WO2012030152A2 - Composition for preventing and treating prostate cancer including alpinia katsumadai hayata extract - Google Patents

Abstract

The present invention relates to a novel usage of Alpinia katsumadai Hayata extract, and more particularly, to a composition for preventing and treating prostate cancer containing Alpinia katsumadai Hayata organic solvent extract. The composition for treating prostate cancer, according to the present invention, effectively suppresses the growth of prostate cancer cells and induces apoptosis, and thus can be effectively used for treating and preventing prostate cancer.

Description

Composition for the treatment of prostate cancer comprising the head extract

The present invention relates to a novel use of the scalp extract. In particular, the present invention relates to a therapeutic composition containing as an active ingredient a head nut extract showing an excellent prophylactic or therapeutic effect against prostate cancer.

The prostate is an organ that exists only in males that makes up part of the semen and is located below the bladder and adjacent to the rectum. Prostate cancer is a malignant tumor that occurs in the prostate gland, and is one of the most common cancers among men in the West, and it is the fastest growing cancer in men due to recent dietary habits in Korea. do. In developed countries such as North America and Western Europe, it is the most common cancer that accounts for about 20% of male cancers, and the United States has the highest frequency of male cancers occurring annually, and ranks second after lung cancer among cancer deaths. In Korea, the incidence of prostate cancer has increased significantly in recent years due to an increase in life expectancy, an increase in the elderly, westernization of a diet, development of diagnostic technology, and increased awareness of prostate cancer. According to the central cancer registration data, the cancer incidence fraction of prostate cancer was 6th in 2001, 2.7% of male cancers and 3.0% in 2002, and it is the fastest growing in recent years and is expected to increase further. to be. Prostate cancer is known to be caused by hormones, dietary habits, and chemicals in addition to genetic predisposition.

Most prostate cancers are cancers of gland cells in the prostate, which spread well to lymph nodes and bones. About 90% of prostate cancers are proliferated by male hormones that are produced in your body. For this reason, hormonal therapies that inhibit cancer growth and kill some of the cancer cells by inhibiting the action of male hormones are most commonly used in the treatment of prostate cancer (Greenlee, R. T et al., Cancer statistics.CA Cancer J. Clin 50: 7 (2000).

Treatments for prostate cancer include (a) radical prostatectomy (b) radiation (c) chemotherapy (d) hormonal therapy. The most widely used treatment is hormonal therapy, which is a so-called androgen removal method that inhibits male hormone secretion or blocks hormone production by surgery, since a significant portion of prostate cancer cells multiply androgen-dependently. This method has a temporary therapeutic effect in more than 80% of patients, such as cancer suppression or lesion reduction. However, even patients who responded well to transient hormonal therapy progressed to hormone-refractory prostate cancer (HRPC) after a certain period of time, and those who die within one year of the mean diagnosis. In the case of HRPC, conventional anticancer drugs, chemotherapy, or radiation therapy do not show a great effect, so a new treatment method is required (Fong, L. et al., Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization: implications for immunotherapy of prostate cancer.J. Immunol. 159: 3113. (1997).

Recently, with the possibility of immunotherapy using cytokines and dendritic cells, immunotherapy using dendritic cell therapy (DC vaccine) has emerged as the next generation treatment for HRPC patients (Fong, L. et al., Dendritic cells in cancer immunotherapy. Annu. Rev. Immunol. 18: 245. (2000); Xue BH. Et al., Induction of human cytotoxic T lymphocytes specific for prostate specific antigen.Prostate. 30: 73.78. (1997)). For the clinical trial of immunotherapy using dendritic cells, the effectiveness and safety of the animal model should be confirmed first. However, there is no animal model for prostate cancer to evaluate the efficacy of dendritic cell vaccine against human prostate cancer.

On the other hand, Alpiniae Katsumadaii Semen (Alpiniae Katsumadaii Semen; Alpinia Katsumadai) is a herb belonging to the family Zingiberaceae. Chodugu contains essential oils such as d-4-terpinyl acetate and d-alpha-terpineol. It is distributed in various parts of the tropics, and has the effects of detoxification, constitution improvement, oral hygiene, and anti-inflammatory. Edinosa is a perennial herb, with thick and short roots, with greenish stem. When the fruit turns yellow in autumn, harvest it and dry it for 7 to 8 days in the sun, peel off the skin and dry it again in the sun. The shape of the head nut is a spherical round shape of seeds, and is 15 to 27 mm in diameter. The outer side is grayish brown, and in the middle, it is divided into three parts by the gray-white diaphragm, and the seeds are divided. Seeds are smooth and do not fall easily. Oval polyhedron, 3 to 5 mm long, about 3 mm in diameter, covered with a light brown membranous apiary, and hard.

Chodugu is spicy and warm in nature, acting on the spleen and stomach. In addition, it is warm and directional, warming the stomach, warming the inside, eliminating all the chills, lowering the soaring flag, and eliminating the scorn. Chodugu is a dampness of the stomach, and the abdomen is cold, painful, vomiting, anorexia, thin stool, heartburn caused by the accumulation of diarrhea, nausea is used. The pharmacological action of chodogu is known as fungi, intestinal excitement, intestinal suppression, wrinkle improvement, oral hygiene, hormone replacement treatment effect. As such, various pharmacological activities have been reported for cephalospheres, but there have been no reports and studies on the treatment or prevention of prostate cancer.

Accordingly, the present inventors completed the present invention by confirming that the extract of the head nutmeg can effectively kill prostate cancer cells during the study of herbal medicine on the head nutmeg.

It is an object of the present invention to provide a composition for treating prostate cancer, which contains a head nut extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of prostate cancer containing an organic solvent extract of Alpiniae Katsumadaii Semen as an active ingredient.

Hereinafter, the present invention will be described in detail.

In the composition for preventing and treating prostate cancer of the present invention, the organic solvent is preferably ethanol, wherein the ethanol extract is more preferably extracted at 50 ° C. for 24 hours or dried and concentrated at 45 ° C. under reduced pressure, and also Most preferably, the ethanol is 95%. The composition of the present invention comprises a nutmeg extract as an active ingredient, and may additionally include a pharmaceutically acceptable carrier or diluent.

Although the turmeric extract of the present invention is possible at any part of the turmeric, it is preferable to extract from the flower. The extraction solution may be obtained by extraction with water or an organic solvent, and examples of the organic solvent may include lower alcohols, acetone, chloroform, methylene chloride, ether, ethyl acetate, and hexane. Lower alcohols include methanol, ethanol, propanol and butanol, with ethanol being most preferred.

Specifically, 1 to 5 times, preferably 3 times, 95% ethanol is added to the dried or powdered granulosa and 10 to 100 hours, preferably 15 at a temperature of 20 to 100 ° C, preferably 40 to 60 ° C. To 40 hours, more preferably, 24 hours after the extraction can be filtered to prepare the ethanol extract of the head. Preferably, the filtrate obtained by filtering the extract may be concentrated under reduced pressure. In the above extraction methods, the extraction process may be repeated two or more times as necessary, and the extract obtained after filtration may be lyophilized or dried under reduced pressure to obtain a powder form.

The anticancer composition of the present invention includes chondula as an active ingredient, and may further include a pharmaceutically acceptable carrier or diluent. The "pharmaceutically acceptable carrier" is a pharmaceutically acceptable substance such as a liquid or solid filler, diluent, excipient or solvent which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body. , Composition or vehicle.

In one embodiment of the present invention, the composition of the present invention alone or in radiation therapy or chemotherapy (cell growth arrest or cytotoxic substance, antibiotic-type substance, alkylating agent, anti-metabolic substance, hormone, immuno-agent, interferon type Substances, cyclooxygenase inhibitors (e.g. COX-2 inhibitors), metallomatrix protease inhibitors, teromerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor materials, anti-HER substances, anti-EGFR substances, anti Other anticancer agents such as angiogenesis agents, farnesyl transferase inhibitors, ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binders, topoisomerase I inhibitors, topoisomerase II inhibitors, etc. It can be administered in combination with treatment. By way of example, the compositions of the present invention may include one or more chemotherapeutic agents optionally contained in liposome formulations (e.g., taxanes, taxane derivatives, encapsulated taxanes, CPT-11, camptothecin derivatives, anthracycline glycosides, doxorubicin, idarubicin). Leucine, epirubicin, etoposide, navelbine, vinblastine, carboplatin, cisplatin, estramustine, celecoxib, schgen SU-5416, schgen SU-6668, herceptin and the like). have. When formulated at a constant dose, such combinations use the compositions of the invention within the dosage ranges described below and other pharmaceutically active substances within the approved dosage ranges. The compositions of the present invention can be used sequentially with known anticancer agents when the combination formulation is inappropriate. The effective daily dose of the active ingredient in the anticancer composition of the present invention may be determined according to the age, sex, site of application, frequency of administration, administration time, formulation, type of adjuvant, and the like.

The composition of the present invention can be administered via a route commonly used for anticancer treatment. In addition, the anticancer composition of the present invention may be prepared in various formulations depending on the dosage form. For example, it may be administered in oral form of tablets, capsules, dragees or film encapsulated tablets, solutions or suspensions, parenteral forms of intramuscular, intravenous and / or intrathecal and / or intrathecal injection or infusion. For example, oral solids may be used in combination with the active compound in diluents (e.g. lactose, dextrose, sucrose, cellulose, corn starch or potato starch), lubricants (e.g. silica, talc, stearic acid, Magnesium or calcium stearate and / or polyethylene glycol), binders (e.g. starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone), disintegrants (e.g. starch, alginic acid, Alginate or sodium starch glycolate), formal mixtures, dyes, sweeteners, wetting agents (eg lecithin, polysorbates, laurylsulfate) and pharmacologically inert substances generally used in pharmaceuticals. These agents can be prepared by known methods, for example by means of mixing, granulating, tableting, dragging or film-coating processes. Liquid dispersions for oral administration can be, for example, syrups, emulsions and suspensions. Suspensions and emulsions may contain, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol as carriers. Suspensions or solutions for intramuscular injection, together with the active compound, may be used as a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols (e. G. Propylene glycol) and, if necessary, in suitable amounts of lidocaine hydrochloride. It may contain. Solutions for intravenous injection or infusion may, for example, contain sterile water or preferably in the form of sterile, aqueous, isotonic saline solutions or carrier propylene glycol.

In a specific embodiment of the present invention, the ethanol extract of the cephalosphere kills 88% and 97% of PC-3 or DU 145 prostate cancer cells at 100 μg / ml, respectively. The above results demonstrate that the chondroitin extract of the present invention has excellent killing activity of PC-3 or DU 145 prostate cancer cells and further has prostate cancer treatment and prophylactic activity.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to a patient's sexually transmitted disease, age, severity, and drug activity. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The method of administering the composition comprising the extract or compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. In general, when the effective dose is oral administration, it is usually 1 to 1 time per adult. 500 mg / kg is preferred, and in the case of intravenous administration, 1 to 100 mg / kg is preferred, and may be administered 2-3 times a day. Dosage levels for a particular patient may vary depending on gender, age, ultraglomerular state, diet, time of administration, method of administration, drug mixture, patient's condition and the extent of neurological disease.

In the present invention, as a result of injecting the chondula extract of the present invention into PC-3 or DU 145 prostate cancer cells, it was confirmed that PC-3 or DU 145 prostate cancer cells are killed (see FIGS . 1 and 2 ). In addition, as a result of flow cytometry and Western blotting, chondura extract extracts prostate cancer cells of human prostate cancer cell lines PC-3 and DU 145 cells in a specific cell cycle to stop cell division and apoptosis process (apoptosis). apoptosis) process (see FIGS. 3, 4 and 7 ). In addition, chondula extract affects Myc and AP-1 cell signals of prostate cancer cells (see FIGS . 5 and 6 ). Thus, it can be seen that the extract of the scalp has a prostate cancer therapeutic effect.

As described above, the chondrocyte extract of the present invention inhibits the growth of prostate cancer cells and induces apoptosis. Therefore, the composition for treating prostate cancer according to the present invention will be very effective for the treatment of patients with prostate cancer.

1 is a result of the Alamar Blue analysis to determine the effect of the introduction of the scalp extract obtained in Example 1 on the growth of prostate cancer cells in PC-3 cells, a human prostate cancer cell line, wherein the X-axis is the concentration of And the Y axis represents the survival rate of surviving human prostate cancer PC-3 cells.

Figure 2 is the result of Alamar Blue analysis to determine the effect of the introduction of the scalp extract obtained in Example 1 on the growth of prostate cancer cells in DU 145 cells, a human prostate cancer cell line, wherein the X-axis is the concentration of , Y axis shows survival rate of surviving human prostate cancer DU 145 cells.

FIG. 3 is a flow cytometry result for determining the effect of the introduction of the scalp extract obtained in Example 1 in PC-3 cells, which are human prostate cancer cell lines, to stop the prostate cancer cells' cell cycle and apoptosis.

Figure 4 is a flow cytometry result to determine the effect of the introduction of the scalp extract obtained in Example 1 in the human prostate cancer cell line DU 145 to stop the prostate cancer cells cell cycle and apoptosis.

Figure 5 is the result of analyzing luciferase activity by the Myc reporter assay method to determine the effect of the introduction of the scalp extract in PC-3, a human prostate cancer cell line on Myc cell signal of prostate cancer cells.

Figure 6 shows the results of analyzing luciferase activity in the AP-1 reporter assay method to determine the effect of the introduction of the scalp extract on the AP-1 cell signal of prostate cancer cells in PC-3, a human prostate cancer cell line.

Figure 7 shows the results of Western blotting analysis of the quantitative change of Bax induced by the introduction of the scalp extract in PC-3, a human prostate cancer cell line, causes apoptosis of prostate cancer cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Preparation of Cereal Extract

3 kg of Choduk-gu (Chinese) purchased from Seoul medicinal herb was dried and ground for 5 days at the shade and room temperature. The pulverized cedar was immersed in 30 L of 95% ethanol and extracted at 50 ° C. for 24 hours. This was filtered through filter paper, dried and concentrated under reduced pressure at 45 ° C. to obtain 475 g of the total extract, and stored at −20 ° C.

Example 2 Preparation and Treatment of Human Prostate Cancer Cell Line

The human prostate cancer cell lines PC-3 and DU 145 used in the present invention were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and used for experiments. Specifically, PC-3 and DU 145 cell lines were treated with DMEM (Dulbeco's Modified Eagle's Medium) medium containing 10% fetal bovine serum (FBS) (Welgene) at 37 ° C. and 5% CO ₂ . It was kept as it was, and it was passaged for 2-3 days.

Example 3 Effect of Cultivated Extracts on Prostate Cancer Cells

In order to determine the effect of the extract from Example 1 on the growth of prostate cancer cells, PC-3 and DU 145 cells, which are human prostate cancer cells, were treated with ethanol extracts of the cerebellum for 48 hours and subjected to Alamar Blue analysis. It was. The Alamar Blue assay is a modified form of the MTT assay, in which a specific enzyme degrades a living cell and then measures the fluorescence intensity of the product as the compound breaks down to determine the relative number of living cells after treatment. I am an experimental method. It will be described in more detail below.

4 x 10 per each well in a 96 well plate³ PC-3 and DU 145 cells were seeded and incubated for 24 hours. Ultracereal head of Example 1 dissolved in DMSO (dimethyl sulfoxide) When the ethanol extract was treated at concentrations of 0 to 100 μg / ml (specifically, concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 μg / ml, respectively) for 48 hours, the degree of inhibition of cell growth was confirmed. (Table 1 and Table 2). After treatment of each concentration of extract, 20 μl of Alamar Blue reagent was added to 0.2 ml of cell culture filled in each well in a 96-well plate, and the plates were incubated for 2 hours in an incubator. The plate was slowly shaken to evenly react the cells in each well, and the intensity of fluorescence was measured by Fluorescence Microplate Reader (Molecular Devices Corp.) at 590 nm while irradiating irradiated light at a wavelength of 544 nm. Survival rates of -3 and DU 145 human prostate cancer cells are also shown in FIGS. 1 and 2.

Table 1

Ultracranial Concentration of Example 1	Experiment 1	Experiment 2	Experiment 3	PC-3 cell viability (average)	Standard Deviation
0 μg / ml	100%	100%	100%	100%	0%
3.125 μg / ml	113%	102%	102%	106%	6%
6.25 μg / ml	98%	108%	111%	105%	7%
12.5 μg / ml	91%	99%	96%	95%	4%
25 μg / ml	64%	63%	63%	63%	0%
50 μg / ml	27%	28%	32%	29%	3%
100 μg / ml	12%	10%	13%	12%	2%

TABLE 2

Ultracranial Concentration of Example 1	Experiment 1	Experiment 2	Experiment 3	DU 145 cell viability (average)	Standard Deviation
0 μg / ml	100%	100%	100%	100%	0%
3.125 μg / ml	101%	90%	96%	96%	5%
6.25 μg / ml	97%	100%	96%	97%	2%
12.5 μg / ml	78%	74%	80%	77%	3%
25 μg / ml	38%	34%	36%	36%	2%
50 μg / ml	11%	8%	8%	9%	2%
100 μg / ml	3%	2%	3%	3%	0%

As a result, as shown in Table 1 and Table 2 (see Figs. 1 and 2), the higher the treatment concentration of the head nut, the growth of cancer cells was reduced, from which it can be seen that the head nut has an anticancer effect.

Example 4 Effect of Cultivated Turmeric Extracts on Cell Division Cycle of Prostate Cancer Cells

Flow Cytometry was performed to investigate the effect of S. aureus extract on the cell division cycle of cancer cells. Flow cytometry is a method to quickly identify the characteristics of a cell as it passes through the detection zone. When staining the cell nucleus with a fluorescent material, the flow cytometry can be determined. .

Specifically, PC-3 and DU 145 cells were seeded in 6-well plates at about 2.4 × 10 ⁵ cells per well and incubated for 24 hours. The cells were administered with 100 μg / ml of cephalosphere extract and allowed to stand for 24 hours. The culture medium in each well was removed from the 6-well plate and washed twice with PBS (phosphate-buffered saline). The attached cells were suspended by treatment with trypsin-EDTA solution, transferred to microtubes and centrifuged to recover the cells. The recovered cells were washed with PBS, centrifuged and suspended in cold ethanol. Cells suspended in ethanol were stored at 4 ° C to fix the cells, centrifuged and washed with PBS. The washed cells were suspended in 500 μl of PI solution (50 μg / ml of Propidium Iodide, 10 μg / ml of RNase A) and stored at 37 ° C. for 30 minutes to stain nuclei, and 2 ml of PBS was added thereto. Cells suspended in solution were analyzed by flow cytometry (FACS; BD biosciences), and cell cycles and apoptosis of PC-3 and DU 145 human prostate cancer cells were shown in FIGS. 3 and 4, respectively.

As a result, as shown in Fig. 3 and 4, the cancer cells treated with the chondula extract were found to be in a stationary state in a specific cell cycle compared to the control group, and the apoptosis process, which is an apoptosis process, was in progress. From this, it can be seen that the cerebellum output stops the division of cancer cells and leads to apoptosis.

Example 5 Effect of Cultivated Turmeric Extracts on Cell Signals of Prostate Cancer Cells

In order to investigate the effect of C. edodes extract on the cell signal leading to the growth of cancer cells, cell signal inhibition activity in actual cells was confirmed using a reporter assay.

Specifically, the reporter assay transfects human prostate cancer cell PC-3 with luciferase vectors designed to measure transcriptional activity of Myc and AP-1, which have a major effect on cancer cell growth. After the transfection, 6 hours later, the treatment was carried out by treating the cephalosphere extract at a concentration of 0-100 μg / ml and measuring luciferase activity after 24 hours. Table 3 shows the degree of inhibition of the cellular signal of Myc and AP-1 expressed in human prostate cancer cells.

TABLE 3

	Myc signal		AP-1 signal
density	Average	Standard Deviation	Average	Standard Deviation
0 μg / ml	100%	0%	100%	0%
100 μg / ml	5%	One%	17%	2%

As a result, as shown in Figures 5, 6 and 7 was treated with ultra-real head cancer cells decreased the transcriptional activity of Myc and AP-1 leading the growth of cancer cells compared to the control group, The extract was found to inhibit the growth of cancer cells.

<Example 6> Effect of Cultivated Extract of Cultivated Shrub on Apoptosis of Prostate Cancer Cells

In order to investigate the effect of C. aeruginosa extract on apoptosis of cancer cells, Western blotting was used to determine the quantitative changes of Bax that induced apoptosis after treating C. aureus extract on human prostate cancer cells.

Specifically, PC-3 cells were cultured to grow 60 to 70% in a 60 mm dish, and then the cedar extract was treated at a concentration of 0 to 100 μg / ml. After 24 hours, the culture medium was removed from the cells in culture, washed with PBS, and treated with NP-40 solution to obtain cell lysate. The cell lysate was centrifuged to recover the supernatant in which the cellular protein was dissolved, and the sample was prepared for electrophoresis after measuring the concentration. The prepared samples were electrophoresed and Western blotting was performed to analyze the amount of Bax protein with Bax antibody. The results are shown in FIG. 8.

As shown in Figure 7, the turmeric extract confirmed that the amount of Bax leading to the apoptosis of cancer cells was increased, and through this, the turmeric was effectively induced to the apoptosis of cancer cells, indicating an anticancer effect.

Preparation Example 1 Preparation of Prostate Cancer Therapeutic Agents Containing Extracts from Scutellum Fructus

The present inventors have confirmed that the prostate cancer treatment efficacy of the choledophil extract is excellent through the above embodiment, and prepared a prostate cancer therapeutic agent containing the chondula extract as an active ingredient as follows.

<1-1> Soft Gelatin Capsules Containing Cereal Extract

Green Bean Extract 20%

Vitamin C 4.5%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

10% beeswax

Palm oil 25%

Safflower Seed Oil 30.399%

 <1-2> Preparation of Intravenous Formulation Containing Cereal Extract

Green Bean Extract 0.2%

Mannitol 0.3%

Saline 9.5%

<1-3> tablet containing turmeric extract

Green Bean Extract 35%

Vitamin C 10%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

Crystalline cellulose 25.0%

Lactose 17.999%

Magnesium Stearate 2%

Claims (6)

1. A composition for preventing and treating prostate cancer, comprising an organic solvent extract of Alpiniae Katsumadaii Semen as an active ingredient.
2. The composition for preventing and treating prostate cancer according to claim 1, wherein the organic solvent is ethanol.
3. According to claim 2, wherein the ethanol extract is a composition for preventing and treating prostate cancer, characterized in that extracted for 24 hours at 50 ℃.
4. The composition for preventing and treating prostate cancer according to claim 2 or 3, wherein the ethanol extract is dried and concentrated at 45 ° C under reduced pressure.
5. 4. The composition for preventing or treating prostate cancer according to claim 2 or 3, wherein the ethanol is 95%.
6. According to claim 1, wherein the composition is a composition for preventing and treating prostate cancer, characterized in that it comprises a pharmaceutically acceptable carrier or diluent.

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