KR20040093917A - Method for preparing taraxinic acid from Taraxacum coreanum Nakai and anticancer agent containing taraxinic acid as an effective ingradient - Google Patents

Abstract

PURPOSE: A method for preparing taraxinic acid from Taraxacum coreanum nakai and an anticancer agent containing taraxinic acid as an effective ingredient are provided, which taraxinic acid has improved inhibiting activity to various cancers, and cell differentiation inducing activity Therefore, taraxinic acid can be useful for prevention or treatment of cancer. CONSTITUTION: The method for preparing taraxinic acid of the formula(1) from Taraxacum coreanum nakai comprises the steps of: (1) extracting Taraxacum coreanum nakai with alcohol; (2) extracting the extract of step (1) with dichloromethane; (3) extracting the extract of step (2) with ethyl acetate; (4) subjecting the extract of step (3) to the column to isolate taraxinic acid beta-glucopyranosyl ester; and (5) hydrolysis of the isolated taraxinic acid beta-glucopyranosyl ester, wherein the alcohol is selected from methanol, ethanol, propanol and butanol; the column is silica gel and Sephadex columns. The anticancer agent contains 1 to 50 mg/kg of the taraxinic acid as an effective ingredient, wherein the cancer is selected from leukemia, colon cancer, liver cancer and lymphoma.

Description

Method for preparing taraxinic acid from Taraxacum coreanum Nakai and anticancer agent containing taraxinic acid as an effective ingradient}

The present invention relates to an anticancer agent comprising peraxic acid, and more particularly to an anticancer agent containing peraxic acid isolated from dandelion as an active ingredient.

The imbalance between proliferation, apoptosis and differentiation of cells leads to the proliferation of malignant cells. Recently, the method of inducing cell differentiation and natural death based on the understanding of tumor biology about the cell population dynamics has emerged as a new approach for the prevention and treatment of cancer. As with natural death of cancer, differentiating malignant or premalignant cells into cells such as mature cells or normal cells may be usefully applied to prevent cancer. Thus, compounds that are effective in inducing differentiation of cells can be usefully used to treat or prevent cancer.

Many types of cancers are known to be caused by abnormal differentiation or stop of differentiation of cells, and researches on the development of anticancer drugs by cancer cell differentiation inducing substances that specifically act on cancer cells by using the characteristics of these cancers are being actively conducted. Differentiation and proliferation of cells have a mutually opposite relationship. As the differentiation progresses faster, the proliferation is inhibited. Differentiation of cells is an essential step in cell maturation, but for cancer cells, these cells continue to accumulate, with this differentiation ceases and remains only at the cell growth stage (HP Koeffler et al ., Blood , 62, 709-721). , 1983).

HL-60 cell lines isolated from patients with acute myeloid leukemia are useful cell lines for differentiation studies at the cellular and molecular level. In addition, recent attempts have been made to treat leukemia by using substances such as interferon, retinoids and vitamin D ₃ (1α, 25-dihyroxyvitamin D ₃ ), which are known to induce differentiation of HL-60 cells. (PE Harris et al ., Cancer Research , 45, 3090-3095, 1985; HP Koeffler et al ., Cancer Research , 44, 5624-5628; VK Ostem et al ., Proc. Natl. Acad. Sci. USA , 84, 2610-2614, 1987).

On the other hand, hay of Taraxacum coreanum Nakai has been used for long time as a traditional medicine for diuretic and anti-inflammatory treatments (Andeokgyun, Korea Herbal Medicine, 134-135, 1998). In the 1970s, research on the chemical composition of white dandelions included studies on abscisic acid and essential oils (Jae-Kil Kim, Primitive Color Natural Medicine Dictionary (Sangwon), 77, 1974; 4), 2414-2420, 1985), and thereafter, no studies on active ingredients in white dandelion have been conducted.

The inventors of the present invention, while researching to develop a useful material for the prevention and treatment of cancer, the dexacin acid isolated from ethyl acetate extract of white dandelion shows a proliferation inhibitory effect on various cancer cell lines, in particular the differentiation of human leukemia cell line The present invention has been completed by confirming that there is a potent inducing activity, and thus, can be effectively used for the prevention or treatment of cancer.

It is an object of the present invention to provide a method for separating taracic acid from white dandelion and an anticancer agent containing the taracic acid as an active ingredient.

1 is a graph showing the extent of inhibiting the proliferation of HL-60 cells when the treatment of taracic acid by concentration.

■: no treatment, ●: 15 μM of taracic acid,

◆: 30 μM of taracic acid, ▲: 60 μM of taracic acid

Figure 2 shows the results of FACS analysis of the effect of taracic acid on the expression of CD14 and CD66b, the cell membrane surface antigens of HL-60 cells.

In order to achieve the above object, the present invention provides a method for separating the taracic acid represented by the formula (1) from white dandelion.

The present invention also provides an anticancer agent containing the taracic acid as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

1) extracting the white dandelion with alcohol;

2) extracting the extract of step 1 with dichloromethane;

3) extracting the extract of step 2 with ethyl acetate;

4) applying the extract of step 3 to the column to separate the taracic acid beta-glucopyranosyl ester; And

5) It provides a method for separating the taracic acid represented by the formula (1) comprising the step of hydrolyzing the taracic acid beta-glucopyranosyl ester separated in step 4.

In step 1, the alcohol may be selected from the group consisting of methanol, ethanol, propanol and butanol, preferably methanol.

In step 3, extraction with ethyl acetate is a step of extracting the water layer obtained by extraction in step 2 several times with ethyl acetate.

In step 4, the column may be a silica gel and Sephadex column, and the ethyl acetate layer obtained in step 3 is separated by the developing solvent using the fraction obtained by evaporation. The compound isolated after the step 4 is a taracic acid beta-glucopyranosyl ester (taraxinic acid β-D-glucopyranosyl ester). In a preferred embodiment of the present invention, the fraction obtained by evaporating the ethyl acetate layer extracted from the dandelion was separated by passing through column chromatography using silica gel (column chromatography, 2 x 30 cm). At this time, dichloromethane / methanol / water mixed solvent (100/10 / 0.5 → 40/10/1 → 70/30/3) was eluted from the column using a developing solvent, and divided into four fractions. The second fraction of the obtained fractions was developed on a Sephadex LH-20 column using methanol as the developing solvent, and then silica gel column chromatography using dichloromethane / methanol / water (100/20 / 1.5) as the developing solvent. Was developed to separate taracic acid beta-glucopyranosyl ester, a compound of colorless crystals.

In step 5, hydrolysis is carried out by adding beta-glucosidase to the taracic acid beta-glucopyranosyl ester separated by step 4, wherein the taracic acid beta-glucopyranosyl ester is Is converted to decomposing acid. In a preferred embodiment of the present invention, the taracic acid beta-glucopyranosyl ester was dissolved in NaOAc buffer (pH 5.2), and then beta-glucosidase was added and stored at 37 ° C for two days. The precipitate was filtered from the reaction solution, extracted with chloroform, and subjected to thin layer chromatography (TLC) using a residue obtained by evaporating the solvent. As a result, it was possible to separate taracic acid.

The present invention also provides an anticancer agent containing the taracic acid as an active ingredient.

The dexacin acid of the present invention is P388 (mouse leukemia cell line), L-1210 (mouse leukemia cell line), SNU-C5 (human colorectal cancer cell line), HL-60 (human myeloid leukemia cell line), U-937 (human) Histologic lymphoma cell line), HepG2 (human liver cancer cell line) shows a cell proliferation inhibitory effect (see Fig . 1 ). In addition, the dexacin acid of the present invention exhibits a proliferation inhibitory effect on HL-60 cells isolated from patients with acute myeloid leukemia, as well as nitroblue tetrazolium reduction test, esterase activity, macrophage activity, morphological changes, and CD14. Expression test of the and CD66b surface antigen was confirmed to strongly induce differentiation of cells (see Table 2 and Figure 2 ). Therefore, the taracic acid of the present invention can be used for the prevention or treatment of various kinds of cancer cells.

The taracic acid of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

In the actual clinical administration of the taracsin acid of the present invention can be administered in a variety of oral and parenteral formulations, when formulated using diluents or excipients such as fillers, extenders, wetting agents, disintegrating agents, surfactants, etc. commonly used It is prepared. Solid form preparations for oral administration include tablets, pills, powders, rapeseeds, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, or It may be prepared by mixing lactose, gelatin and the like. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of taracsin acid of the present invention is 1 to 50 mg / kg, preferably 5 to 20 mg / kg, and may be administered 1 to 3 times a day.

The anticancer agent containing the taracic acid of the present invention as an active ingredient can be used to treat or prevent various cancers, and preferably can be used to treat or prevent leukemia.

In addition, the taracic acid of the present invention is a safe compound having an oral minimum lethal dose (LD50) of 500 mg / kg or more since it does not show toxicity in rats in the oral toxicity test.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Decaynic Acid Beta-Glucopyranosyl Ester from White Dandelion

The white dandelion (Taraxacum coreanum Nakai) outpost collected from Namyangju-si, Gyeonggi-do was dried in the shade and finely chopped. The fine dandelion 850 g was immersed in 20 L of methanol, extracted for 4 to 5 days at room temperature, and filtered. The methanol solution extracted three times was concentrated in a rotary concentrator at 40 ° C. to obtain a methanol extract. The methanol extract of the white dandelion was suspended in 1 L of water, extracted three times with 600 mL of dichloromethane (CH ₂ Cl ₂ ), and the obtained water layer was extracted three times with 600 mL of ethyl acetate (EtOAc). 6.0 g of the fraction obtained by evaporating the obtained ethyl acetate layer was separated by passing through column chromatography using silica gel (2 × 30 cm). At this time, dichloromethane / methanol / water mixed solvent (100/10 / 0.5 → 40/10/1 → 70/30/3) was eluted from the column using a developing solvent, and divided into four fractions. The second fraction of the obtained fraction was developed with Sephadex LH-20 using methanol as a developing solvent, followed by silica gel column chromatography using dichloromethane / methanol / water (100/20 / 1.5) as a developing solvent. It developed to separate the compound of colorless crystals.

The chemical properties of the isolated compound are as follows, and as a result of the identification, it can be seen that it is a taracic acid beta-glucopyranosyl ester (Hansel et al ., Phytochemistry , 19, 857-861, 1980).

<Chemical Properties of Taracic Acid Beta-Glucopyranosyl Ester>

Melting point: 85-90 ℃,

¹ H NMR (600 MHz, CDCl ₃ ): δ 6.18 (d, 1H, J = 3.6 Hz), 5.84 (dd, 1H, J = 12.8, 3.7 Hz), 5.63 (d, 1H, J = 3.3 Hz), 5.53 (d, 1H, J = 7.7 Hz), 4.98 (dd, 1H, J = 10.2, 1.3 Hz), 4.73 (dd, 1H, J = 10.24, 8.7 Hz), 3.84 (dd, 1H, J = 12.0, 1.5 Hz), 3.68 (dd, 1H, J = 12.0, 4.4 Hz), 3.30-3.50 (m, 4H), 3.32 (m, 1H), 2.90 (m, 1H), 2.72 (m, 1H), 2.03- 2.41 (m, 5H), 1.63 (s, 3H),

IR _max (KBr): 3400 (OH), 1755 (lactone C = O), 1725 (C = O), 1630 (C = C) cm ^-1

Example 2 Preparation of Taraxic Acid

After dissolving 36.4 mg of the taracic acid beta-glucopyranosyl ester isolated in Example 1 in 5 ml of NaOAc buffer (pH 5.2), 3.4 mg of beta-glucosidase was added thereto, followed by two days at 37 ° C. Stored for a while. The precipitate was filtered from the reaction solution, followed by extraction with chloroform and evaporation of the solvent.

As a result, the compound having the following ¹ H NMR spectrum results were isolated, it was confirmed that it was a decomposing acid (Hansel et al ., Phytochemistry , 19, 857-861, 1980).

< ¹ H NMR Spectrum Result of Taraxic Acid>

¹ H NMR (300 MHz, CDCl ₃ ): δ 6.27 (d, 1H, J = 3.6 Hz), 5.81 (dd, 1H, J = 12.4, 4.0 Hz), 5.53 (d, 1H, J = 3.2 Hz), 4.93 (br d, 1H, J = 10.2 Hz), 4.61 (m, 1H), 3.43 (m, 1H), 2.93 (m, 1H), 2.58 (m, 1H), 2.42-1.93 (m, 6H), 1.64 (s, 3 H)

Example 3 Cytotoxic Effects of Taraxic Acid on Cancer Cell Lines

It was confirmed that the taracic acid and the taracic acid beta-glucopyranosyl esters obtained in Examples 1 and 2 had a toxic effect on cancer cell lines. Specifically, mouse leukemia cell line P388 (Korea Cell Line Bank), mouse leukemia cell line L-1210 (Korea Cell Line Bank), human colon cancer cell line SNU-C5 (Korea Cell Line Bank), myeloid leukemia cell line HL- 60 (Korea Cell Line Bank), human tissue lymphoma cell line U-937 (Korea Cell Line Bank), human liver cancer cell line HepG2 (Korea Cell Line Bank), respectively, 10% FBS (Life Technologies, Inc), 100 units / ml penicillin ( Life Technologies, Inc), RPMI medium containing 100 μg / ml streptomycin sulfate (Life Technologies, Inc), was cultured in a humid environment with 37 ° C., 5% carbon dioxide supplied. After incubation, each of the cancer cells was obtained by centrifugation or scraper, and then, 100 μl of RPMI 1640 medium containing 10% FBS was added to a 96 well plate so that the number of cells was 1 × 10 ⁵ cells per well. Taracic acid and taracic acid beta-glucopyranosyl esters were dissolved in dimethyl sulfoxide (DMSO) solvent and the concentration of DMSO in all experiments did not exceed 0.1%.

After overnight cell culture, various concentrations of 0 to 200 μM or more were added and incubated for 24 hours. After incubation, the cells were washed once, and then 5 mg / ml of MTT (β-Glycosidase, 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl-tertazolium bromide) (Sigma Chemical Co.) 50 μl of medium containing FBS was added and incubated at 37 ° C. for 4 hours. After incubation, the medium was removed, and 100 μl of DMSO was added to dissolve formazan blue formed in the cells by the MTT reaction. Then, after measuring the absorbance at 540 nm, the IC ₅₀ value was calculated to determine the proliferation inhibitory effect of the taracic acid and the taracic acid beta-glucopyranosyl ester on cancer cells. IC ₅₀ value refers to a concentration (IC ₅₀ ) that shows a 50% reduction in the number of cells compared to the case where no compound was treated, and expressed as an average value of three times.

As a result, the fall acid beta-glucopyranosyl ester, while treatment concentration is increased up to 200 μM also does not exhibit cytotoxicity, fallen acid inhibited the growth of cancer cells in 34.5 ~ 135.9 μM concentration for a variety of cancer cell lines (Table 1 ). Therefore, it was found that the anti-cancer effect was effectively exhibited when there was no glucosyl ester group in the taracic acid.

Cancer cell line IC ₅₀ (μM) Decay acid Taracic acid beta-glucopyranosyl ester P-388 67.5 > 200 L-1210 59.4 > 200 U-937 46.5 > 200 HL-60 34.5 > 200 SNU-C5 115.8 > 200 HepG2 135.9 > 200

Example 4 Inhibition of Proliferation of Taraxic Acid on HL-60 Cells

The purpose of this study was to analyze the proliferation inhibitory effect of taracic acid on HL-60 cells. Specifically, HL-60 cells were cultured in a humid environment with 10% FBS, 100 units / ml penicillin, RPMI medium containing 100 μg / ml streptomycin sulfate, 37 ° C., 5% carbon dioxide. Cells were stored in larval cultures at intervals of 2 to 3 days at a concentration of 1 × 10 ⁵ cells / ml. Taracic acid dissolved in DMSO was added to the cultured cells at concentrations of 0, 15, 30 and 60 μM and then incubated for 1 to 4 days. Cell viability after cell culture was measured by trypan blue exclusion method (Kyung-Tae Lee et al . , Biol. Pharm. Bull ., 24, 1117-1121, 2001). The experiment was performed three times, and the experimental results are expressed as the average and standard deviation of the three experiments.

As a result, the growth of HL-60 cells was inhibited depending on the concentration and treatment time of the treated taracic acid. The proliferation inhibitory effect of taracic acid on HL-60 cells was apparent when treated with 15 μM and 30 μM, and no cytotoxicity was observed at this concentration ( FIG. 1 ).

Example 5 Induction of Cell Differentiation of Taraxic Acid

To determine whether taracic acid induces cell differentiation, the effect of inducing cell differentiation was compared using vitamin D ₃ , a cell differentiation inducer of HL-60 cells. The nitroblue tetrazolium reduction test, the phagocytosis test, the esterase activity test and the expression analysis of CD14 and CD66b surface antigens were performed. The effect of differentiation induction on cell differentiation was analyzed using HL-60 cells treated with 0, 15 or 30 μM of taracic acid, and as a comparison group, vitamin D ₃ (1α, 25 (OH) 2D3) (Sigma Chemical Co. HL-60 cells treated with 0.02 μM concentration were used. All results are expressed as mean values performed three times.

1) Nitroblue Tetrazolium Reduction Test

Treated HL-60 cells with 15 μM or 30 μM concentrations of taracic acid were treated with 1.0 mg / ml of nitroblue tetrazolium (abbreviated as "NBT") (Sigma Chemical Co.) at 37 ° C. The degree of reduction of NBT to formazan was measured by incubation for 30 minutes. TPA (12-O-tetradecanoylphorbol-13-acetate) (Sigma Chemical Co.) was used as a stimulant to form formazan.

As a result, about 28.9% and 79.7% of HL-60 cells treated with 15 and 30 μM concentrations of taracic acid were stained with NBT so that the cells not treated with taracic acid were stained with NBT by 13.2%. It was found that the number of cells to be stained increased. In addition, the comparative drug vitamin D ₃ (20 nM) was stained 58.4% ( Table 2 ).

2) macrophage activity test

1 × 10 ⁶ cells / ml of HL-60 cells treated with taracic acid were suspended in RPMI 1640 medium containing 0.2% latex particles (average diameter 0.81 μM) and serum-free, and then 4 hours at 37 ° C. Incubated for After incubation, the cells were washed once with PBS (phosphate-buffered saline), and the cells containing 10 or more latex particles were counted as phagocytic cells.

As a result, the macrophage activity of the cells treated with taracic acid was significantly increased ( Table 2 ).

3) Esterase Activity Measurement

HL-60 cells treated with taracic acid at a concentration of 15 μM or 30 μM were treated with α-naphthyl acetate esterase kit (Sigma Chemical Co.) and naphthol AS-D chloroacetate esterase ( Esterase activity was measured using the naphthol AS-D chloroacetate esterase kit (Sigma Chemical Co.).

As a result of treatment with taraxic acid in HL-60 cells, α-naphthyl acetate esterase activity was increased, but the effect on naphthyl AS-D chloro acetate esterase activity was relatively insignificant ( Table 2 ).

compound Treatment concentration (μM) NBT Reduction Rate (%) Naphthyl AS-D Chloroacetate Esterase Activity (%) α-naphthyl acetate esterase activity (%) Macrophage activity (%) Decay acid - 13.2 ± 0.8 12.0 ± 0.8 11.0 ± 0.9 2.7 ± 0.5 15 28.9 ± 1.1 * 13.8 ± 0.9 39.6 ± 2.8 * 22.4 ± 1.0 * 30 79.7 ± 2.5 * 14.9 ± 0.2 75.1 ± 5.3 * 36.9 ± 1.8 * Vitamin D ₃ 0.02 58.4 ± 5.4 * 10.5 ± 3.2 49.6 ± 4.8 * 40.4 ± 3.5 *

In the above, * indicates that the value increases effectively.

4) Expression Analysis of CD14 and CD66b Surface Antigens

HL-60 cells (2 × 10 ⁵ cells / ml) treated with taracic acid were obtained and rinsed twice with cold PBS. Then, anti-CD14 antibody (Pharmingen) or anti-CD66b antibody (Pharmingen) attached to the FITC was left on ice for 30 minutes and rinsed twice with PBS again. Antibody-attached cells were quantified with a Fluorescent-Activated Cell Sorter (FACS).

As a result, there was no change in the cell membrane surface antigen CD66b after treatment with taracic acid, but CD14 expression was significantly increased ( FIG. 2 ). These results demonstrate that HL-60 cells are induced to differentiate into multicellular / macrophages.

Through the above results, it was found that taracic acid effectively induces differentiation of HL-60 cells. In addition, these results suggest that dexacin acid induces differentiation of human leukemia cells into monocytes / macrophage. Therefore, it can be seen that the dexacin acid of the present invention can be usefully used for treating or preventing various cancers including leukemia.

As described above, the taracic acid of the present invention isolated from the white dandelion shows a proliferation inhibitory effect on various cancer cells and has an activity of strongly inducing the differentiation of cells, and thus can be effectively used for the prevention or treatment of cancer.

Claims (9)

1) extracting the white dandelion with alcohol; 2) extracting the extract of step 1 with dichloromethane; 3) extracting the extract of step 2 with ethyl acetate; 4) applying the extract of step 3 to the column to separate the taracic acid beta-glucopyranosyl ester; And 5) A method of separating taracic acid represented by Formula 1, comprising hydrolyzing the taracic acid beta-glucopyranosyl ester separated in step 4. <Formula 1> The method of claim 1 wherein the alcohol of step 1 is selected from the group consisting of methanol, ethanol, propanol and butanol. The method of claim 2 wherein the alcohol is methanol. The method of claim 1 wherein the column of step 4 is characterized in that the silica gel and Sephadex column. An anticancer agent containing taracic acid represented by the formula (1) as an active ingredient. 6. The anticancer agent according to claim 5, wherein the cancer is selected from the group consisting of leukemia, colorectal cancer, liver cancer and lymphoma. 7. The anticancer agent according to claim 6, wherein the cancer is leukemia. [Claim 6] The anticancer agent according to claim 5, wherein the effective dose of taracsin acid represented by Formula 1 is 1 to 50 mg / kg. [Claim 10] The anticancer agent according to claim 8, wherein the effective dose of taracic acid represented by Formula 1 is 5 to 20 mg / kg.