KR19980076449A - Urushiol fraction having anticancer effect isolated from sap of lacquer tree and its separation method - Google Patents

Abstract

The present invention separates the crude urushiol component by adding acetone to the sap of lacquer, and adds the crude urushiol component to the column in an amount of 1/6 to 1/4 of the weight of silica gel, and then the ratio of hexane: acetone is 80:20. Anti-cancer containing urushiol from the sap of lacquer tree, which comprises collecting a fraction of a residence time of 90 to 160 minutes by separating silica gel adsorption column chromatography using a mixed solvent having a concentration of from 95: 5 (v / v) as an eluent. A method of separating the active fractions; A urushiol-containing fraction having anticancer activity isolated from sap of lacquer trees by the above method; And it relates to an anticancer composition comprising it as an active ingredient.

Description

Urushiol fraction having anticancer effect isolated from sap of lacquer tree and its separation method

The present invention relates to a urushiol fraction excellent in anticancer effect among urushiol (urushiol) components contained in sap (seven liquors) collected from lacquer trees, and a method of separating the same.

In the Orient, the lacquer has been collected from various lacquer trees for thousands of years.In Korea, Japan and China, lacquer ( Rhus verniciflua ), vietnamese lacquer ( Rhus succedanea ) in Vietnam and Taiwan, and melanorrhoea usitate in Myanmar, etc. It has been used as a coating material. Among them, Rhus verniciflua in Korea, Japan and China is believed to have been transferred from China to Korea and Japan.

The lactose consists of urushiol, rubber, laccase (oxidase), nitrogenous substance and water, and separation methods for these components have been reported in the literature (松井, 化 漆化學, p177 (1963)). .

Urushiol is a major component of about seventy percent of the solution, which is a complex of three-substituted catechols with C ₁₅ -alkyl or alkenyl groups, the main component being 3- (8'Z, 11'E with three double bonds). , 13'Z-pentadecatenyl) catechol (3- (8'Z, 11'E, 13'Z-pentadecatrienyl) catechol). To date, 13 components of 3-substituted alkylcatechols having 0, 1, 2 or 3 double bonds in the C ₁₅ side chain have been identified (Yumin Du and Ryuichi Oshima, J. of Chromatography , 284 , 463-473 (1984) Urushiol is a mixture of these monomers and polymers in which the monomers are naturally polymerized by LaCase (Yamauchi, Y., T. Murakami and J. Kumanotani, J. of Chromatography , 214 , 343-348 (1981); Yamauchi, Y., R. Oshima and J. Kumanotani, J. of Chromatography , 243 , 71-84 (1982); and Y. Du, R. Oshima and H. Iwatsuki, J. of Chromatography , 295 , 179-186 (1984).

In addition, urushiol is the main ingredient of Rhus toxicodendron radicans, and has been studied urushiol for immunological or dermatitis studies without studying lacquer as a paint or medicinal resource due to severe lacquer rise in Westerners with sensitive skin. (David W. Roberts and Claude Benezra, Contact Dermatitis , 29 , 78-83 (1993); and Markiewity KH and CR Dawson, J. Org. Chem. , 30 , 1610-1613 (1965)).

Cancer is an incurable disease of humans, so a specific anticancer drug is effective in some cancers, but not active in other cancers. Anticancer drugs can be divided into direct killing of cancer cells and inhibition of cancer cell proliferation by enhancing the body's immune function. In the former case, since most commercial anticancer drugs have side effects not only to cancer cells but also to other parts of the human body, it is difficult to conquer cancer. Therefore, it is important to develop an anticancer drug that can increase the anticancer effect without side effects on the human body.

Natural-derived anticancer drugs have a lower risk of side effects than synthetic anticancer drugs. Especially, ingredients extracted from plants that have been edible since ancient times have no risk of side effects and are considered to have optimal conditions as anticancer drugs (Hideji, Itokawa, J. Oriental Bot. Res. , 5 (2), 73-84 (1992).

In the private sector, lactose edible and edible lacquer stalks have been eaten for a long time, and the lacquer bark has been medicinal for its effects on gastrointestinal diseases.

It is an object of the present invention to provide a method for separating urushiol-containing fractions having anticancer activity from lacquer trees.

Another object of the present invention is to provide a urushiol fraction isolated from the sumac by the above method.

Still another object of the present invention is to provide an anticancer composition containing the urushiol fraction.

1 (A) and (B) show HPLC chromatograms of crude urushiol and fraction 7, respectively.

Figure 2 shows the cytotoxicity of the urushiol fractions of the present invention and tetraplatin (Tetraplatin) against the mouse blood cancer cell line L1210.

Figure 3 shows the cytotoxicity of the urushiol fractions of the present invention and tetraplatin against human lung cancer cell line PC-9.

Figure 4 shows the cytotoxicity of the urushiol fractions of the present invention and tetraplatin against human gastric cancer cell line KATO III.

Figure 5 shows the cytotoxicity of the urushiol fraction 7 of the present invention and tetraplatin against human lung cancer cell line A427.

Figure 6 shows the results of MTT analysis of the anti-cancer effect of the urushiol fractions and tetraplatin mouse blood cancer cell line L1210 of the present invention.

Figure 7 shows the proliferation inhibitory effect on the human lung cancer cell line PC-9 of the urushiol fraction 7 of the present invention.

The method of the present invention for separating an urushiol-containing fraction having anticancer activity from lacquer trees comprises separating a crude urushiol component from a lacquer solution and subjecting the crude urushiol component to silica gel column chromatography.

In the step of separating the crude urushiol component from the lacquer, 2-4 times, preferably 3 times, volume of acetone is first added to the lacquer, and then the mixture is added at 3-5 ° C., preferably at 4 ° C. for 30 minutes to 4 minutes. It is allowed to stand for 1 hour, preferably 1 hour, to separate the urushiol component and other saline components, ie rubbery, nitrogenous substance, lacquer and the like. At this time, tetrahydrofuran, toluene, methanol, etc. can also be used as an extraction solvent. The supernatant containing urushiol is centrifuged and filtered, and the solvent is evaporated from the filtered extract to prepare crude urushiol.

Silica gel column chromatography is applied to the column filled with silica gel in an amount of about 1/6 to 1/4, preferably 1/5 of the weight of silica gel, followed by the addition of a crude solvent of hexane: acetone or hexane: methanol as eluent. The ratio of the two components is used in a ratio of 80:20 (v / v) to 95: 5 (v / v), preferably 90:10 (v / v), and the eluate has a flow rate of 0.9 to 1.1 ml / min. It flows to and obtains the urushiol containing fraction which is excellent in anticancer activity. The urushiol fraction is eluted at a residence time of about 80 to 190 minutes. Most preferably, to a column filled with activated silica gel, crude urushiol is added in an amount of 1/5 of the weight of silica gel, and a hexane: acetone (90:10 (v / v)) mixed solvent is flowed at a flow rate of 1 ml / min. The urushiol containing fraction (fraction 7) is collected between 90 and 160 minutes residence time.

Further, the urushiol main component can be separated by purifying the urushiol-containing fraction (fraction 7) obtained above by high pressure liquid chromatography. At this time, acetonitrile: water (80:20) mixed solvent containing 0.002% acetic acid can be used as the mobile phase, and Novapak-C18 (Waters Co., USA) and μBondapak-C18 (Waters Co., USA) as column fillers. Or Deltapak-C18 (Waters Co., USA).

The urushiol containing fraction obtained by the method of the present invention (fraction 7) is a collection of urushiol monomers as shown in FIG. 1 by HPLC analysis and the main urushiol monomers are as follows:

[Formula 1]

C15

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH = CHCH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH = CHCH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH = CH ₂

2-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH = CH ₂ CH ₃

2-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH = CH ₂

C17

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH ₂ CH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH = CHCH ₃

1-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH = CHCH ₂ CH = CHCH ₂ CH = CH ₂

The fraction (fraction 6) obtained by HPLC as a main component of the urushiol containing fraction (fraction 7) of the present invention contains only the monomers indicated in bold above.

The urushiol-containing fraction (fraction 7) of the present invention is about 17 times as compared to the conventional anticancer drug platinum anticancer drug tetraplatin (Traplatin; Bristol-Myers Squibb, USA) about the mouse blood cancer cell line L1210 (ATCC CCL 219), human body The anticancer effect is about four times higher for the lung cancer cell line PC-9 (acquired from the Japan National Cancer Institute) and about 105 times higher for the human lung cancer cell line A427 (acquired from the Japan National Cancer Institute).

In addition, the urushiol-containing fraction (fraction 7) of the present invention is investigated for acute toxicity according to the general toxicity test standard, for example, the National Institute of Health Safety Toxicity Test Standard (National Institute of Safety Notice No. 94-3 (1994)). LD ₅₀ was 2.75 g / kg with little toxicity.

The anti-cancer composition may be prepared by mixing the urushiol fraction of the present invention with a pharmaceutically acceptable carrier as an active ingredient. The anticancer composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, and the like, and preparations for tablets, capsules, powders, granules, suspensions, emulsions or parenteral administration by conventional methods It may be formulated as a unit dosage form or several dosage forms such as a pharmaceutical preparation.

Antibiotics containing the urushiol fraction of the present invention as an active ingredient can be parenterally or orally administered as desired, and can be administered in one to several times a day. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1 Separation of the Urushiol Fraction

(Step 1) Separation of crude urushiol

After adding three times the volume of acetone to the columnar lactose sampled in 1994, it was left to stand at 4 ° C for 1 hour, whereby urushiol and other lactate components, ie, rubbery, nitrogenous substance, and lacquer, were added to the upper layer. The other seven liquid components were separated into the lower layer. Only the supernatant containing urushiol was centrifuged at 4,000 rpm for 15 minutes and then filtered with filter paper (Whatman No. 2). Zourushiol was prepared by evaporating the solvent from the filtrate using a rotary vacuum evaporator (Labo rota 300, Resona Co., Switzerland).

(Step 2) Silica Gel Adsorption Column Chromatography

40 g of silica gel (230-400 mesh, for chromatography, Merck Co.) was activated at 130 ° C. for 3 hours and then n-hexane was added to make a slurry. This silica gel slurry was charged to a glass column (2.9 x 45 cm), and then 8 g of zourushiol obtained in (step 1) corresponding to 1/5 of the silica gel weight was added. The hexane: acetone mixed solvent was used as the eluent, increasing the ratio of the two components in steps of 90:10 (v / v), 50:50 (v / v) and 0: 100 (v / v) in steps of 1 ml / min. Fractions (fraction 7) with a retention time of 90 minutes to 160 minutes eluted with a solvent having a hexane: acetone concentration ratio of 90:10 (v / v) and a fraction of hexane: acetone at a flow rate of 50:50 (v / v). v) fraction of fraction of 175 minutes to 245 fractions eluted with solvent (fraction 8) and fraction of fraction of fraction of 255 minutes to 330 fractions eluted with solvent with a hexane: acetone ratio of 0: 100 (v / v). 9) was obtained.

(Step 3) High Pressure Liquid Chromatography (HPLC)

In order to separate only urushiol main component using HPLC, fraction 7 obtained in (Step 2) was mixed with methanol 1: 1 (v / v) and then pre-treated with Sep-pak (C ₁₈ type, Waters, USA). It filtered by the scanning filter (syringe filter, 0.2 micrometer, Satorius). The filtrate was analyzed by HPLC (Waters 'Nova-Pak C ₁₈ , 3.9 x 300 mm) and semi-prep columns (Waters' Delta Pak C ₁₈ -300 mm, 7.8 x 300 mm, 15 μ, spherical). The urushiol main component fraction (fraction 6) was isolated using chromatography 441) under the following conditions:

Mobile phase: 0.002% acetic acid containing acetonitrile: water (80:20)

Sample injection volume: 20 μl (analysis column), 40 μl (semi-prep column)

Flow rate: 1.0 ml / min (analytical column), 1.6 ml / min (semi-prep column)

Detection: 441 UV detector, Wavelength: 254 nm

The chromatogram obtained as a result of the HPLC is shown in FIG. 1 (B).

On the other hand, the zourushiol obtained in (Step 1) was pretreated with C ₁₈ Sep-pak as described above, filtered through a 0.2 μm injection filter and analyzed by HPLC, and after 50 minutes of residence time as shown in FIG. 1 (A) Many components were eluted, but when fraction 7 was analyzed under the same conditions, peaks after 50 minutes did not appear as shown in FIG. This is similar to the result shown in the analysis of HPLC after separation of urushiol, in which 95% or more are monomers (Y. Du, R. Oshima and H. Iwatsuki, J. of Chromatography , 295 , 179-186 (19984)). Do. Therefore, the component eluted after 50 minutes in the urushiol is considered to be a dimer or oligomer in which two or more urushiol monomers are polymerized.

As can be seen in FIG. 1, fraction 7 was a combination of only urushiol monomers (FIG. 1B), and fraction 8 was the main component of thishiol component found in the literature (Y. Du et al., Supra). (FIG. 1A).

Example 2: Anticancer Effect of Urushiol

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test method (J. Carmicheal et al . , Cancer Res. , 47) , 936 (1987)), and subject cell lines such as L1210 (mouse blood cancer cell line; ATCC CCL 219), PC-9 (Japan National Cancer Institute), KATO III (ATCC HTB 103) and A427 (Japan National Cancer Institute). Suspension was suspended in 0.2% Trypane Blue to make a single cell suspension, and the suspension was placed in a cytometer (Hemacytometer, Superior Co., Germany) and the viable cells counted by an inverted microscope.

To each well of a 96-well cell culture plate, a mixed medium of 90% RPMI-1640 medium (Gibco) and 10% fetal bovine serum was added and each cell line was added in an amount of ³ × 10 ³ cells / well. As a test group, each solution of 10-fold dilutions of fractions 6, 7, 8, and 9 and the comparator Tetraplatin from 0.0003 μg / ml to 30 μg / ml in phosphate buffered saline (PBS) was added to each well. In the control, PBS was added to the wells. Cell culture plates were incubated at 37 ° C., 5% CO ₂ for 3 days for L1210 cell lines and 4 days for other cell lines. Thereafter, 50 μl of 2 mg / ml MTT solution was added to each well, followed by further incubation for 4 hours at 37 ° C. and 5% CO ₂ . Next, the culture supernatant was aspirated off using a multiple channel aspirator so that only 30 μl of the culture solution was left, and 150 μl of dimethyl sulfoxide (DMSO) was added to each well, followed by shaking for 15 minutes, or After addition of acidified isopropanol without removing the culture, the optical density (OD) was measured at 540 nm with an ELISA reader (ELISA reader, Dynatech, MR5000).

Tetraplatin, used as a comparator, is a second-generation platinum complex anticancer agent currently being developed by Bristol-Myers Squibb in the United States, which is more toxic than commercially available carboplatin or cisplatin. And anti-cancer effects are about 10 times higher (Noh Kim et al., 3rd generation platinum anticancer drug development, 3rd generation anticancer drug development report, Seoul National University College of Medicine and Sunkyong Industry Research Institute, p229, 1992).

IC ₅₀ (50% inhibitory concentration) refers to the concentration at which the average optical density of the test well is 50% of the average optical density of the control well, as measured after color reaction with MTT dye. The experiment was repeated three times, and the cytotoxicity (%) of the test group was determined by the following formula, and then the 50% Inhibition concentration (IC ₅₀ ), ㎍ / mL was linear regression equation. Obtained by Background wells are wells containing only PBS.

[Equation 1]

Cytotoxicity (%) =

The test results are as follows.

(1) Anticancer Effect on L1210 Cell Line (Mouse Blood Cancer Cell Line)

The cytotoxicity (%) of fractions 6, 7, 8, 9 and tetraplatin for the L1210 cell line showed the same trend as in FIG. 2, and IC ₅₀ was 1.82 μg / ml, fraction 7 as shown in Table 1. 0.11 μg / ml of silver, 30 μg / ml of fraction 8, 20.56 μg / ml of fraction 9, and 0.34 μg / ml of tetraplatin. 3.1 times better than. In addition, fraction 7, which is a combination of monomers alone, was 17.1 times higher in cytotoxicity than the single component of fraction 6, the main component of urushiol. This is thought to be a synergistic effect between the ingredients frequently found in natural products. FIG. 6 shows MTT test results for L1210 cell lines, the darker the color of the wells in which the cancer cells grew actively, and the lighter the color of the cancer cells, the more proliferation of the cancer cells was suppressed.

(2) anticancer effect on PC-9 cell line (human lung cancer cell line)

Cytotoxicity (%) of fractions 6, 7, 8, 9 and tetraplatin for PC-9 cell lines showed the same trend as in FIG. 3, and IC ₅₀ was 12.12 µg / ml, as in Table 1, Fraction 7 had 1.01 μg / ml, Fraction 8 had 30 μg / ml or more, Fraction 9 had 30 μg / ml or more, and tetraplatin had 3.96 μg / ml. It was 3.9 times higher than platinum. In addition, the cytotoxic effect of fraction 7 was 12 times higher than that of fraction 6. Compared to the cytotoxicity results for the L1210 cell line, all components showed a 10-fold decrease in effect, and the relative cytotoxicity index for the comparative tetraplatin was similar to that of L1210.

FIG. 7 shows 30 μg / ml, 3 μg / ml and 0.3 μg / ml of fraction 7, respectively, in a mixed medium of 90% RPMI-1640 medium (Gibco) and 10% fetal bovine serum, where PC- 9 cell lines were incubated for 4 days at 37 ° C. and 5% CO _2, and then the effect of inhibiting cell proliferation was confirmed under an optical microscope. As a result of measuring the cell proliferation inhibition by the ELISA reader, cancer cell proliferation was inhibited more than 90% in wells treated with 30 μg / ml fraction 7, 32% inhibited cancer cell proliferation in 3 μg / ml treated wells, 0.3 μg / Cancer cells proliferated in ml treated wells.

(3) anticancer effect on KATO III cell line (human gastric cancer cell line)

The cytotoxicity (%) of fractions 6, 7, 8, 9 and tetraplatin for the KATO III cell line showed the same trend as in FIG. 4, and IC ₅₀ was 11.47 μg / ml, fraction 6 as shown in Table 1. 7 is 1.85 μg / ml, Fraction 8 is at least 30 μg / ml, Fraction 9 is at least 30 μg / ml, and tetraplatin is 0.47 μg / ml. It was 3.9 times lower than in Latin. In addition, the cytotoxic effect of Fraction 7 was 9.6 times higher than that of Fraction 6, supporting the fact that there is a synergistic effect among the components.

(4) anticancer effect on A427 cell line (human lung cancer cell line)

For the A427 cell line, only fraction 7 with the best anticancer effect was tested in comparison with tetraplatin. Cytotoxicity (%) of fraction 7 and tetraplatin showed the same trend as in FIG. 5, and IC ₅₀ was 0.018 µg / ml in fraction 7, and 1.9 µg / ml in tetraplatin, as shown in Table 1. As in 2, the cytotoxic effect of fraction 7 was 105.5 times higher than that of tetraplatin. Thus, fraction 7 has a significant meaning in revealing the cancer cell proliferation inhibitory mechanism because it shows a very good cancer cell proliferation inhibitory effect on the A427 cell line that is characterized by growing on the base wall.

TABLE 1

TABLE 2

According to the method of the present invention, it is possible to separate the urushiol fraction having excellent anticancer effect from the lacquer. The urushiol fraction isolated by the method of the present invention exhibits superior anticancer activity than conventional anticancer agents but does not cause adverse effects on the human body.

Claims (4)

(A) adding acetone to sap of lacquer to separate crude urushiol component, and (B) adding the crude urushiol component to the column in an amount of 1/6 to 1/4 of the weight of silica gel, and adding hexane: acetone. From the sap of the lacquer tree comprising the step of collecting a fraction of the residence time 90 to 160 minutes by separating the silica gel adsorption column chromatography using a mixed solvent having a ratio of 80:20 to 95: 5 (v / v) as an eluent A method for separating an anticancer active fraction containing urushiol. The method according to claim 1, wherein in the step (a), 2-4 times the volume of acetone is added to the sap of the lacquer tree, and the mixture is left at 3 to 5 ° C. for 30 minutes to 3 hours, and the supernatant is separated and centrifuged. Separating, centrifuging the supernatant, and evaporating the solvent from the filtrate to obtain crude urushiol. An urushiol-containing fraction having anticancer activity, isolated from the sap of lacquer by the method of claim 1. An anticancer drug composition comprising an urushiol-containing fraction having anticancer activity isolated from sap of lacquer trees by the method of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.