KR100416120B1 - Anti-tumor lignan compound isolated from anthriscus sylvestris hoffmann(umbelliferae) - Google Patents

Abstract

PURPOSE: An anti-tumor lignan compound isolated from Anthriscus sylvestris HOFFMANN(Umbelliferae) is provided, which compound has similar or improved anti-tumor activity to the prior anti-tumor compounds and no toxicity. CONSTITUTION: The anti-tumor lignan compound isolated from Anthriscus sylvestris HOFFMANN(Umbelliferae) represented by formula (1) is provided. The method for isolating the Anti-tumor lignan compound isolated from Anthriscus sylvestris HOFFMANN(Umbelliferae) of formula (1) comprises the steps of: cutting roots of Anthriscus sylvestris HOFFMANN(Umbelliferae); extracting the cut roots of Anthriscus sylvestris HOFFMANN(Umbelliferae) with methanol in 5 times; fractioning the methanol extract of Anthriscus sylvestris HOFFMANN(Umbelliferae) with solvent of hexane, chloroform, acetic acid ethyl, butanol and water to obtain an active fraction; subjecting the active fraction to silica gel, Sephadex and RP-18 chromatography to obtain anti-tumor compounds; and isolating and purifying an anti-tumor compound, Angeloyl podophyllotoxin, by subjecting the anti-tumor compounds to prep HPLC.

Description

New Lignan System Antitumor Substances Isolated from Natural Products

The present invention relates to a novel compound of the lignan line isolated from Anthriscus sylvestris HOFFMANN (Umbelliferae). Specifically, the present invention relates to Angeloyl podophyllotoxin of the following Chemical Formula 1 having antitumor activity isolated from the root of Jeonho and to a method for extracting the substance.

[Formula 1]

Various treatment methods for the treatment of malignant tumors are being developed, and local therapies such as surgery, radiation therapy, and systemic therapy such as chemotherapy and immunotherapy are used. Among them, chemotherapy plays a big role in increasing survival rate. Doing However, chemotherapeutic agents have a strong toxicity and side effects for actively dividing cells such as cancer cells as well as bone marrow cells, causing opportunistic infections such as anemia and other fungi, digestive disorders, and renal disorders.

Attempts to create new drugs from natural products, especially herbal medicines, are common because of the high likelihood and probability that new compounds with new actions, skeletal structures, and new substances with less toxicity and side effects are found in traditionally used natural products than synthetic compounds. In response to this trend, the present inventors have begun research.

Jeonho is a perennial herb that is wild in Europe and Asia. Young leaves are edible, and its roots are Jeonho in Korea. Boiled premises have been used to treat asthma, bruises and edema.

The present inventors found that the hexane and chloroform extracts of the foregoing were cytotoxic to human tumor cell lines, and traced the active substances using a series of solvent extraction methods and ktomatoprey, and thus, cancer cells of animals (rats). Lignan lineage with compounds known as components of other natural products, as well as anthricin, a compound known to be cytotoxic to oleic acid, Molelensin, Falcarindiol and Bursehernin Angeloyl podophyllotoxin, a novel compound of was isolated. In addition, as a result of assaying the activity of human tumor cell lines of these substances, it was found that the activity is excellent, and completed the present invention. Grape filotoxin, the mother of angeloyl grapephytotoxin, is a compound that has anticancer activity but is highly toxic and cannot be developed as a medicine. In order to solve this problem, many derivatives have been developed. Thus, it is well known that etoposide and the like have been developed, but derivatives having angeloyl groups are not yet known.

Cancer is caused by internal or external causes of normal cell DNA to cause somatic mutations, cells with genetic abnormalities are replicated, and genetic abnormalities are promoted by various factors, such as hormones or chemicals. It is known that aberrant proliferation of cells occurs when cells in good condition to proliferate due to the change of gene expression are preferentially selected and grow. Thus, cancer treatment is initiated by exposure to chemotherapy (anticancer) and carcinogen, a method of selectively destroying cancer cells that abnormally proliferate from normal cells (carcinogenesis). initiation, chemoprevention, which blocks further progression in the promotion phase. Among the compounds that can be used as anticancer agents, anticancer agents such as vinblastine, vincristine, bleomycin, doxurubicin and etoposide, which are currently used in the clinic, and chemosuppressants ( Retinoids, vitamins, polyphenols, flavonoids, etc., which are spotlighted as chemopreventive agents, are materials developed from natural products. An experimental approach to the development of anticancer chemopreventive agents in natural products is bioassay-directed fractionation. The plant extracts selected by various routes were tested in an appropriate bioassay system, followed by fractionation of the active fractions, followed by fractionation, followed by activity in the same assay system. It can play an important role either as a substance or as a precursor.

Applicants of the present invention showed a high activity in the Jehoho extract as a result of measuring the activity of 200 plant extracts using the MTT search method as a bioassay system (bioassay system). After selecting Jeonho as a research material, a new lignan-based substance called angeloylpodophyllotoxin was isolated and purified by bioassay-directed fractionation. This new substance is expected to have high development potential as an anticancer drug, as it shows activity equivalent to that of the toxin rubicin and etoposide currently used in clinical trials and MTT screening method.

Accordingly, it is an object of the present invention to provide a novel lignan-based compound of formula 1 extracted from Jeonho which is a natural product. The present invention also provides a method for extracting the compound of Formula 1.

1 is a diagram showing the determined structure of compound V.

2 is a graph showing the correlation between the C-H obtained from the HMQC and HMBC spectrum of the compound V.

3 is a diagram showing the NOESY correlation of Compound V.

Hereinafter, the present invention will be described in detail.

Separation and purification of active substance from the previous issue

The roots of the fine Jeonho were extracted five times with methanol using an extractor equipped with a cooling circuit. The methanol extracts were solvent fractionated with hexane, chloroform, ethyl acetate, butanol and water and then subjected to cytotoxicity tests on human tumor cell lines of the extracts and fractions. The active fractions obtained here were purified by chromatography using silica gel, Sephadex, RP-18, etc., and cytotoxicity test results by MTTT screening on cancer cell strains at double intervals in vitro. The species was found to be present and the five antitumor substances described above were purified by preparative high performance liquid chromatography (prep HPLC).

Structure Determination of Compounds

The structure of Compound V (Angeloyl podophyllotoxyl) in the anti-tumor material of the present invention was determined by mass spectroscopy, infrared (IR) spectroscopy, and 2D nuclear magnetic resonance (NMR). .

C ₂₇ H ₂₈ O ₉ , IR ν _max cm _-1 : 2923 (CH strech), 1774 (C = O), 1712 (C = O), 1488 (C = C aromatic ring), 1234 (CO), 1126 ( CO)

는 -109.1(클로로포름)의 값을 갖는다.The mineralization of this substance

Has a value of -109.1 (chloroform).

Angel in addition to three methoxy (3.68 and 3.73 ppm) peaks by known podophyllotoxin in ¹ H-NMR (C.Fred Brewer at al., J. Medicinal Chwmiarey, 22, 1979, 215-221) Two methyl groups (1.86 and 1.96 ppm) characteristic by acid and hydrogen bound to tertiary carbon (6.14 ppm) were identified.

The overall structure of this compound was confirmed by experiments of ¹³ C-NMR and 2D NMR, ¹ H- ¹ H COZY, ¹ H- ¹³ C HMQC, and ¹ H- ¹³ C HMBC. It was determined by the NOESY experiment. In addition, the distinction between (Z) -2-methyl-2-butenoic acid (angelic acid) and the isomer (E) -2-methyl-2-butenoic acid (tiglylic acid) is distinguished between two methyl groups in the NOESY experiment. It is clearly confirmed that no NOE signals are present at each other.

EXAMPLES

By the following examples will be described in detail the present invention. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1

Separation and purification of active substance from the previous issue

Plant taxonomically validated Umbelliferae wars were obtained commercially. Finely chopped and dried 2kg Jeonju root was extracted more than 5 times with methanol using an extractor equipped with a cooler. The methanol extract was filtered at on time, and then concentrated under reduced pressure at 40 ° C. or lower to form a precipitate. After the filtrate was filtered, the precipitate was repeatedly recrystallized to obtain Compound I. The filtrate was almost completely concentrated and suspended in water, and then the suspension was solvent fractionated sequentially using hexane, chloroform, ethyl acetate, and butanol. Since activity was shown in the hexane and chloroform fractions of the fractions, each fraction was subjected to column chromatography (C.C) using silica gel and RP-18. Hexane fractions were fractionated into 18 by C.C using silica gel. The elution solvent was carried out in a solvent gradient using hexane and ethyl acetate (hexane: ethyl acetate = 10: 0 → 0: 10). Of the 22 fractions, Compound II was obtained as a result of the separation and purification of the active substance using MPLC (Medium Pressure Liquid Chromatography), VCC (Vaccum Column Chromatography) and prep HPLC. In addition, fraction 24 was used to separate and purify the active material using MPLC, VCC and prep HPLC to obtain Compound III.

The chloroform fractions were fractionated into 31 by column chromatography using silica gel. The elution solvent was carried out in a solvent gradient using hexane and ethyl acetate (hexane: ethyl acetate = 10: 1 → 0: 10). Compound 22 and Compound V were obtained by separating and purifying the active substance using MPLC, VCC, and prep HPLC. The active substances obtained were Compound I as anthricin, Compound II as Falcarindiol, Compound III as Molelensin, Compound IV as Bursehernin and 4 known substances as Compound V Angeloyl podophyllotoxin (Angeloyl podophyllotoxin) was the new material of the present invention.

Example 2

Cytotoxicity Test on Cancer Cells

In vitro cytotoxicity to whole extracts, solvent fractions and purified anti-tumor substances was measured by MTT screening for cancer cell strains at double intervals. The MTT method is an experimental method that determines the extent to which the cell death or suppression of proliferation by anticancer drugs by measuring the degree of absorbance of the MTT reduced to formazan crystals by mitochondrial succinate dehydrogenase of living cells. The concentration of each test drug (IC ₅₀ ) that inhibits growth by 50% was determined, and the cytotoxicity was compared with etoposide and doxorubicin. Each test drug was dissolved in DMSO (dimethylsulfoxide) at a concentration of 20 mg / ml, filtered through a 0.22 μm filter, and titrated with an azeotropy of 5 from 40 μg / ml to 0.00256 μg / ml using RPMI 1640 medium. Diluted to concentration and used. A549 human lung cancer cells, K562 human blood cancer cells, and Colo205 human colon cancer cells were used as the test cancer cells in this test. 100 μl (10,000 cells / well) of each suspension of cancer cells suspended in RPMI 1640 medium was inoculated in a 96 well microplate, incubated for 24 hours at 37 ° C., 5% CO ₂ , and then diluted in each well. 100 μl was added to a final drug concentration of 20 μg / ml to 0.00128 μg / μl and the control group was inoculated with the same amount of RPMI 1640 medium. Here, RPMI 1640 medium containing 10% (V / V) fetal bovine serum, penicillin and streptomycin was used for cell culture. After exposure of the cancer cells to the drug for 72 hours, 25 μl of MTT solution (2 mg / ml saline solution) was added to each well, followed by incubation for 4 hours, followed by centrifugation (1,000 rpm, 10 minutes) to remove the supernatant. The resulting formazan crystals were dissolved in 100 μl of dimethylsulfoxide. This was measured by absorbance at 540nm using a microplate reader to calculate the concentration of the test drug (IC ₅₀ ) that inhibits the growth of cancer cells by 50% compared to the control.

Experimental results of cytotoxicity of human cancer cells by the MTT detection method are shown in Table 1 below. Etoposide and doxorubicin were used as control.

Example 3

Structure Determination of Compound V

When measuring the infrared (IR) spectrometer, the sample was measured by dissolving in chloroform and thinly coated on KBr plate. The photoluminescence was measured at 20 ° C by dissolving 27.5 mg of this substance in 10 ml of chloroform. NMR spectra of 400 MHz (1H) and 100 MHz ( ¹³ C) were measured and the chemical shift of each peak was expressed as a relative value of the chemical shift value (7.2 ppm, 77.0 ppm) of the residual solvent chloroform.

The physicochemical properties of this compound obtained as amorphous solids in the present invention are as follows:

EIMS m / z (% rel.int): 496 [m] ⁺ (100.0), 397 [MC ₅ H ₇ O ₂ (angelic acid)] + (14.34), 263 (8.7), 229 (7.9), 185 (22.5) ), 168 (15.6), 135 (7.6), 83 (17.27), 55 (18.86)

C ₂₇ H ₂₈ O ₉ , IR ν _max cm ^-1 : 2923, 1774, 1712, 1488, 1234, 1126

: -109.1(클로로포름)Optical intensity

: -109.1 (chloroform)

¹ H-NMR spectral data is shown in Table 2 and Table 1.

The overall structure of the grape phylotoxyl angelic acid ester of the present invention is the result of analysis of known phytophytotoxin (see C. Fred Brewer at al., J. Medicinal Chemistry, 22, (1979), 215-221) and ¹ H-. ¹ H COSY, NOESY, HMQC, HMBC experiment results. The structure determined in FIG. 1 is shown.

All results observed in HMQC and HMBC of this material were in good agreement with the structure shown in FIG. 1, and the correlated C-H obtained from the HMBC spectrum is shown in FIG. 2.

The pairing constant of H-1 and H-2 is J = 4.4Hz, and these hydrogens have eclipsed cis form and H-2 and H-3 have J = 14.7, which is anti trans form, H-3 and H -4 is J = 9.1, it can be seen that the anti trans form of each other. In addition, since there is no correlation signal between Me-4 ″ and Me-5 ″ in the NOESY experiment, it was confirmed that the two methyl groups form trans forms with each other.

Example 4

Acute Toxicity Test

Toxicity experiments were performed on mice with the compounds obtained in the above examples. Compounds were suspended in 0.5% Na-CMC and administered to each of the isolated animal groups (5 per group) and observed for 14 days to determine mortality. At this time, the compound was administered in five steps of 1 g / kg, 500 mg / kg, 250 mg / kg, 125 mg / kg and 62.5 mg / kg. As a result, the 50% lethal dose (LD ₅₀ ) of the compounds was 1 g / kg.

Thus, the compounds according to the invention have been found to be very safe drugs with little toxicity.

The compound according to the present invention is expected to be useful as a new anticancer agent because it exhibits the same or more activity compared to the control drugs currently used in clinical practice and is not toxic as a substance extracted from natural products.

Claims (2)

A compound of formula [ Formula 1 ]

Extracting the roots of fine Jeonho with methanol and fractionating the extract with chloroform and purifying with chromatography to extract the compound of Formula 1. [ Formula 1 ]

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