KR20030016774A - Compositions Comprising Antofine And The Use Thereof - Google Patents

Abstract

PURPOSE: A composition containing an effective amount of antofine derived from an extract of Cynanchum paniculatum Kitagawa is provided which exhibits enhanced cytotoxicity toward tumor cells and can be used as an anticancer or a cancer cell growth inhibitor. CONSTITUTION: Cynanchum paniculatum Kitagawa is extracted with water or alcohol, the extract is subjected to silica gel column chromatography, and the silica gel column chromatography eluent is subjected to thin-layer chromatography to separate 7-demethoxytylophorine(antofine) of formula(I). After extracting in water or alcohol or before being subjected to silica gel column chromatography, it can be extracted in methylene chloride. A fraction containing a high concentration of antofine is screened by measuring cytotoxicity toward human tumor cells and/or human cancer cell growth inhibiting activity.

Description

Compositions Comprising Antepine and Uses thereof {Compositions Comprising Antofine And The Use Thereof}

The present invention relates to a new anticancer agent. More specifically, it is derived from the natural herbal herbal extracts, and relates to the compound antofine and its derivatives represented by the following general formula (I) which is toxic to human lung cancer cells and human colon cancer cells and inhibits the growth of these cancer cells will be.

According to recent data released by the Korea National Statistical Office, the mortality rate from cancer increased by 3.9% over the past 10 years, from 110.4 per 100,000 population in 1990 to 114.7 in 1999. As of 1999, mortality rates increased from 14.4 in 1990 to 22.1 in 1999, colorectal cancer (4.5 in 1990 → 7.9 in 1999), and pancreatic cancer (3.3 in 1990 → 1999). 5.4), female breast cancer (3.5 in 1990 → 4.8 in 1999) and prostate cancer (1.6 in 1990 → 1.8 in 1999). Lung cancer has increased significantly due to the aging of the population, the increase in the smoking population, and the deepening of air pollution.In addition, colon cancer, pancreatic cancer, breast cancer, and prostate cancer are also affected by high-fat diets, environmental pollutants, and alcohol consumption. It is steadily increasing due to the increase.

Because cancer cells have many similar properties to normal cells, it is not easy to remove cancer cells without damaging normal cells. However, cancer cells are distinguished from several normal cells, firstly, cancer cells are not regulated in cell proliferation, secondly, they are relatively lacking in differentiation characteristics, and thirdly, they invade and metastasize to surrounding tissues. . Normal cells proliferate by receiving signals from growth factors as needed, while cancer cells have low dependence on growth factors and do not have contact inhibition, which inhibits growth by contact with surrounding cells. Secrete angiogenic factor to promote metastasis. In addition, cancer cells are not differentiated, do not cause apoptosis or programmed cell death, and have genetically unstable characteristics. Genetic instability of cancer cells is very important in cancer progression and is known to induce resistance to chemotherapeutic agents (Folksman et al., Science, 235, 442-447 (1987); Liotta et al., Cell , 64, 327-336 (1991)).

As a cancer treatment method, surgery, chemotherapy and radiation therapy are widely used. Among them, chemotherapy is the most studied. Currently used chemotherapeutic agents include alkylating agents, metabolic inhibitors, DNA-binders, drugs that act on tubulin, and topo. Although there are isomerase inhibitors and other drugs that act on DNA, the development of new anticancer drugs is still urgently needed because they are limited in treating various types of cancers, especially solid cancers with slow growth.

There are about 150,000 plants on earth. The two plants are an important resource in the development of cancer is, and its examples are separated from Cantabria Lantus Rosedale mouse (Catharanthus roseus) is the empty bra Destin (vinblastine), grape pilrum Pell tatum (Podophyllum peltatum) with the alkaloids (alkaloid) separated from the lignans ( Taxol, Camptotheca accuminata , isolated from etonan, teniposide, and taxus brevifolia , a semisynthetic derivative of lignan podophyllotoxin ) separated from the camptothecin (camptothecine), three arms TAXUS haring Estonian bar drupa Asia (Cephalotaxus harringtonia var. drupacea) the Queer haring tonin (homoharringtonin), Fusarium solani (Fusarium solani) separated from the separation from 4- Ipomeanol (4-ipomeanole) and the like (Cordell et al., Bioactive Natural Products, CRS Press, 195-220 (1993); Cragg et al., Human Medicinal Agents from Plants, American Chemical Society, 534-553 (1993)).

Accordingly, the present inventors have studied the anticancer agent of the herbal medicine origin that is widely used in herbal medicine, and as a result, antopine isolated from the herbal components confirmed excellent anticancer activity and came to complete the present invention.

The present invention provides an anticancer pharmaceutical composition comprising an effective amount of antepine and a method for preventing or treating cancer by administering the pharmaceutical composition to a patient.

The present invention provides a method for separating anthine from gourds.

The present invention provides a method for screening a fraction containing high concentrations of anthopines, characterized by measuring apoptosis of cancer cells or measuring growth inhibition of cancer cells.

1 is a graph showing the effect of antagonist human lung cancer cytotoxicity.

Figure 2 is a graph showing the cytotoxic effect of anthofine in human colon cancer.

Figure 3 is a micrograph showing the growth inhibitory effect of antagonist human colon cancer cells.

Figure 4 is a flow cytometer analysis graph showing the effect of antagonist on the cell growth cycle of human colon cancer cells.

The present invention relates to the prevention and treatment of cancer. Specifically, the present invention provides an anticancer pharmaceutical composition comprising an effective amount of antepine and a method for preventing or treating cancer by administering the pharmaceutical composition to a patient. Antepine is a natural substance represented by the following general formula (I).

[Formula (I)]

Antepine is an alkaloid found in a few species of the Asclepiadaceae plant. In the present invention, the antepine was isolated from the extract of acid seabacterium which is used as a natural product herbal. Cynanchum paniculatum Kitagawa is a perennial herbaceous genus of Asclepiadaceae, which grows in the mountains of Korea and is distributed in Japan, Manchuria, and China. There are deceived bakjugarigwa baekmikkot 100 species worldwide and about 10 species distributed in the country, but the roots of herbal original baekmikkot rice country in baekmikkot (Cynanchum atratum Bunge) is used as a medicine, civil baekmikkot (Cynanchum ascyrifolium Matsmura) and sanhae night (Cynanchum paniculatum The roots of Kitagawa are all mixed with white rice, and the roots of Sanhaebak are marketed as white rice (Kim Yoon-sik and others, Book of Korean plants, Academy books, 446-449 (1985)).

The root herb of Sanhaebak is called white rice, while the outpost herb is called Seo Jang-kyung (Lim, Rok-jae and other medicinal plants, Korean Cultural History (1999)). Although there are few records of classics about root herbal medicines, Seo Jang-kyung, an outpost herb, cures, refreshes, and arouses the symptoms of classic detoxification, epidemics, quartet, warmth, or confusion. It is said to cure stiffness in the lower back and knees, to release snake venom, and to cure swelling symptoms, shingles, cirrhosis, ascites, dysmenorrhea, dysmenorrhea, and dysmenorrhea. Chinese Medicine Dictionary, Jungdam, (1999)). Seo Jang-kyung is also known as bamboo leaf seed, bamboo, seonju, cheon bamboo, mountain stream, sedge or ryoza bamboo. .

Recently, there have been literature reports of over 1% paeonol as an active ingredient in wild seaweed outposts, showing analgesic and sedation (You, Chinese Medica, Harwood Academic Publishers, 270-278 (1998); Lee et al., Journal of Pharmacognosy, 11 , 66-68 (1980)), roots and rhizomes are known to contain saponins, steroids, diterpenoids (Sugama et al. , Chem. Pharm. Bull., 34 , 4500-4507 (1986); Qui et al., Abst. Chi.Med., 1 , 113-129 (1986)), and acidic peppers are also tumor necrosis factors. (Cho et al., Nat. Prod. Sci., 5 , 12-19 (1999)), blood cholesterol lowering effect and antibacterial activity (You, Chinese Medica, Harwood Academic Publishers) (1998). However, no cancer cytotoxicity and cancer cell growth inhibitory substances have been isolated from acid seabacterium.

The present inventors have experimentally demonstrated that the anthopine of the formula (I) isolated from Sanhaebak has cancer cytotoxic effect and cancer cell growth inhibitory activity. Antepine showed high cancer cell toxicity activity as IC ₅₀ was 3.2 pg / ml as a result of toxicity evaluation on human lung cancer cells in vitro (Fig. 1), and also in the toxicity evaluation result on human colon cancer cells. ₅₀ was 4.3 pg / ml, indicating high cancer cytotoxic activity (FIG. 2). This includes existing anticancer agents such as taxol (IC ₅₀ = 0.007 μg / ml, human lung cancer cells; IC ₅₀ = 0.003 μg / ml, human colon cancer cells) and ellipsine (IC ₅₀ = 0.5 μg / ml, human lung cancer cells; IC ₅₀ = 0.3 μg / ml, human colon cancer cells).

On the other hand, using human colon cancer cells to examine the mechanism of action on cytotoxicity, it was found that anthofine stops the growth of cancer cells rather than killing cancer cells directly (Fig. 3), G2 / M in the cancer cell growth cycle It has been shown to inhibit cancer cell growth by stopping the phase (arrest) (FIG. 4).

Accordingly, the present invention provides an anticancer composition comprising an antepine in an amount sufficient to treat cancer, and a method for treating cancer cell cancer by administering the anticancer composition. The composition of the present invention may comprise a pharmaceutically acceptable carrier. Carriers include solvents, dispersion media, absorption retardants, and the like commonly used in the pharmaceutical art. The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Thus, the compositions of the present invention may be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. can do. Preferred formulations are injections. Dosage is determined in consideration of the skill of the art depending on the type and extent of the disease, age, sex, etc. of the patient.

Furthermore, the present invention provides a cancer prevention to cancer treatment composition comprising an effective amount of an antepine and a method for treating cancer by administering the same. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Carriers include solvents, dispersion media, absorption retardants, and the like commonly used in the pharmaceutical art. The pharmaceutical compositions of the present invention may be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. Can be. Preferred formulations are injections. Dosage is determined in consideration of the skill of the art depending on the type and extent of the disease, age, sex, etc. of the patient. The pharmaceutical composition of the present invention can be used to treat diseases such as brain cancer, ovarian cancer, chest cancer, prostate cancer, lung cancer, liver cancer, colon cancer, melanoma, cervical cancer, uterine cancer, gastric cancer, kidney cancer, bladder cancer, epidermal cancer and lymphoma Can be.

The present invention provides a method for separating anthopines from plants, preferably gourds, and more preferably from domestic seaweed ( Cynanchum paniculatum Kitagawa). Antepine may be separated from the plant containing the same by an extraction method. The method for separating an antepine of the present invention comprises the steps of: a) extracting a dry plant sample, for example acacia, with water or alcohol; b) silica gel column chromatography of the extract; And c) TLC the silica gel column chromatography eluate. The anthopine separation method of the present invention may optionally include extracting with methylene chloride after step a) and before step b).

The present inventors have separated the anthopines of the present invention from the acidic bacterium using the bioactivity assay based on the anticancer effect (Examples 1 and 2). The isolated compounds were identified as anthopines with phenanthroindolizidine alkaloid backbone through various physicochemical properties and instrumental analysis data. Antofine derivatives have recently been reported to be isolated from the same genus plant, Cynanchum vinvetoxicum (Staek et al., J. Nat. Prod., 63 , 1584-1586 (2000)). There have been no reports of separation from gourds.

It has been proved that the anthopine of the present invention can inhibit the growth of cancer cells. The present invention provides a food composition for cancer prevention or cancer cell growth containing anthopine. The food used as a base material is not specifically limited, Various foods, such as drinks, confectionery, bread, noodles, seasonings, such as water, a soft drink, or a fruit drink, can be based on. The food of this invention is easily made by adding the process of adding the composition of this invention mentioned above to the manufacturing process of the base food, or by adding the process of adding the composition of this invention mentioned above after manufacture of the food of base materials. You can get At this time, you may add a taste and odor correction agent as needed. The content of the above-mentioned composition in the food of the present invention varies depending on the type and preference of the food of interest, but the plant extract containing anthpine is sufficiently utilized to fully utilize the cancer prevention effect and health treatment effect that antine originally had. It is preferable to set it as 0.1 weight% or more. The food of this invention obtained in this way is a food which can fully utilize the health prevention effect and health treatment effect which antepine originally had.

The present invention provides a method for screening fractions containing high concentrations of anthopines by measuring cytotoxicity against human cancer cells and / or human cancer cell growth inhibition. The bioactivity assay of an antepine provided by the present invention can be usefully used to find a fraction in which an antepine is concentrated at a high concentration. The bioactivity assay of the present invention is preferably a sulfohodamine B (SRB) assay (Lee et al, Chemico-Biol. Interact., 115 , 215-228 (1998)) or flow cytos as described in the literature. Flow cytometer analysis.

The following examples are intended to illustrate the invention. Therefore, the scope of the present invention should not be limited by the following examples.

<Example 1>

Isolation and Purification of Antofine

3 kg of dried acidic seaweed was extracted with 100% methanol three times for 3 hours in a water bath. The extract was filtered and concentrated under reduced pressure to give 370 g of acidic methanol extract. By dispersing the methanol extract in distilled water and extracted with hexane (hexane), methylene chloride (methylene chloride), ethyl acetate (ethyl acetate), n- butanol (n -buthanol) in order to obtain a soluble fraction and aqueous layer of each solvent in each fraction The activity experiment was carried out according to the method of Example 3.

The most active methylene chloride fraction (73 g) in the solvent fraction was subjected to silica gel column chromatography with a chloroform / acetone (50: 1-1: 1) gradient and divided into 10 fractions. Take 9 fractions with the highest activity (IC ₅₀ = 1.0 μg / ml, human lung cancer cells; IC ₅₀ = 1.3 μg / ml, human colon cancer cells) and take a gradient of chloroform / methanol (20: 1-1: 1) Silicagel column chromatography was performed to obtain five fractions. Preparative TLC; methylene chloride: methanol = 8: 1, for the second most active fraction (IC ₅₀ = 0.007 μg / ml, human lung cancer cells; IC ₅₀ = 0.004 μg / ml, human colon cancer cells) Pure active ingredient I (5 mg) was isolated by Rf = 0.55) and prep HPLC (preparative HPLC, methanol: water = 97: 3). The separated compound was a pale yellow powder, which was well soluble in methylene chloride and methanol solvent but insoluble in water and acetone.

Example 2

Determine the structure of the compound

Analytical HPLC was used to evaluate the purity of the components isolated in Example 1. Various spectroscopic experiments are performed in the state identified as a single component, that is, H ¹ -NMR, C ¹³ -NMR, DEPT, H ¹ -H ¹ COZY, H ¹ -C ¹³ COZY, HMBC, and positive FABMS. The structure was determined. In order to determine the structure, 1D spectra of ¹ H and ¹³ C were first obtained, and carbon multiplicity was examined by DEPT (Distortless Enhancement Polarization Transfer) experiment. Complexity of the isolated compounds is shown in Table 1. The position of methoxy groups of 55.5, 55.9 and 56.0 ppm was clearly identified in ¹³ C-NMR. In addition, the chemical shift of carbon shows an aromatic ring between 120 and 160 ppm, and the CH-group in the aromatic ring is present over 103 to 115 ppm. It can be seen that the peak corresponding to the CH ₂ -group in the aliphatic cyclic ring appears in the upfield, and also the peak shifted to the downfield by adjacent nitrogen (N). It can be seen that it exists. Heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) two-dimensional (2D) experiments were also obtained with 2k x 256 (t2 x t1) data values.

The value obtained from the 1D 1H-NMR spectra was consistent with the structure reported by Baumgartner et al. (Phytochemistry, 29 , 3327-3330 (1990)), and appeared to be nearly identical to antofine. All H numbers and J values match, and their multiplicity also matches. Also, the positive FAB mass-spectroscopy measured M + 1 as 364.3 and molecular weight as 363. The alkaloid reaction test was performed to confirm that the above material was an alkaloid, and when it was sprayed with platinum chloride, it showed a light pink positive reaction. Therefore, by combining the above results, the present inventors confirmed that the single substance separated from acid seaweed is anthopine (antofine), which is a kind of alkaloid.

Table 1. NMR spectral data of compound I (CDCl ₃ , 600 MHz)

Example 3

Determination of Efficacy of Human Cancer Cell Toxicity in Vitro

The effect of anthopines on cell growth was measured by sulfohodamine B (SRB method) (Lee et al, Chemico-Biol. Interact., 115 , 215-228 (1998)). Human cancer cell lines (A549, human lung cancer cells; Col2, human colon cancer cells) were passaged in MEME medium containing 10% FBS, 1% PSF and the like. In each well of a 96-well plate, 10 μl of sample dissolved in 10% DMSO and 190 μl of the cell suspension (5 × 10 ⁴ cells / ml) (MEME medium) were added and incubated for 3 days. 190 μl of the cell suspension was added to at least 16 wells and incubated for 30 minutes to be used as a zero-day control before the experiment. The cultured cells were fixed with 10% trichloroacetic acid (TCA) and stained with SRB solution, and the dye solution was dissolved with 10 mM Tris-base, and the absorbance was measured at 515 nm. In the case of incubation in 10% DMSO as a control, the cell viability according to each test substance treatment was converted according to the following equation.

When the control group was not treated with the sample at 100%, the value of the sample treatment group was expressed as a percentage of the control group, and each test substance treatment was calculated as the average value ± SEM of the double or triple test. IC ₅₀ value is the concentration of test substance for 50% survival. 1 and 2 show the effects of anthopine on human cancer cells.

Antepine has been shown to be cytotoxic to human cancer cells in a concentration-dependent manner. The IC ₅₀ for human lung cancer cells was 3.2 pg / ml, and the IC ₅₀ for human colon cancer cells was 4.3 pg / ml, indicating that the antitumor taxol (IC ₅₀ = 0.007 μg / ml, human lung cancer cells; IC ₅₀ = 0.003 μg / ml, human colon cancer cells) and ellipticine (IC ₅₀ = 0.5 μg / ml, human lung cancer cells; IC ₅₀ = 0.3 μg / ml, human colon cancer cells) I have it.

Example 4

Observation of Cell Morphology and Cell Cycle in Vitro

Col2 cells, which are human colon cancer cells, were suspended in 10% FBS-MEME at 2 x 10 ⁵ cells / ml. 10 ml of the cell suspension was attached to a 10 cm cell culture dish. Test substance (50 pg / ml concentration) was added and then incubated for a certain time at 37 ℃, 5% CO ₂ . First, the cells were cultured for 24 hours to observe the morphological changes of the cells, and then the culture medium was removed and washed twice with PBS (phosphate buffered saline). The morphology of the cells was observed at 100 times magnification under fluorescence microscope. The results are shown in FIG.

The effect of the test substances on the cell cycle was measured by the following method. Test substance (50 pg / ml concentration) was treated with the cells. After a certain time (24 hours, 48 hours, 72 hours, 96 hours), trypsin treatment to detach the cells attached to the dish, centrifuged cells suspended in the cell medium and cells detached by the trypsin treatment The cells were washed twice with 10 ml of tris-saline solution (pH 7.0) and then fixed with 1.5 ml of 90% ethanol. After washing twice at room temperature with 1 ml of phosphate-citrate buffer and shading staining with propidium iodide (PI) buffer for 30 minutes, the PI buffer was removed and suspended in 1 ml of PBS. The cell suspension was transferred to a 5 ml polystyrene round bottom tube and subjected to flow cytometer analysis. The results are shown in FIG.

As a result of the experiment, it can be seen that the anthopine has a mechanism of stopping cell growth in the G2 / M phase in the cell cycle for colon cancer cells.

Antepine of the present invention can be used as an anticancer agent or a cancer cell growth inhibitor that can kill cancer cells because of its high cytotoxicity against human cancer cells. The present invention also provides a method for separating the antepine from an acidic herb, a natural herbal substance, and a bioactivity assay for screening the activity of the antepine.

Claims (6)

An anticancer pharmaceutical composition comprising an antepine represented by formula (I) [Formula I] Food composition for cancer prevention or cancer cell growth, comprising an antepine represented by the formula (I) The composition according to claim 1 or 2, wherein the anthopine is isolated from the acidic seaweed. A) extracting acidic watermelon belonging to the cabbage family with water or alcohol; b) silica gel column chromatography of the extract; And c) TLC the silica gel column chromatography eluate. The method of claim 4, comprising extracting with methylene chloride after step a) and before step b). Screening fractions containing high concentrations of antagonists by measuring cytotoxicity against human cancer cells and / or human cancer cell growth inhibition