KR20010084363A - Apoptosis-inducing pharmaceutical composition comprising lavandulylflavonoids - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for inducing apoptosis containing lavandulyl flavonoids derived from Sophora flavescens Aiton is provided, which is useful for the treatment and prevention of cancer by inhibiting cell proliferation and inducing apoptosis. CONSTITUTION: The pharmaceutical composition for inducing apoptosis contains lavandulyl flavonoids of formula 1 and a pharmaceutically acceptable carrier. In formula, R1 is H, OH or C1-3 alkoxy, R2 is H or OH, R3 is H, OH or C1-3 alkoxy. The lavandulyl flavonoids are one or more selected from sophoraflavanone G, kurarinone, 2-methoxykurarinone and leachianone A.

Description

Apoptosis-inducing pharmaceutical composition comprising lavandulylflavonoids

The present invention relates to a pharmaceutical composition for inducing apoptosis, which contains lavandulylflavonoids as an active ingredient.

Apoptosis is a process for achieving physiological control of cell numbers in developing organs and organisms. Apoptotic cell death is characterized by cell contraction, membrane vesicle formation, and chromatin enrichment, which is caused by several stimuli such as high fever, oxidative stress, and growth factor removal (Takasu T., Lyons JC, Park). HJ and Song CW (1998) Apoptosis and perturbation of cell cycle progression in an acidic environment after hyperthermia. Cancer Research 58, 2504-2508). In recent years, it has been known that cancer treatment and prevention agents play their pharmacological role by inducing apoptotic cell death or cell cycle transition, so induction of apoptosis in tumor cells is a precursor to the tumor treatment response. Parker BW, Kaur G., Nieves-Neira W., Taimi M., Kohlhagen G., Shimizu T., Losiewicz MD, Pommier Y., Sausville EA and Senderowicz AM (1998) Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 91 , 458-465). Many compounds derived from natural substances have been reported to exert anticancer effects mediated by apoptotic cell death (Dirsch VM, Gerbes AL and Vollmar AM (1998) Ajoene, a compound ofgarlic, induces apoptosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor kappa B. Molecular Pharmacology 53, 402-407).

Previous studies have also revealed that certain flavonoids, both synthetic and natural, are antiproliferative in human cancer cell lines (Kuntz S., Wenzel U. and Daniel H. (1999) Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines.European Journal of Nutrition 38 , 133-142). In most cases, the effect of flavonoid cytotoxicity depends on the nature of the side chain and the location and number of hydroxyl groups. Although the mechanisms of the anticancer effects of flavonoids have not been determined so far, their ability to inhibit protein kinase C, block cell cycle progression or induce apoptosis is considered as a possible explanation (Richter M., Ebermann R., and Marian B. (1999) Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling. Nutrition and Cancer 34 , 88-99). Here, the specific structure was not necessarily required with respect to the induction of apoptosis. Polymethoxylated flavonoids nobiletin and tangeretin showed antiproliferative and apoptotic effects in human cancer cells, supporting the fact that the less hydrophilic the flavonoids, the easier they cross the cell membrane. Some prenylated flavonoids have been reported to have antiproliferative effects in cancer cell lines, but no apoptosis induction is known (Miranda CL, Stevens JF, Helmrich A., Henderson MC, Rodriguez RJ, Yang YH, Deinzer ML, Barnes). DW and Buhler DR (1999) Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines.Food and Chemical Toxicology 37 , 271-285).

On the other hand, the roots of red ginseng ( Sophora flavescens AITON) have been reported to have antitumor activity in sarcoma 180, lymphocytic leukemia 1210 and melanin melanoma (Abbott BJ, Hartwell JL, Leiter J., Perdue RE Jr. and Schepartz). SA (1966) Screening data from the Cancer Chemotherapy National Service Center screening laboratories. Cancer Research 26, 1461-1656). Flavonoids derived from red ginseng include Sophoraflavanone G, kurarinone, 2'-methoxykurarinone, leachianone A, kurarinol and kurarinol. Kushenol H, or kushenol K and the like are known. The structure of the flavonoids is represented by the formula (1). However, the apoptotic action of these flavonoids and related cancer treatment and prophylactic effects are not known.

compound R ₁ R ₂ R ₃ R ₄ Sophoraflavanone G (SR-A) Curarinone (SR-D) 2'-methoxycurinone (SR-G) Richyanone A (SR-I) Curalinol Kushenol H Cusholol K AAAABBB OHOMeOMeOHOMeOMeOMe HHHHHOH (α) OH (β) OHOHOMeOMeOHOHOH

Disclosure of Invention An object of the present invention is to provide a pharmaceutical composition for inducing apoptosis, which can be used as a cancer treatment and prevention agent by inhibiting cell proliferation and inducing apoptosis.

The present inventors have conducted studies on the biological activity of natural-derived flavonoid derivatives, and as a result, specific ravandulyl flavonoids derived from wild ginseng act to inhibit cell proliferation and induce apoptosis in human cancer cells. The present invention was completed by discovering that apoptosis inducing ability is due to lavandulyl side chain.

1 shows DNA extracted from HL-60 cells treated with lavandulylflavonoids [SR-A (FIG. 1A), SR-D (FIG. 1B), SR-G (FIG. 1C) and SR-I (FIG. 1D)). Agarose gel electrophoresis results are graphs. However, line 1 is a marker, line 2 is a control group, and lines 3 to 5 are cases in which the concentrations of lavandulyl flavonoids are 10, 30, and 50 μM, respectively, and line 6 is a comparative group.

FIG. 2 shows the amount of DNA fragments in cells after treatment with the lavandulylflavonoids [SR-A (FIG. 2A), SR-D (FIG. 2B), SR-G (FIG. 2C) and SR-I (FIG. 2D)]. It is a graph showing the results measured by enzyme-linked immunosorbent assay (ELISA). However, ** means that there is a significant difference from the control group (p <O.O1).

3 is the lavanddulyl flavonoid [SR-D ( ) And other flavonoids derived from red ginseng [Curalinol ( ), Cushenol H ( ) And Cusholol K ( )] Is a graph showing the results of comparative cytotoxicity experiments.

The present invention relates to a pharmaceutical composition for inducing apoptosis (apoptosis) containing lavandulylflavonoids represented by the following formula (2) as an active ingredient.

In formula, R <_1> is hydrogen, a hydroxyl group, or a C1-C3 alkoxy group, R <_2> is hydrogen or a hydroxyl group, R <_3> is hydrogen, a hydroxyl group, or a C1-C3 alkoxy group.

It is preferable that the lavandulyl flavonoid in the present invention is extracted from red ginseng.

In addition, in the pharmaceutical composition for inducing apoptosis according to the present invention, lavandulylflavonoids are Sophoraflavanone G (SR-A), Kurarinone (kurarinone (SR-D), 2'-methoxykura More preferably, it is at least one selected from the group consisting of 2'-methoxykurarinone (SR-G) and leachianone A (SR-I).

Another aspect of the present invention relates to a cancer treatment and prevention agent comprising the pharmaceutical composition for inducing apoptosis according to the present invention. This is because the pharmaceutical composition for inducing apoptosis according to the present invention may exhibit cancer treatment and prophylactic effects by inhibiting cell proliferation of cancer cells and inducing apoptosis.

The administration method of the pharmaceutical composition is a method such as administration using injection, orally made tablets and orally administered according to the condition and other circumstances of the patient and a method used as a transdermal agent.

Dosage will vary depending on the patient's condition, weight, age, etc. However, a person skilled in the art of medicine can easily predict the dosage by referring to the activity of the lavandulyl flavonoid. For reference, the preferred dosage is about 0.1 ~ 300 mg / kg (weight) / day (day).

The pharmaceutical composition may be prepared in various forms, namely, creams, ointments, injections, granules, and external skin liquids depending on the administration method. In order to make a suitable medicament, the labwandulla flavonoids may contain stabilizers, emulsifiers, buffers, salts, high ginseng extracts or small amounts of antibiotics that can inhibit further infection.

Hereinafter, the present invention is described in more detail by some preferred embodiments. However, the present invention is not limited to the following examples.

Example 1 Extraction of Lavandulyl Flavonoids

Sophoraflavanone G, curarinone, 2'-methoxycurarinone, lycanone A, curarinol, Cushenol H and Cushenol K, respectively, were extracted from methanol extracts of red ginseng using Sephadex LH-20 and silica gel column chromatography. It was separated and used in the following experimental example.

Experimental Example 1. Cytotoxicity of Lavandulyl Flavonoids Against Human Cancer Cell Lines

First, a human myeloid leukemia HL-60 cells and human hepatoma HepG2 cells (American Type Culture Collection, Rockville, MD) the temperature 37 ℃, 5% CO ₂ - a 95%, heating the fire in the air and humidified incubator activated with 10% fetal bovine serum (Gibco BRL, Grand lsland, NY) and 2mM L-glutamine (Sigma Chem. Co. St. Louis, Mo.) supplemented in a logarithmic expansion of RPMI 1640 medium (Gibco BRL, Grand lsland, NY).

Cultured HL-60 cells and HepG2 cells were seeded in 96-well plates at 1 × 10 ⁴ cells / well, respectively, and at various concentrations of Sophoraflavanone G (SR-A), Kurarinon (SR) obtained in Example 1 -D), cells treated with 2'-methoxycurinone (SR-G), liccianone A (SR-I) and cisplatin (Cisplatin, CDDPU; United Pharmaceutics, Seoul, Korea) for 96 hours Survival rate was measured. The viability of the cultured cells was determined by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide reduced to formazan according to Monks et al .; Sigma Chem. Co., St. Louis , MO).

In order to calculate the concentration (IC ₅₀ ) required for 50% growth inhibition from the MTT analysis, first, logarithm of the% survival rate was taken for the concentration using a linear regression or a linear-secondary function regression model. Estimate the regression model using the statistical software program S + 4.5 (Mathsoft, 1997, S-Plus 4 User's Guide, Mathsoft lnc., Seattle), and solve the IC ₅₀ value by solving the linear or quadratic equations using the estimated regression model. Got it. The obtained IC ₅₀ values are shown in Table 1.

As can be seen in Table 1, it was confirmed that the lavandulyl flavonoids of the present invention exhibit a potent cell proliferation inhibitory effect on human cancer cell lines, and this effect is dose dependent.

Cytotoxicity of Lavandulyl Flavonoids Against Human Cancer Cell Lines compound IC ₅₀ (μM) (R ² ) ^* HL-60 HepG2 SR-A 12.5 (0.958) 13.3 (0.967) SR-D 18.5 (0.963) 36.2 (0.952) SR-G 13.7 (0.891) 21.1 (0.893) SR-I 11.3 (0.977) 13.3 (0.949)

* R ² is a measure of goodness of fit. When R ² = 1.0, all points are exactly on the curve without scattering.

Experimental Example 2. Apoptosis effect Experiment Ⅰ: DNA fragment analysis by electrophoresis

To elucidate the mode of cell death by lavandulyl flavonoids, the effect of flavonoids on internucleosomal DNA fragmentation was tested.

Sophoraflavanone G (SR-A) obtained in Example 1 of 10, 30 and 50 μM, respectively, of HL-60 cells cultured at a concentration of 2 × 10 ⁶ cells / ml according to the same method as Experimental Example 1 , Curarinone (SR-D), 2'-methoxycurarinone (SR-G), and lycyanone A (SR-I). The control group was treated with no drug, and the control group was treated with 33 μM of comparative drug cisplatin (CDDPU). Genomic DNA was extracted from each group of cells using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wis.). This DNA was precipitated with isopropanol, separated with 1.5% agarose gel, stained with ethidium bromide and visualized by UV irradiation. The results are shown in Figures 1a to 1d.

SR-A (FIG. 1A), SR-D (FIG. 1B), SR-G (FIG. 1C), and SR-I (FIG. 1D) which are the lavandulyl flavonoids of this invention are all a control (2 of FIGS. 1A-D). Compared with, the degree of internucleosomal DNA cleavage was not large at the concentration of 10 μM (3 in FIGS. 1A-D) but gradually increased at 30 μM (4 in FIGS. 1A-D) and 50 μM (5 in FIGS. 1A-D). It was confirmed that the increase in the degree of internucleosomal DNA cleavage of the comparative drug cisplatin. Therefore, it can be seen that the cell proliferation inhibition and apoptosis effects of the lavandyl flavonoids of the present invention are achieved through DNA cleavage to some extent.

Experimental Example 3. Apoptosis Effect Experiment II: Quantitative Evaluation of Apoptosis by Sandwich Enzyme Immunoassay

In order to confirm the induction of apoptosis by lavandyl flavonoids and to quantify the degree of DNA fragments, sandwich enzyme immunoassay was performed as follows.

The HL-60 cells cultured according to the same method as Experimental Example 1 were first labeled with 5-bromo-2'-deoxyuridine (5-bromo-2'-deoxyuridine; BrdU) for 18 hours to give a final concentration. Was 10 μM. Sophoraflavanone G (SR-A), Curarinone (SR-D), 2'-methoxycurarinone (SR-G) and Lithionone A obtained at 1, 5, 10, 25 μM Cells were treated with (SR-I) for 24 hours each. Separately, no drug treatment was used as a control group, and the control group was treated with 33 μM of comparative drug cisplatin (CDDPU). After treatment, the culture medium and the cells were separately collected, lysed and centrifuged at 250 x g for 10 minutes. Enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of BrdU labeled DNA fragments in culture medium and cells using the Cellular DNA fragmentation kit (Boehringer Mannheim, Mannheim, Germany). It was. The kit uses mouse monoclonal antibodies against DNA and 5-bromo-2'-deoxyuridine.

The results are shown in Figures 2a, 2b, 2c and 2d for SR-A, SR-D, SR-G and SR-I, respectively. The amount of DNA fragment in the cell fraction exposed to the test drug is shown. Each bar represents the mean and standard deviation after 3 replicates, and ** indicates significant differences from the control (p <O.O1).

The increase in the amount of DNA fragments compared to the control group was 2.2 and 2.7 times in the treatment of SR-A (FIG. 2A) and SR-I (FIG. 2D) at 10 μM treatment, and SR-A (FIG. 2a), SR-D (FIG. 2B), SR-G (FIG. 2C) and SR-I (FIG. 2D) all increased significantly and appeared 3 to 4.5 times. In particular, SR-G (Fig. 2c) was higher than the comparative drug cisplatin at the treatment concentration of 25 μM.

In addition, until this concentration, no DNA fragments were observed in the culture medium (and therefore not shown). Therefore, it was confirmed that the flavonoid cell death pattern was apoptosis and not necrosis.

Therefore, it was confirmed that the lavandulyl flavonoids of the present invention have an excellent apoptosis effect.

Experimental Example 4. Comparison of cytotoxicity against human cancer cell lines

Cytotoxic effects through cytostatic inhibition and apoptosis ability to the human cancer cell line exhibited by the labandulyl flavonoids in Experimental Examples 1 to 3 were confirmed. This experiment was compared with the cytotoxic effects on human cancer cell lines exhibited by other flavonoids from Ginseng.

For HL-60 cells, the test drug was the same as in Experiment 1 except that the test drug was used as SR-D, curarinol, cuschenol H and cuschenol K at concentrations of 0, 10, 20, 30, 40 and 50 μM, respectively. Each survival rate was determined by MTT analysis. The mean and standard deviation after three replicates were determined. The results are shown in FIG.

SR-D, a Labandulyl Flavonoid ( ) Has shown potent cytotoxicity against human cancer cell lines, but other flavonoids, ie, curanol ( ), Cushinol H ( ) And Cusholol K ( ) Did not show cytotoxicity against human cancer cell lines. As shown in formula (1), Curarinol, Cusholol H and Cusholol K are C4 ^... -C5 ^…. The double bond of is broken and the hydroxyl group is reloaded, and the labanyl side chain is modified. This modification of the lavandulyl side chain results in the complete loss of the cytotoxic effect through cytostatic and apoptosis activity, thus demonstrating that the lavandulyl side chain is an integral part of the effect.

The novel apoptosis inducing pharmaceutical composition comprising the lavandulyl flavonoids of the present invention is useful for inhibiting cell proliferation and inducing apoptosis to treat or prevent cancer.

Claims (4)

A pharmaceutical composition for inducing apoptosis, comprising a lavandyl flavonoid represented by the following formula and a pharmaceutically acceptable carrier. (Wherein R ₁ is hydrogen, a hydroxyl group or an alkoxy group having 1 to 3 carbon atoms, R ₂ is hydrogen or a hydroxyl group, and R ₃ is a hydrogen, a hydroxyl group or an alkoxy group having 1 to 3 carbon atoms.) The pharmaceutical composition for inducing apoptosis according to claim 1, wherein the ravandulyl flavonoid is extracted from red ginseng. The pharmaceutical composition for inducing apoptosis according to claim 1, wherein the lavandulyl flavonoid is at least one member selected from the group consisting of sophoraflavanone G, curarinone, 2'-methoxycurarinone, and lycanone A. The pharmaceutical composition for inducing apoptosis according to claim 1, which is an agent for treating and preventing cancer.