KR101923781B1 - The preventing prostate cancer composition containing high platycodin aged Doraji extract methods - Google Patents

Abstract

The present invention relates to a method for producing an aged blackflower extract containing platycodin, which is a kind of saponin contained in platycodon, which is excellent in the prevention of prostate cancer, and more specifically, , The yeast is cultured in a culture medium after three kinds of mixed lactic acid bacteria are cultured. Then, the extract is concentrated to prepare a health food for prevention of prostate cancer containing an aged black platycodone extract containing high purity platycodine through an ion exchange resin column .
According to the present invention, the aged Blackberry extract having high purity of platycodin A and D can be effectively used for the treatment and prevention of prostate cancer because it has an excellent effect of inhibiting the growth of prostate cancer cells and inducing apoptosis.

Description

[0001] The present invention relates to a method for preparing a platycodin-containing platycodin,

The present invention relates to a method for producing an aged blackberry extract having high purity of platycodin, which is a kind of saponin contained in platycodon, which is excellent in the effect of preventing prostate cancer.

Recently, the food industry is rapidly changing with the fusion of various BT technologies based on the food material processing technology, and going to the environment-friendly LOHAS trend that can maintain the health more than the existing well-being concept one by one . In addition, chronic or intractable diseases such as cancer, heart disease, diabetes and arthritis are increasing due to the improvement of the quality of life and the development of medical technology, so that the food industry has high added value for prevention and improvement of various diseases Food materials and functional foods are attracting much attention.

Over the past decade, cancer has been the leading cause of mortality worldwide, and continues to grow despite continued medical advances. Prostate cancer is one of the most commonly occurring urological tumors worldwide, appear. In Korea, the number of prostate cancer patients has increased by 21 times in 20 years, which is recognized as one of the most common diseases among modern people. Most of the currently marketed prostate cancer medicines are synthetic medicines. These medicines have various side effects such as tolerance for long-term administration. Therefore, recent research directions are to use natural resources derived from plants and microorganisms which are guaranteed to be safe It is required to develop a new therapeutic agent derived from natural products which does not affect normal cells by specifically acting only on cancer cells while reducing side effects.

The cancer treatment induces apoptosis of cancer cells and inhibits the development and progression of cancer. The prostate gland causes defects in signal transduction involved in cell apoptosis, leading to unbalance of cell proliferation and apoptosis, leading to tumor development and metastasis . The general pathway of apoptosis is mediated by caspase enzyme activity, and caspases are activated by various cell death signals. Caspase-3 is activated by caspase-9 as an activator of apoptosis, and then it is damaged by inactivating poly (ADP-ribose) polymerase (PARP) Induce apoptosis. As caspase-3 plays a key role as a final executor in cell apoptosis-related signal transduction pathways, caspase-3 is a major target protein and effective material for prostate cancer cell death has been discovered.

While studying the physiological activity of natural products without cytotoxicity, the present invention has been applied to a variety of medicines including bradykinin extracts such as inhibition of lung cancer, antibacterial, anti-inflammatory and alcoholic liver diseases, A composition for prevention and treatment of prostate cancer is disclosed. Patent Publication No. 0830236 discloses a composition for prevention and treatment of prostate cancer, which contains a bellflower extract. In the patent publication No. 2012-0117197, a composition having an immunological activity and an anticancer activity effect is disclosed However, in the case of the above-described known techniques, in the case of the platycodon grandiflorum extract or fraction, there is no significant difference in that a solvent having a different polarity is sequentially used as an extraction solvent in order to extract an active ingredient from a natural product.

Accordingly, the present invention relates to a method for producing a prostate cancer cell line LNCap, which is obtained by harvesting platycodon (Rhodophyta) of 5 years or more over a period of 5 years or more, and platycodin- Dependent activation of caspase-3, and it also shows the action of enhancing caspase-3 activity, which is a core executor of cell apoptosis. Thus, the present invention has been completed.

The present invention has been made to develop a material effective for the self-destruction of prostate cancer cells derived from natural products without side effects, and as a result, it has been found that, when a platycodon (platycodon) of 5 years or more is harvested and cultured at a constant temperature, , The present inventors have completed the present invention by confirming the excellent effect of aged blackberry extract having high purity of platycodin in inducing magnetic cell apoptosis of prostate cancer cell line and enhancing caspase-3 activity as a key executor of cell apoptosis .

Accordingly, an object of the present invention is to provide a method for producing a high-purity platycodin-containing aged blackrox extract which is excellent in preventing prostate cancer.

In order to accomplish the above object, the present invention provides a method for preparing an aged blackrox extract having high purity platycodin which is excellent in preventing prostate cancer.

Hereinafter, the present invention will be described in detail.

Platycodon grandiflorum is a perennial herb that belongs to Campanulaceae. Its roots are used as traditional herbal medicines or foods. It is known to have various pharmacological actions such as gadolin, vinegar, fever and analgesia. Other known pharmacological effects include anti-inflammatory, hypotensive, hemolytic, and central inhibitory effects.

In particular, it has been reported that active substances such as saponin such as triterpene are contained according to a study report on the immunological effect on the bellflower, and reported ingredients include platycodigen (glycyrrhizin), platycodigenin Platycodin) A, C, D, and 10 kinds of triterpene saponins have been reported to contain trace amounts.

Accordingly, the present inventors have succeeded in obtaining a platycodin ingredient having high pharmacological activity over a 5-year-old muscle or more and exhibiting various pharmacological activities contained in the platycodon, (a) ; (b) extracting the aged blackflower with any one solvent selected from the group consisting of a mixture of water and water; (C) Lactobacillus plantarum , Lactobacillus acidophilus , and Pediococcus acidilactici , which are three kinds of three kinds of kimchi-derived lactic acid bacteria, %, Cultured at 35 to 37 ° C for 48 hours, inoculated with Saccharomyces cerevisiae yeast isolated from makgeolli, and cultured at 30 to 35 ° C for 48 hours. (d) concentrating the cultured extract; (e) adsorbing the concentrate on a column filled with an ion exchange resin, and then purifying the material adsorbed on the column by eluting with a solvent.

The food composition for preventing and treating prostate cancer of the present invention has an effect of inducing an effective cell death by increasing caspase-3 activity which is a main executor of cell death. The food composition of the present invention may further contain another herb medicine or other extract which is pharmacologically acceptable to enhance its effect. In this case, the herbal medicine extract extracted by the above method may be added, or the other herbal medicines may be mixed and then extracted by the above-mentioned method to be contained in the food.

As described above, the present invention provides a method for producing an extract containing platycodin of high purity in a bellflower and a high-purity platycodine-containing maturation black rot flower extract prepared by the method. The extract of the present invention contains platycodin of high purity and is excellent in the effect of prostate cancer.

FIG. 1 is a schematic view showing a process for producing an aged blackrox extract containing high-purity platocyte according to the present invention.
FIG. 2 is a result of LC / MS analysis of a high-purity platycodine-containing maturing black lotion extract purified through an aged blackrod berry extract prepared by the present invention and an ion exchange resin.
FIG. 3 shows the results of the concentration-dependent effect evaluation of the LNCap cell line on the apoptosis of the aged Blackflower extract according to the present invention.
FIG. 4 is a time-dependent effect evaluation result on the cell death of the LNCap cell line of the aged Blackflower extract according to the present invention.
FIG. 5 shows the results of the evaluation of the effect on the caspase-3 expression of the LNCap cell line of the aged Blackflower extract according to the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> High purity Platycodine Aging Black Bellflower  Preparation of compositions containing extracts

The bellflower used in the present invention is obtained by harvesting (about) bellflower cultivated for 5 years or more in Gyeongbuk province, washing the bellflower at 90 ~ 95 ℃ for 2 hours, drying at 50 ~ 60 ℃ for 55 ~ 70 hours, And then dried at 50 to 65 ° C for 55 to 70 hours to prepare aged blackened porridge having a moisture content of 8 to 13%, and then 500% by weight of purified water was added thereto Heat was applied with stirring, and the mixture was extracted at 55 to 65 ° C for 5 hours. Lactobacillus plantarum , Lactobacillus acidophilus , and Pediococcus acidilactici , which are three kinds of lactic acid bacteria derived from kimchi, were inoculated at 1 to 10% After incubation for 48 hours at 37 ° C, Saccharomyces cerevisiae yeast isolated from makkolli was inoculated and cultured at 30-35 for 48 hours. Then, the extract was transferred through a transfer tube, To 100mesh. The mixture was concentrated at low temperature of 50 brix (bx) using an evaporator and adsorbed on a column filled with ion exchange resin. The material adsorbed on the column was purified by eluting with a solvent to obtain a resin The extracts were prepared with 5.6 - fold enhancement of the ticodin component. The resulting extract was concentrated under reduced pressure, and then lyophilized to obtain dried solid, which was powdered to complete the preparation of the aged blackberry extract having enhanced platycin component.

< Example  2> Agar-bellflower extract and high purity Platycodine  contain Aging Black Bellflower  Identification of cell killing effect of extract

<2-1> Cell culture

LNCap cell line of human prostate cancer was cultured in DMEM medium supplemented with heat-inactivated 10% fetal bovine serum, 100 units / penicillin, 100 g / streptomycin Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; and 5% CO2.

<2-2> Concentration-Dependent Cell Death Experiment

Cell Counting Kit-8 (CCK-8) assay was performed to determine the cell killing effect of the aged blackberry extract and the high-purity platycodine-containing aged blackberry extract. The samples were treated with LNCap prostate cancer cell lines in 96-well plates at 0, 10, 25, 50, 100 and 200 ug / ml for 24 hours. CCK-8 was added at a concentration of 10%, and reacted at 37 for 2 hours. Absorbance was measured at 450 nm using a microplate spectrophotometer (Biorad, Hercules, USA) and compared with the control group Relative cell viability (%) was calculated.

As a result, as shown in FIG. 3, it was confirmed that the aged blackberry extract and the high-purity pliticone-containing aged blackberry extract induced cell death in a concentration-dependent manner. As a result, it was found that the treatment of 25 ug / ml, 90 ug / ml treatment group, 82 ug / ml treated group and 75 ㎎% treatment group, respectively, Ml of platycodin containing high-purity platycodine was 86.84%, 79.04% at 25ug / ml, 68.38% at 100ug / ml and 51.03 / % Survival rate. In the case of the high purity platycodin containing aged blackberry extract, the highest concentration of 200 ug / ml treated group showed about 50% of the prostate cancer cell line killing effect.

<2-3> Time-dependent cell death experiments

Cell Counting Kit-8 (CCK-8) assay was performed to determine the cell killing effect of the aged blackberry extract and the high-purity platycodine-containing aged blackberry extract. LNCap prostate cancer cell line was treated with 200 ug / ml of the sample in a 96 well plate and cultured for 24, 48, 72 hours. CCK-8 was added at a concentration of 10% and reacted at 37 for 2 hours. Absorbance was measured at 450 nm using a microplate spectrophotometer (Biorad, Hercules, USA) and compared with the control group Relative cell viability (%) was calculated.

As a result, as shown in FIG. 4, it was confirmed that the aged blackberry extract and the high-purity platycodine-containing aged blackberry extract induce apoptosis in a time-dependent manner. As a result, the survival rate of the fermented black platycodon extracts was 80.2%, 71.16% and 72.33% in 24 hours, 72 hours and 67.33%, respectively. The survival rate was 64.04% in the 24 hours treatment group, 51.49% in the 48 hours treatment group and 44.59% in the 72 hours treatment group, confirming the time-dependent effect of prostate cancer cell line killing, In the case of Platycodon grandiflorum extract, the effect of prostate cancer cell line killing was found to be about 1.5 times higher under the same concentration conditions.

< Example  3> caspase -3 expression evaluation experiment

LNCap prostate cancer cell lines were treated with high purity platycodin-containing aged blackberry extracts at concentrations of 0, 10, 25, 50, 100 and 200 ug / ml and cultured for 24 hours. Cells were collected and lysed by adding lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM Nacl, 0.1% SDS, 1.0% Triton X-100, 0.25% Deoxycholate, 1 mM EDTA, 1 mM PMSF) Respectively. Cells were lysed and the obtained homogenate was centrifuged at 12,000 rpm for 15 minutes to obtain a protein layer. The expression level of Caspase-3 protein was measured using a western blot method. 50 g of protein was electrophoresed on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membrane was reacted with blocking solution (5% skim milk, 10 mM Tris, pH 7.5, 10 mM NaCl, 0.1% Tween 20) for 1 hour to exclude nonspecific blinding. Primary antibody of Caspase-3 protein was added and reacted at room temperature for 2 hours . After washing 3 times for 10 minutes with blocking solution, secondary antibody was added and reacted for 1 hour and washed as above. After the ECL solution was exposed to X-ray film, the amount of protein change was confirmed. The amount of change was calculated by optical density using an image analyzer.

As shown in FIG. 4, when caspase-3 protein expression levels were examined, the expression of caspase-3 was increased in a concentration-dependent manner by treating the high-purity platocidin-containing aged blackberry extract with concentration. This means that aged blackrod grains increased cell death by increasing the expression of major proteins of cell apoptosis.

Claims (5)

a) 5-year old Rhododendron at 90 ~ 95 ° C for 2 hours and drying at 50 ~ 60 ° C for 55 ~ 70 hours;
b) extracting the dried bellflower with purified water;
c) culturing the extract in a Lactobacillus plantarum , a Lactobacillus acidophilus , or a Pediococcus acidilactici with three kinds of lactic acid bacteria, Lactobacillus plantarum , Lactobacillus acidophilus and Pediococcus acidilactici ;
d) Culturing with three kinds of lactic acid bacteria and culturing with one kind of Saccharomyces cerevisiae :
e) filtering and concentrating the cultured extract; And
f) adsorbing the concentrate of step e) on an ion exchange resin, and then purifying the concentrate containing high purity platycodin A, D as an eluting solvent in an aqueous solution containing the alcohol. Excellent aging for prevention Black rosewood extract








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