang Saeng Doraji GF ₂ and GF ₃ shows the content of the general bellflower GF ₈ relatively higher in GF ₅ has been found that if asked a significant difference in the structure of inulin. In the above, G is glucose, F is fructose, which represents a component of inulin, and the number represents the number of fructose. As such, the fructose degree of fructose is detected with higher oligofractose with a lower fructose as the number of years of cultivation increases. These results are inferred to be related to the results of clinical trials that Jangsaeng Bellflower is more effective in hypertension, bronchus, diabetes, etc. than the common bellflower.

As inorganic salts, the contents of calcium (Ca), magnesium (Mg), and copper (Cu) are detected 3 to 10 times higher in Jangsaeng Bellflower than the common bellflower.

General and Jangsaeng bellflowers were extracted with 80% ethanol and 30% methanol, and analyzed by HPLC. The peaks, which are present only in Jangsaeng Doraji, were divided into solvents according to their polarity, and their physiological activities were examined. The effects were recognized in the petroleum ether and diethyl ether fractions. [Hashibong, Master Thesis, Gyeongsang National University (1996)].

Jangsaeng Doraji not only has a significant difference in the composition and composition, but also in pharmacological effects such as antibacterial activity, fat metabolism improvement, cancer cell adhesion inhibitory activity, immunopotentiation, hypoglycemic action, hypoglycemic action, etc. Excellent

80% ethanol and 30% methanol extract of Jangsaeng Doraji has free radical scavenging and antifungal activity and no cytotoxicity. Jangsaeng Bellflower shows broader and superior antibacterial spectrum than the common bellflower. Ethanol extract of Jangsaeng Doraji has a minimum inhibitory concentration (MIC) value of 5 mg / ml for bacteria and 2.5-10 mg / ml for fungi. Antimicrobial actives are most easily extracted at 50 ° C. and 95% ethanol concentration. When the 95% ethanol extract of Jangsaeng Doraji was again fractionated with solvent, the butanol fraction showed the highest antimicrobial activity, which is believed to be due to the saponin component, and the activity was detected in other solvent fractions. It is inferred to be abnormal. Among the antimicrobial active substances of Jangsaeng Doraji, it was isolated from ethyl acetate fraction and analyzed by structural analysis using several instruments. It was identified as glyceryl linoleate.

In addition, the blood glucose and lipid content of serum and liver are significantly reduced when dieting Jangsaeng Dorage powder or extract in hyperlipidemic rats. In particular, the 5% diet of Jangsaeng Doraji powder significantly reduced the total cholesterol and triglyceride levels in serum and liver, and the atherosclerosis index was also lower than that of the control group treated with Jangsaeng Doraji. These results suggest the possibility of prescribing Jangsaeng Doraji in the prevention and treatment of hypercholesterolemia or hyperlipidemia.

Jangsaeng bellflower extract of the present invention is effective in the treatment of diabetes mellitus is superior to the general bellflower lipids, such as blood sugar, serum, liver. Jangsaeng Doraji extract acts to significantly reduce serum total cholesterol and LDL levels in the body and increase serum HDL-cholesterol levels. Serum free cholesterol, cholesterol esters, triglycerides and phospholipids are also reduced to low levels and atherosclerosis is low.

When Jangsaeng Doraji extract is treated to cancer cell lines, the cancer cell adhesion inhibitory activity is excellent. When Jangsaeng Doraji methanol extract was treated to B16-F1 mouse melanoma cells to measure the binding capacity of laminin (laminin) when the concentration of the extract is more than 0.125 mg / ml will show 100% cancer cell adhesion inhibitory activity in cancer cell lines. It can be seen that Jangsaeng Doraji extract can inhibit cancer metastasis even at low concentrations.

In addition, the extract of Jangsaeng Doraji extract according to the polarity of the solvent to investigate the immune enhancing function shows the best effect in the aqueous layer. HPLC analysis of this aqueous layer showed the best immunostimulating activity in the inulin fraction of 20 inulin fructose polymerization. It is estimated that Jangsaeng Doraji correlates with inulin's fructose polymerization more than 5 times higher than the general bellflower.

Jangsaeng Doraji extract can be used for the treatment of inflammatory diseases such as rheumatoid arthritis because it has the ability to inhibit edema. It shows edema inhibitory activity similar to that of predisolone, an existing edema inhibitor. Incorporation of an acceptable herbal medicine or herbal extract that can enhance the efficacy of Jangsaeng Dorage will increase the inhibitory activity of edema due to synergy.

The present invention provides health foods and drugs using the excellent pharmacological activity of Jangsaeng Bellflower. Extracts that can be provided as health foods or drugs using Jang Saeng Doraji of the present invention are treated according to methods commonly used in the art. Jangsaeng Doraji extract can usually include hot water extract or organic solvent extract, these extracts may be used alone or may be prepared by adding a herbal medicine.

Generally, Jangsaeng Bellflower powder is prepared by drying Jangsaeng Bellflower so as to have a moisture content of 5% or less, pulverizing it, and then selecting a particle size of 0.6mm or smaller. The hot water extract is prepared by adding about 5-10 times of water to Jangsaeng Bellflower powder and selectively adding an acceptable herbal or herbal extract to the filtrate obtained by extracting twice for 4-6 hours at a temperature of 90-100 ° C. . In the case of the organic solvent extract, about three times the organic solvent is added to Jangsaeng Bellflower powder, and the filtrate obtained by extracting twice at room temperature is concentrated under reduced pressure to be prepared by selectively adding an acceptable herbal or herbal extract to the extract.

Alcohol may be used as the organic solvent, and methanol, ethanol, or the like may be preferably used. Acceptable herbal medicines include creation, mountain lions, gardens, sewage, baekbokyeong, and casualties.

Jangsaeng Doraji extracts of the present invention may be formulated or formulated as a dietary supplement by known methods using food hygiene or pharmacologically acceptable prostheses and pressurized into unit dosage forms or multi-dose containers. The dietary supplement or formulation may be in the form of an aqueous solution, suspension or emulsion, or may be in the form of extracts, powders, granules, tablets, capsules, and may contain conventional dispersants, suspensions or stabilizers.

Pharmacological effects, such as antimicrobial activity, fat metabolism improvement, hypoglycemic activity, cancer cell adhesion inhibitory activity, immune enhancing action, anti-arthritis activity, etc. of Jangsaeng Doraji extract prepared by the above method or other acceptable herbal medicines Experiments for are shown in the following examples.

The present invention will be embodied by the following examples, which are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example

Example 1 Preparation of Jangsaeng Bellflower Extract Herbal Medicine

Jangsaeng bellflower is dried and pulverized to 5% or less of water content, and then the particle size is selected to be 0.6mm or less to prepare a powder. 10 times of water was added to Jangsaeng Bellflower powder and extracted twice at 4-100 hours at 90-100 ° C. to prepare a liquid product. In addition, the filtrate was further concentrated to prepare an extract, powder, granules, capsules and cyclic herbal medicines.

The ethanol extract was ground 1kg of Jangsaeng Bellflower, extracted twice with 4L of ethanol at room temperature for 2 days, and the filtrate was collected and concentrated under reduced pressure to obtain 125g of extract. The extract was used to prepare extracts, powders, granules, capsules and cyclic herbal medicines.

The extract of Jangsaeng Doraji may be prepared by adding an acceptable herbal medicine such as creation, mountain lion, raw paper, sewage, baekbokyeong, casualty and the like, or extract and concentrated herbal medicine extract as described above to increase the efficacy.

Example 2 Antibacterial Activity Test of Jangsaeng Doji

The ethanol extract of Jangsaeng Doraji was tested for antimicrobial activity by measuring the minimum inhibitory concentration (MIC) of 14 kinds of food decay (food poisoning) microorganisms and pathogenic microorganisms. Using a 96-well plate, the absorbance of each strain was adjusted to 0.5 at 660 nm, and then the bellflower extract was added to the medium according to the concentration, and the growth rate was measured by culturing at 37 ° C. for 24 hours. Are listed in Table 1. Muller Hinton medium was used for bacteria and malt extraction medium for other fungi.

Strain 4 year old bellflower minimum inhibitory concentration (mg / ml) Jangsaeng Bellflower Minimum Inhibitory Concentration (mg / ml) Broth Agar Broth Agar B. subtilis M. luteusS. epidermidis E. coliK. pneumoniaeP. aeroginosaS. typhiV. parahaemolyticus C. albicansS. cerevisiae A. flavusA. nigerM. audouiniiT. mentagophytes 20.0020.0040.0080.0080.00160.0020.0040.00160.0040.00160.0080.0080.0080.00 10.0010.0020.0040.0040.0040.0010.0020.0020.0020.0040.00160.0040.0080.00 5.005.0010.0010.0010.0020.002.505.0020.0010.0020.0010.0010.0010.00 2.502.505.005.005.005.002.505.005.005.005.005.005.0010.00

Example 3:

Effect of Feeding Jangsaeng Doraji on Serum and Hepatic Lipid Metabolism in Hyperlipidemic Rats

Sprague Dawley rats weighing 70 ± 5 g and 4 weeks old were fed a 20% casein-containing diet containing 20% lipid and 0.25% sodium cholate for 2 weeks to induce hyperlipidemia. Six animals were divided into three groups, A, B, and C, for three weeks. Group A was fed only general feed (control), group B was fed a mixture of 5% common bellflower powder, and group C was fed a mixture of 5% Jangsaeng Bellflower powder. To compare the effects, normal diets were fed to 6 normal rats (group D) weighing 70 ± 5 g and 4 weeks old. Jangsaeng and Bellflower used in the experiment were lyophilized and powdered to less than 0.59mm, and then mixed and fed with conventional feed. The weight gain (food intake) as a result of the three-week spike was shown in Table 2. On the last day of 3 weeks of experiment breeding, after choking for 7 hours, blood was collected by cardiac bleeding under ether anesthesia. Soy sauce was perfused with 0.9% physiological saline through the portal vein, and then drained by filter paper. After standing, centrifuged at 3,000 rpm for 15 minutes to obtain a serum. Blood glucose, cholesterol, triglyceride (triglyceride), phospholipid, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaninase (GPT) and lactate dehydrogenase (LDH) in serum and liver were measured, and the results are shown in Table 2. These blood glucose, cholesterol, lipids, and enzyme activities were measured using a kit kit for measurement commonly used in the art.

ingredient Experimental group A group B group C group D group Weight gain (g / 3 weeks) 352 ± 6.1 330 ± 2.8 329 ± 5.2 346 ± 5.2 serum Blood sugar (mg / dl) 158 ± 5.5 156 ± 5.5 141 ± 1.4 142 ± 3.8 Total cholesterol (mg / dl) 262 ± 6.4 243 ± 2.3 166 ± 3.8 168 ± 6.0 LDL-cholesterol (mg / dl) 137 ± 4.3 124 ± 5.0 74.5 ± 3.0 94.4 ± 2.8 HDL-cholesterol (mg / dl) 62.7 ± 2.5 61.4 ± 2.5 65.9 ± 2.6 52.5 ± 2.3 Free cholesterol (mg / dl) 45.0 ± 0.6 44.2 ± 5.3 37.2 ± 1.5 31.8 ± 0.9 Triglycerides (mg / dl) 270 ± 12.0 256 ± 4.4 129 ± 5.0 174 ± 5.3 Phospholipids (mg / dl) 188 ± 3.3 171 ± 3.8 118 ± 5.4 160 ± 4.7 Arteriosclerosis index 3.2 ± 0.3 2.8 ± 0.2 1.5 ± 0.1 2.2 ± 0.2 GPT (Karmen unit / ml) 45.7 ± 2.1 40.2 ± 2.5 29.5 ± 5.1 35.6 ± 8.3 GOT (Karmen unit / ml) 90.1 ± 2.5 89.3 ± 3.1 80.7 ± 1.3 82.5 ± 3.3 LDH (Wroblewski unit / ml) 1,067 ± 23.7 1,347 ± 51.2 1,329 ± 53.5 1,474 ± 40.6 Soy sauce Total cholesterol (mg / g) 17.8 ± 2.8 16.1 ± 2.8 12.4 ± 3.2 8.0 ± 1.7 Free cholesterol (mg / g) 4.8 ± 0.4 5.4 ± 2.1 5.1 ± 0.4 4.0 ± 0.6 Triglycerides (mg / g) 88.1 ± 2.1 78 ± 3.5 46.0 ± 3.5 73.5 ± 2.3 Phospholipids (mg / g) 25.1 ± 4.1 25.0 ± 1.4 22.4 ± 3.0 23.7 ± 3.5

A group: Induce hyperlipidemia and feed group

Group B: Feed feed group containing 5% common bellflower powder

Group C: Feed feed group containing 5% Jangsaeng Bellflower powder

Group D: group not causing hyperlipidemia

Total cholesterol and triglyceride levels in serum and liver were significantly lower in the group that induce hyperlipidemia and dieted Jangsaeng Doraji powder (Group C) than the control group (Group A) or the group that fed General Bellflower Powder (group B). The figure was close to the normal group. Atherosclerosis index was lower than rats that did not cause hyperlipidemia. Therefore, it can be seen that Jangsaeng Doraji powder is effective in preventing and treating hyperlipidemia.

Example 4:

Effect of Jangsaeng Doraji Extract on Blood Glucose and Lipid Components in Diabetic Rats

Four-week-old Sprague Dawley rats weighed 70 ± 5 g were treated with I.P. Experimental animals were divided into three groups, A, B, and C, six by the egg mass method, and freely ingested according to the diet of each experimental group for three weeks. Group A was fed only general feed (control), group B was fed feed mixed with 6 years old bellflower extract, and group C was fed feed mixed with Jangsaeng Bellflower (22 years old) extract. To compare the effects, normal diets were fed to 6 normal rats (group D) weighing 70 ± 5 g and 4 weeks old. The bellflower extract used in the experiment was prepared by adding water to 200 g of lyophilized bellflower, making it 1L and concentrating to 700ml. Then, 800ml of water was added to the residue, followed by hot water extraction until it became 300ml. On the last day of 3 weeks of experiment breeding, after choking for 7 hours, blood was collected by cardiac bleeding under ether anesthesia. The liver was perfused with 0.9% physiological saline through the portal vein, and then drained by filter paper. After standing, centrifuged at 3,000 rpm for 15 minutes to obtain a serum. Blood glucose, cholesterol, triglyceride (triglyceride), phospholipid, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in serum and liver were measured, and the results are shown in Table 3. These blood glucose, cholesterol, lipids, and enzyme activity were measured using a kit kit for measurement commonly used in the art.

ingredient Experimental group A group B group Group C D group serum Weight gain (g / 3 weeks) 30 ± 1.4 338 ± 5.1 328 ± 4.5 352 ± 4.3 Blood sugar (mg / dl) 306.8 ± 19.7 271.6 ± 13.2 227.5 ± 14.1 162.4 ± 9.4 Total cholesterol (mg / dl) 127.4 ± 5.1 104.7 ± 4.5 95.9 ± 3.9 86.5 ± 3.3 LDL-cholesterol (mg / dl) 203.8 ± 12.8 181.6 ± 13.1 163.3 ± 12.7 140.5 ± 9.6 HDL-cholesterol (mg / dl) 16.7 ± 1.6 17.3 ± 1.8 19.0 ± 1.5 23.3 ± 1.3 Arteriosclerosis index 6.6 5.1 4.0 2.7 Free cholesterol (mg / dl) 20.0 ± 0.8 15.0 ± 0.9 14.6 ± 0.7 12.9 ± 0.5 Cholesterol ester (mg / dl) ¹⁾ 107.4 ± 3.2 89.7 ± 3.4 81.3 ± 2.9 73.6 ± 2.5 Cholesterol ester ratio (%) ²⁾ 84.3 85.6 84.8 85.1 Triglycerides (mg / dl) 104.6 ± 3.4 92.5 ± 3.0 86.1 ± 2.9 75.3 ± 2.6 Phospholipids (mg / dl) 148.9 ± 5.2 136.5 ± 3.8 129.4 ± 4.2 115.2 ± 4.7 Soy sauce Total cholesterol (mg / g) 8.4 ± 0.3 7.3 ± 0.3 6.4 ± 0.2 5.1 ± 0.2 Triglycerides (mg / g) 41.5 ± 2.2 35.2 ± 2.1 32.4 ± 2.2 28.7 ± 1.4 Phospholipids (mg / g) 20.8 ± 1.0 18.2 ± 1.1 17.5 ± 1.2 12.6 ± 0.7

1) Cholesterol Ester = Total Cholesterol-Free Cholesterol

2) Cholesterol ester ratio = cholesterol ester / total cholesterol * 100

Group A: Diabetic group induced by alloxane and not treated with bellflower

Group B: Group treated with 6 years old Bellflower Hot Water Extract

C group: 22 years old

Group D: Group not treated with Aloxane

As shown in Table 3, the blood glucose concentration was significantly higher in the oxalose group (A, B and C group) than the D group which did not receive the oxalate, but was treated with the bellflower extract compared to the control group A group. And C group were low, especially C group treated with Jangsaeng Doraji extract.

Serum total cholesterol and LDL concentrations were lower in group B and C than group A. Among them, group C was lower than group D, which was not treated with alloxan. Serum HDL-cholesterol concentration was higher in the bellflower extract group (B and C) than in group A. Among these groups, group C had higher values, which was closest to group D without administration of alloxan. Atherosclerosis index was lower in group B and C than group A. Serum free cholesterol, cholesterol esters, triglycerides and phospholipids were significantly lower in all experimental groups than in group A. The contents of total cholesterol, triglyceride and phospholipids in liver were significantly lower in all experimental groups than in group A. Serum AST, ALT and LDH enzyme activities were highest in group A and relatively low in other experimental groups. From the above results, the bellflower extract was found to be effective in improving lipids of blood sugar, serum and liver, and especially the Jangsaeng Bellflower extract was more effective.

Example 5 Cancer Cell Adhesion Inhibition Activity of Jangsaeng Doraji

The cancer cell adhesion inhibitory activity of Jangsaeng Doraji was investigated by quantitatively measuring whether B16-F1 mouse melanoma cell line inhibited adhesion to laminin, a kind of extracellular matrix by treating with methanol extract of Jangsaeng Doraji. The maintenance of the melanoma cell line was maintained in a minimum essential medium (containing minimum essential medium (MEM), Hank's salt) containing fetal calf serum (FCS (fetal calf serum, 10%)). Laminin-coated plates were dissolved in 2 ml of PBS (phosphate buffer saline, NaCl 8 g, KCl 0.2 g, NaHPO ₄ 1.44 g, KH ₂ PO ₄ 0.24 g / L) and stored in a refrigerator. The concentration (20 μg / ml) was determined and diluted with cold PBS solution, and the solution was used by injecting 50 μl into each well of a 96-well microplate and drying overnight.

Preparation of cell suspension was performed by removing 80% confluence grown B16-F1 cells into 3 ml of 2 mM EDTA solution in a T-25 culture flask and adding 5 ml of serum-free MEM for 5 minutes at 800 rpm. The cells obtained by centrifugation were suspended in an appropriate amount of serum-free MEM containing 1% BSA, adjusted to a cell number of 2 × 10 ⁶ cells / ml, and 50 μl of this suspension was used.

Determination of activity was performed three times by washing the laminin-coated wells with 100 μl of PBS and again adding 100 μl of 1% BSA solution (dissolved in PBS) and removing BSA under reduced pressure. 50 μl of serum-free MEM containing 0.1% BSA, samples and cells were added to the plate and incubated for 1 hour at 37 ° C. in the presence of 5% CO ₂ . After incubation, the cells were washed twice with PBS (37 ° C.), and then washed with 100 μl of 0.5% BSA (dissolved in PBS) and 100 μl of PBS, respectively. Immobilized for minutes.

The results of microscopic observation of the binding forms and cell types of cancer cells to laminin when treated at concentrations of 0.031, 0.062, 0.125, 0.25, 0.5 and 1 mg / ml for Jang Saeng Doraji methanol extracts are shown in FIGS. 1A to 1G. It was. As shown in FIG. 1, when DMSO was used as a vehicle, cancer cells were attached to laminin. In the case of methanol extract concentrations of 0.031 and 0.062 mg / ml, cells of about 30% and 70% were attached, respectively. When the concentration of the methanol extract was 0.125 mg / ml or more, 100% of cells did not adhere to laminin and maintained the globular cell morphology. At the concentration of 0.5 mg / ml, 20-30% of the cells were damaged by the cell membrane, but the remaining 70% -80% of the cells did not adhere to laminin and maintained the globular cell morphology. Breakage was observed.

Drawing Methanol extract concentration (mg / ml) Relative optical density at 570 nm Laminin Adhesion Inhibition Rate (%) a Control 1.00 0 b 0.031 0.71 30 c 0.062 0.22 70 d 0.125 0.00 100 e 0.250 0.00 100 f 0.500 0.00 100 g 1.000 0.00 100

Example 6 Immune Enhancement of Jangsaeng Bellflower

Mice transplanted with ascites and solid cancers were bred for three weeks by dividing into groups A (control), B (general bellflower), and C (group). The proportion of immune cells, such as NK-cell, T-cell, B-cell, and Tc-cell, in the blood of the mice belonging to each group was investigated using FAC Scan. In the case of ascites cancer, as shown in Table 5, NK-cells showing direct anticancer effects were 1.14% in group A and only 1.52% in group B, but 6.23% in group C administered Jangsaeng Bellflower. It was about 5.5 times higher. In addition, B-cell also appeared higher in group C than group A or B, and T-cell was slightly lower in group C, but rather Tc-cell showed higher anticancer effect.

In solid cancer, as shown in Table 6, NK-cell was 3.6% in group A, 3.8% in group B, and 4.55% in group C administered Jangsaeng Doraji. B-cell was lower than the control group, but T-cell and Tc-cell showed higher anti-cancer effects from those higher than the control.

Immune Activity of ascites Cancer in Jangsaeng Doraji Treatment group % positive cells B cell (CD19) T cell (CD3e) Tc cell (CD8a) NK cell (%) A 17.93 71.10 18.87 1.14 B 18.21 58.24 18.52 6.23 C 23.04 62.88 23.37 6.23

Immune Activity against Solid Cancer of Jangsaeng Doraji Treatment group % positive cells B cell (CD19) T cell (CD3e) Tc cell (CD8a) NK cell (%) A 43.85 49.77 12.97 3.6 B 34.18 49.82 12.32 3.80 C 36.92 53.19 13.10 4.55

Example 7:

Effects of Jangsaeng Bellflower Extract and Bellflower Herb Herbal Medicine on the Efficacy of Rheumatoid Arthritis

About 20 days after injecting 0.6 mg / kg of heat killed Mycobacterium histolyticm into the right foot of SD-rat, the degree of edema of inflammation was measured by measuring the edema size of both feet. A diet group that did not cause edema as a control group (group A), a predisolone as a edema inhibitor group (group B), and a group of normal ridge extracts (160 mg / day) Group (C group), 14 kinds of herbal extracts were fed in a dose of 20 mg / day (Group D), and Jangsaeng Bellflower extract was fed in an amount of 160 mg / day (Group E) , And herbal extracts (40 mg / day) and Jangsaeng Doraji extract (160mg / day) were divided into two groups (F group) and investigated. Each group included 6 SD-rats. The results are shown in Table 7.

Experimental group Edema inhibition activity of the right foot (%) Edema inhibition activity of left foot (%) A 100 100 B 84.5 83.8 C 12.6 13.2 D 26.7 26.2 E 67.5 68.3 F 85.8 85.0

Inhibition of edema was observed in the Jangsaeng Bellflower Extract fed group (Group E) and the herbal medicine and Jangsaeng Bellflower Extract mixed group (F group). However, increasing the feed amount of Jangsaeng Doraji Extract to 200mg / kg showed no significant activity, so it was most active to use in the range of about 100 ~ 200 mg / kg. The edema inhibition activity according to the feeding amount showed the strongest activity at the 150 mg / kg dose.

Herbal preparations made of Jangsaeng Doraji extract of more than 20 years of the present invention is excellent in pharmacological action and physiological activity compared to the general bellflower of 2-4 years old, and excellent antimicrobial activity against bacteria or fungi. In addition, the anti-inflammatory therapeutic agent prepared with Jang Saeng Doraji extract has an excellent effect of inhibiting edema and has an excellent effect of treating inflammatory diseases such as rheumatoid arthritis. The antimicrobial or anti-inflammatory therapeutic agents of the present invention also have the effect of the invention without toxicity and other side effects in vivo.

Simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Claims (5)

Jangsaeng Bellflower (20 years old or more perennial Bellflower) was dried and pulverized to prepare Jangsaeng Bellflower Powder; Water was added to the Jangsaeng Bellflower Powder and extracted by filtration twice for 4 to 6 hours at a temperature of 90 to 100 ° C; And Selectively adding an acceptable herbal medicine or herbal extract to the filtrate; Jangsaeng Bellflower hot water extract, characterized in that the step is prepared. Jangsaeng Bellflower (20 years old or more perennial Bellflower) was dried and pulverized to prepare Jangsaeng Bellflower Powder; About 3 times the organic solvent was added to the Jangsaeng Bellflower Powder, and extracted twice at room temperature and filtered; Concentrating the filtrate under reduced pressure to prepare an extract; And Selectively adding an acceptable herbal medicine or herbal medicine extract to the extract; Organic solvent extract of Jangsaeng Bellflower, which is prepared in a step. An antimicrobial agent, characterized in that it is prepared from Jangsaeng Bellflower Extract according to claim 1. Inflammatory disease therapeutic agent, characterized in that the preparation of Jangsaeng Bellflower extract according to claim 1 or 2. The agent for treating inflammatory diseases according to claim 4, wherein the inflammatory disease is rheumatoid arthritis.