JP5275241B2 - Cancer cell growth inhibitor containing liquiritigenin - Google Patents

Description

  The present invention relates to a cancer cell growth inhibitor comprising an active substance licorice liquiritigenin (hereinafter abbreviated as L-genin) in natural plant licorice as an essential component.

Licorice is widely used as a component of Kampo medicines or as an additive to foods. In recent years, systematic research has been conducted on various components in licorice, but research on anticancer active ingredients in licorice has been conducted. It is still insufficient. Currently, triterpene compounds and flavone compounds in licorice are considered to be the main active ingredients of antitumor, and among them, there are relatively many antitumor studies of triterpene compounds, especially licorice glycyrrhizin and licorice glycyrrhizic acid. Has been done. On the other hand, as for licorice flavone compounds, it has been announced that the mixture of licorice flavone compounds has anticancer action, antimutation and antioxidant action (Research Takeno et al.). On the other hand, the compound liquiritigenin belonging to the flavanone monomers in licorice has been announced to be effective against antiviral effects such as treatment for HIV infection and hepatitis C virus (Studies by Clance et al.) Antitumor effects and their mechanisms have not been studied.
None

  Therefore, the present inventors have conducted extensive research on flavanone monomers in liquorice: liquiritigenin (liquoritigenin) and liquiritin, and liquiritigenin has been found to be a human liver cancer cell by an in vitro tumor cell experiment. (SMMC7721), human lung cancer cells (A549), human gastric cancer cells (SGC7901), human poorly differentiated gastric adenocarcinoma cells (BGC-823), human colon cancer cells (LOVO) and human early granulocyte leukemia cells (HL-) 60), it has been found that it has the effect of significantly suppressing the growth of cancer cells.

Accordingly, the present invention resides in a human cancer cell growth inhibitor characterized by comprising liquiritigenin represented by formula 1 as an essential component.

  Liquiritigenin represented by Formula 1 can be administered to human liver cancer cells (SMMC7721), human lung cancer cells (A549), human gastric cancer cells (SGC7901), human poorly differentiated gastric adenocarcinoma cells (BGC-823) by administration of the following significant amounts. , Human colon cancer cells (LOVO) and human early granulocyte leukemia cells (HL-60) can be inhibited.

Example 1
The following experimental results were obtained for liquiritigenin represented by Formula 1.
one. Cell line and selection method
Cell line: human lung cancer cell A549
Human poorly differentiated gastric adenocarcinoma cell BGC-823
Human liver cancer cell SMMC-7721
Human early granulocyte leukemia cell HL-60
Method:
1. Create a cell suspension by measuring 2 to 4 x 10 4 / mL, which peels off the digested wall cells by adding a 0.25% trypsin digestion solution with a bottle of cells in good growth period. To do.
2. Cell suspension is ingested into a 96-hole plate and a 180-hole plate, respectively, and placed in a low-temperature CO · 52 · incubator and cultured for 24 hours.
3. The solution is changed and the drug to be tested is placed in 20 / well and incubated for 72 hours.
4). MTT is placed 20 / well in a 96-well plate and incubated for 4 hours in an incubator.
5. Draw off the upper clear solution, put DMSO in 150 / hole, and shake the plate for 5 minutes.
6). Using an oxygen-linked immunosorbent analyzer, the absorbance value of each hole at a wavelength of 570 nm is measured, and the inhibition rate of the cells is calculated.
Cell inhibition rate% = negative target group OD value−experiment group OD value × 100%
Negative target group OD value

[Table 1]
[Table 2]
[Table 3]
[Table 4]
Table 4. The result is that licorice liquiritigenine has a concentration of 250 μmol / L, 5
At 00 μmo / L, the inhibition rates against gastric cancer cells are 54.8% and 58.1%, respectively.
[Table 5]
Table 5. The result is that licorice liquiritigenine has a concentration of 250 μmol / L, 5
At 00 μmol / L, the inhibition rates against liver cancer cells were 42.0% and 89.9, respectively.
%.
[Table 6]
Table 6. The result is that licorice liquiritigenine has a concentration of 125 μmol / L, 5
At 00 μmol / L, the inhibition rates against colon cancer cells were 57.6% and 91.9, respectively.
%.
Looking at Table 2 above, licorice liquiritigenine is especially a liver cancer cell
(SMMC7721) has a marked inhibitory effect. Its inhibitory action is much higher than the monomer (monomer) glycyrrhizic acid of licorice triterpenes, and more
I know that there is.

(Production example)
The liquiritigenin (formula 1) of the present invention can be produced by direct extraction from licorice, but the liquiritigenin represented by the formula 1 is produced by forming an oxygen heterocycle by cyclizing isoliquiritigenin by the action of isomerase. be able to. Isoliquiritigenin can be synthesized as follows.
1. Selection of process conditions for synthesis (1) For easy synthesis of isoliquiritigenin, 2,4-dihydroxyacetophenone and dihydroxybenzaldehyde and raw materials, Lewis base One-step synthesis can be carried out in a condensation reaction in the context of the catalytic action. The chemical reaction formula is:
The molar ratio of 2,4-dihydroxyacetophenone and dihydroxybenzaldehyde is 1: 1.5 to 1: 3, preferably 1: 1.8 to 1: 2.
(2) Selection of solvent
An organic solvent capable of dissolving 2,4-dihydroxyacetophenone and dihydroxybenzaldehyde can be selected and used. Lower alcohols such as isopropyl alcohol, N-butyl alcohol, isopropyl ether, N, N-dimethylformamide, dimethyl sulfoxide Solvents such as sulphoxide, dioxane, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether. Isopropyl alcohol, isopropyl alcohol, n-butyl alcohol, dioxane and the like are preferable.
(3) Selection of catalyst agent A catalytic reaction must be performed using a relatively strong alkaline reagent, and sodium alkoxide, alkali hydroxide, sodium hydride, or the like can be used. Preferably, the sodium salt of a lower alcohol is used.
(4) Reaction time and temperature A relatively long reaction time at the same temperature is advantageous for improving the production rate. The reaction temperature is 80 to 150 ° C., and the reaction time is 24 to 36 hours.
(5) Selection of antioxidant (antioxidant) The raw materials used in the reaction are all phenols, which are easily oxidized and denatured in an alkaline environment. An oxidant must be added. As a commonly used antioxidant, butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), and other antioxidants can be used.
2. Selection of decontamination process (1) Selection of decoloring agent It is possible to decolorize using activated carbon, activated clay, silica gel, activated magnesium oxide or activated aluminum oxide. The decolorizing effect of activated carbon is the best.
(2) Selection of recrystallization solvent Since isoliquiritigenin and its isomer licorice glycyrrhizin are mixed, it is necessary to remove recrystallization contamination using a mixed solvent. Among them, commonly used mixed solvents include benzene-ethyl acetate, chloroform-ethyl acetate, benzene-methyl alcohol, etc. Among them, the volume ratio of benzene and chloroform is 100, ethyl. Acetate, methyl alcohol or ethyl alcohol is 6-10. The effect of benzene-ethyl acetate is the best.
Specific Implementation Method In a four-necked flask through which nitrogen gas passes, add 140 g of N-butyl alcohol and 20 g of dihydroxybenzaldehyde, add 4.0 g of sodium metal sheet and 1 g of antioxidant at room temperature, and slowly bath in a water bath. And heat until all are dissolved. When the temperature rises to 105 ° C., 15 g of 2,4-dihydroxyacetophenone is slowly added dropwise to 80 ml of N-butyl alcohol solution, stirred vigorously, reacted at this temperature for 28 hours, and then cooled.
Add 18% hydrochloric acid to an ice-water bath until neutral. The reaction solution is divided into two layers. The organic phase is taken, decolorized with activated carbon, centrifuged, and the organic layer is washed with water until the washing solution becomes thin. The solvent was recovered under reduced pressure and dried to obtain 16.5 g of salmon pink isoliquiritigenin crude product. It is a mixture of isoliquiritigenin and licorice glycyrrhizin.
200 g of a benzene-ethyl acetate mixed solvent having a volume ratio of benzene: ethyl acetate of 100: 80 was produced, and a crude mixture of isolithicigenin and licorice glycyrrhizin was heated and dissolved in this solution. The insoluble matter was filtered, and the temperature was lowered to precipitate isoliquiritigenin. After filtration, the solution was thoroughly washed with benzene alone and dried to obtain 8.2 g (0.03 mol) of yellow isoliquiritigenin. obtain. Its HPLC analysis content was ≧ 96%, and its ultraviolet, infrared, nuclear magnetic resonance and mass spectral analysis was the same as the standard product.

  The liquiritigenin according to the present invention can be provided as it is or together with the excipient, in the form of liquid, tablet, granule, jelly, etc., or it can be added to beverages and various foods as a food additive, and has a predetermined anticancer effect. Can be demonstrated.

Claims (2)

A human liver cancer cell (SMMC7721) growth inhibitor comprising licorice liquiritigenin represented by formula 1 as an essential component.
[Chemical 1]
The inhibitory effect on human liver cancer cells (SMMC7721) at 10 μg / ml of the licorice liquiritigenin is 1 × 10 ^―5 The human liver cancer cell (SMMC7721) growth inhibitor according to claim 1, which is equivalent to the inhibitory action of adriamycin on mol / liter human liver cancer cells (SMMC7721).