KR100416334B1 - Hydrazine Demethoxycurcumin and Anti-Cancer Formulation Comprising the Same - Google Patents

Abstract

The present invention provides a method for synthesizing hydrazine demethoxycurcumin, a novel derivative of demethoxycurcumin of the formula (1), which is isolated from the herbal material Curcuma aromatica , and hydrazine demethoxycurcumin. The derivative of the present invention has intracellular activity that inhibits angiogenesis, and because of this activity, hydrazine demethoxycurcumin can be used in anticancer functional agents, especially beverages, including the same.

Formula 1

Description

Hydrazine Demethoxycurcumin and Anti-Cancer Formulation Comprising the Same}

The present invention relates to hydrazine demethoxycurcumin, which is a derivative of demethoxycurcumin (DC). More particularly, the present invention relates to hydrazine demethoxycurcumin, a novel derivative of demethoxycurcumin isolated from turmeric, a method for synthesizing it, and an anticancer functional agent comprising the same.

Ulumum is a perennial herbaceous herbaceous plant belonging to Gingeraceae, and the medicinal herbs are Hwangulumum, Blackumumum, White Saumumum and Noksaumumumum. The muscles that make up the turmeric include 6.1% essential oil, 0.8% d-camphor, 2.5% d-camphor, 65.5% sesquiterpene (L-α curcumen and L-β-curcumen), sesquiterpene alcohol 22%, curcumin, demethoxycurcumin, bismethoxycurcumin, turmeron, arturmeron, starch 30-40%, fatty oil 3%, rubber, yellow pigment, carbon and fenlandrene, anti-uterine cancer effect It is known that not only contains curcumol, Kurdion, tetramethylpyridine, camphor, borneo, isobornol, and curserene, but also 20 other ingredients as main components. .

In general, the pharmacological action of turmeric affects lipid metabolism and is known to affect blood cholesterol levels when it is taken as a decoction or powder. In addition, when administered to rabbits or rats, the main and coronary artery formations are formed, lipid deposits are reduced, and the ethyl ether extract of the same plant curcuma amada reduces blood cholesterol levels when administered. Known.

Other pharmacological actions of turmeric include the ability to inhibit various pathogenic fungi when turmeric is used, and essential oil emulsions are known to eliminate gallbladder contractions when using gallbladder contrast. , Book Publishing, p. 144).

On the other hand, as a method of extracting an active ingredient from turmeric, a method of separating curcumin, demethoxycurcumin and bis-demethoxycurcumin, which are phenolic diketones isolated from the root of Curcuma aromatica , is described in Planta Med. . 66 (2000) 396-398. In this document, after curcumin is dissolved in acetone and celite is added, the powder obtained is placed on a dihydrogen phosphate saturated silica gel in a sintered glass funnel and from a 1: 1 mixture of ethyl acetate-heptane A method of recrystallization is disclosed.

In addition, a method of extracting components of turmeric by chilling for several days, for example, 5-7 days using a solvent such as methanol and ethanol, which are widely used for chilling herbal medicines, is disclosed in the art as described below. .

Cancer is a chronic disease caused by abnormalities of cell proliferation control due to congenital (genetic) and acquired (environmental) factors, and it is not fundamentally cured even if it is treated with surgery, radiation, and chemotherapy once it develops into a malignant tumor. It is a disease that causes pain to the patient and ultimately leads to death. Recently, with the development of life sciences, as the understanding of the causes and mechanisms of cancer increases, new treatments for cancer are rapidly detected, which inhibits the development of cancer before metastases to malignant tumors or other tissues. There is a growing interest in the so-called cancer prevention method. In the case of Chinese herbal medicine, since it is less toxic than conventional anticancer drugs and has been applied to the clinic for a long time, its safety is already known, and thus it is highly likely to be developed as a cancer prevention therapeutic agent.

However, in the case of herbal medicines, the scientific basis of pharmacological activity is usually unclear due to the relationship between a plurality of ingredients used in the extract, and the concern of side effects due to the use of the mixed extract cannot be excluded. It has now been found that DC, the active ingredient of turmeric, and the novel derivatives synthesized by the present invention are safe and useful angiogenesis inhibitors.

The inventors have focused on the utility of DC (demethoxycurcumin), an active ingredient of turmeric that inhibits angiogenesis, and has invented a method of extracting and purifying it more efficiently. Further, the novel derivatives thereof synthesized by the present invention are described below. As can be seen, it has been found to inhibit the formation of neovascularization such as DC.

It is therefore an object of the present invention to provide hydrazine demethoxycurcumin, which is a novel derivative of demethoxycurcumin.

Another object of the present invention is to provide a method for synthesizing hydrazine demethoxycurcumin.

Furthermore, another object of the present invention is to provide an anti-cancer functional agent comprising the compound by identifying angiogenesis inhibitory activity essential for the proliferation and metastasis of hydrazine demethoxycurcumin.

DC, which has been found to be an anti-angiogenic component of turmeric that is already used in clinical practice, can be used as a lead compound in the development of new cancer preventive agents and related new drugs.

Figure 1 shows the chemical structure of demethoxycurcumin (DC).

2 is a schematic diagram of a method for separating and purifying DC from turmeric.

3 is a graph showing the toxicity of vascular endothelial cells of DC.

4 is a graph showing proliferation inhibitory activity against vascular endothelial cells of DC.

5 is a photograph showing the blood vessel inhibitory activity of DC.

6 is a bar graph showing the inhibitory activity of the basal tissue infiltration action of vascular endothelial cells of DC.

7 is a bar graph showing the inhibitory activity against chemotaxis of vascular endothelial cells of DC.

Demethoxycurcumin, a component showing angiogenesis inhibitory activity among the components of turmeric, a herbal medicine material disclosed by the present invention, is represented by the following Chemical Formula 1.

Extraction method of the DC according to the present invention is a method including heat treatment before extracting the active ingredient of turmeric. Methanol is used as the solvent, which is used at a concentration of 70-98%. The heat treatment is carried out for about 2-5 hours at a high temperature of 55-65 ℃. According to this method, as shown in the following examples, a large amount of DC was obtained very efficiently by obtaining a yield of about 1.5-2 times as compared to when cold washing was performed for about 5 days using the same concentration of solvent. This will bring about an effect that can be extracted.

In the purification method of the present invention, the fraction obtained by separating the solvent extract by silica gel column chromatography is developed by treating at regular intervals on thin layer chromatography using a developing solvent consisting of hexane, chloroform and ethanol, and each fraction is developed by vascular endothelial cells. The activity is measured by treatment with and the fraction showing the activity is separated and purified by thin layer chromatography over and over again.

The novel dimethylated DC of formula (2) provided by the present invention dissolves DC in acetone, adds methyl iodide and K ₂ CO ₃ , and reacts with stirring, and after the reaction is analyzed through TLC and HPLC analysis Obtained by purifying dimethylated DC.

The novel DC oxime of formula (3) provided by the present invention dissolves DC in methanol, adds benzyl hydroxylamine and triethylamine, and reacts under heating and stirring, and after reaction, DC through TLC and HPLC analysis Obtained by purifying an oxime derivative.

In addition, the novel hydrazine DC of the formula (4) provided by the present invention is dissolved DC in methanol, hydrazine is added, acetic acid and then reacted with stirring, after the reaction to purify the DC hydrazine derivatives by TLC and HPLC analysis To obtain.

The present inventors measured various neovascularization inhibitory activities of the demethoxycurcumin of the formula (1) extracted and purified by the method of the present invention, and furthermore, the biological activity of the neovascularization inhibitory activity against DC derivatives synthesized from DC. Pharmacological activity in vivo was measured and in vitro and in vivo pharmacological activity was confirmed for DCs obtained by the methods herein and the novel DC derivatives prepared by the present invention. These experimental results show that the novel DC derivatives of the present invention have neovascularization inhibitory activity that is equal to or much stronger than that of DC.

Therefore, the novel DC derivatives synthesized according to the present invention can be developed as cancer prevention materials to which their neovascularization inhibitory activity is added when prepared with lactic acid bacteria beverages or other preparations containing them. For example, lactic acid bacteria (Bifidobacteria and Lactobacillus) are recently attracting attention as health functional beverages and preparations as probiotic with formal and anticancer activity (von Wright A., et. al., 1999). DC and a novel DC derivative of the present invention having a neovascularization inhibitory activity equal to or more potent than that of DC and a combination of the Bifidobacteria, the new angiogenesis inhibitory activity of the DC and the Bifidobacteria anticancer activity It can be developed as a cancer prevention material. Oligosaccharides and various vitamins can be added to a formulation containing 5 mg / g of DC using Bifidobacterial 1 × ^{10 8} cells / g of powder, and prepared using a casein as a solid. In addition to Bifidobacteria, the function of these materials is enhanced by adding 0.5-1% of health functional materials with different chemical properties and pharmacological activity, such as chitosan, silk amino acids, and mushroom extracts, which are polysaccharides and have anti-cancer physiological activity. Formulations may be prepared.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention, and are not intended to limit the present invention.

Example 1

Isolation and Purification of DC

1) Extraction Method

end. Analysis of DC Contents According to the Types of Extracted Solvents

In order to effectively extract a large amount of DC in turmeric (20g), the content of DC according to the type of solvent was investigated. Methanol (85%), ethanol (85%) and methanol: dichloromethanol (1: 1) (Planta Medica 66 (2000) 396-398), the most commonly used solvents for cold soaking After extracting the components of turmeric, the content of DC was analyzed by HPLC. As a result, the solvent was selected because the content of DC was the highest in methanol (85%).

DC content in the components of turmeric depending on the type of extraction solvent Constituents of Turmeric Methanol (85%) Ethanol (85%) Methanol: Dichloromethanol (1: 1) Curcumin (CUR) 235 mg 176 mg 59 mg Demethoxy curcumin (DC) 118 mg 106 mg 47 mg Bisdemethoxy curcumin (BDC) 147 mg 129 mg 105 mg

I. DC content analysis with and without heat treatment

In each solvent system, the contents of DC with and without heat treatment at 55-65 ° C. for 2-5 hours were investigated, respectively. As a result, as shown in Table 2, it can be seen that the highest content of DC in the heat-treated methanol (85%) of the present invention. In other words, when the heat treatment is not performed, the DC content is 118 mg when methanol (85%) is used, and when the heat treatment is performed, the content of DC is 347 mg, which is about twice as efficient as the heat treatment. It can be seen. In addition, it can be seen that even when ethanol is used, a greater amount of DC is extracted when the heat treatment is performed.

DC content in the components of turmeric with or without heat treatment If no heat treatment Constituents of Turmeric Methanol (85%) Ethanol (85%) Methanol: Dichloromethanol (1: 1) CUR 235 mg 176 mg 59 mg DC 118 mg 106 mg 47 mg BDC 147 mg 129 mg 105 mg When heat treated (2-5h, 55-65 ℃) Constituents of Turmeric Methanol (85%) Ethanol (85%) Methanol: Dichloromethanol (1: 1) CUR 753 mg 588 mg 71 mg DC 347 mg 235 mg 47 mg BDC 459 mg 353 mg 106 mg

All. DC content analysis according to the ratio of methanol

In order to investigate the DC content according to the ratio of methanol, turmeric was extracted using methanol at a concentration of 100% to 70%. HPLC analysis showed the highest DC content in 95% of methanol.

According to the experiments of the present inventors, when heat-treated at a methanol concentration of 70-98% and a temperature of 55-65 ° C for 2-5 hours, DC can be extracted at about 1.5 times or more compared to the conventional 5-day cooling method. This was confirmed.

On the other hand, the best effect was found to be the most efficient extraction of DC from turmeric by heat treatment (95C) for 3 hours with 95% methanol.

Investigation of the content of DC in the components of turmeric according to the ratio of methanol Constituents of Turmeric Methanol content 100% 95% 90% 85% 80% 70% CUR 764 mg 765 mg 735 mg 753 mg 712 mg 694 mg DC 294 mg 359 mg 353 mg 347 mg 341 mg 335 mg BDC 353 mg 471 mg 459 mg 459 mg 459 mg 459 mg

Example 2

Isolation and Purification of Physiologically Active Substances

Each fraction obtained by separating the ethyl acetate solvent extract of turmeric by silica gel column chromatography was developed by treating at regular intervals on thin layer chromatography (developing solvent conditions; hexane: chloroform: methanol = 4: 8: 1, 3: 9). :One). At this time, the same location of the development mobility was collected to obtain 13 fractions, and then separated by thin layer chromatography, and the separated material was treated by vascular endothelial cells to measure the activity. As a result of measuring activity, activity was shown in fraction 8, and this active material was again subjected to silica gel column chromatography (developing solvent condition; hexane: chloroform: methanol = 7: 6: 1) to collect fractions of the same mobility. Four fractions were obtained. Treatment of these four fractions with vascular endothelial cells showed activity in fraction 1, and preparative silica gel thin layer chromatography (developing solvent conditions; hexane: chloroform: methanol = 5 :) was used to effectively isolate and purify the active substance. 9:10). Preparative silica gel thin layer chromatography gave three fractions, and the activity of these fractions was measured to show activity in fraction two. The next fraction 1 was purified by HPLC (0.1% trifluoroacetic acid: 80% acetonitrile = 40: 60) to obtain a single substance, and the chemical structure was determined by NMR and EI-MS for the single substance. It was confirmed that the DC (same as the developing solvent conditions of Figure 2, reference of the whole process: Figure 2).

In the following Examples 3, 4 and 5 will be described for the synthesis of the derivative of DC.

Example 3

Dimethylated DC

5 mg of DC (14.7 μmol) was dissolved in 0.5 ml of acetone, methyl iodide (18.3 μl, 294 μmol) and K ₂ CO ₃ (20.3 mg, 147 μmol) were added, followed by stirring for 12 hours. I was. After the reaction, 2 mg of dimethylated DC was isolated through analysis of TLC and HPLC.

The dimethylated DC was confirmed molecular weight and molecular structure by ESI MS and ¹ H-NMR. The molecular weight and physicochemical properties were as follows:

Molecular Weight: 365

Chemical Formula: C ₂₂ H ₂₁ O ₅

. Physicochemical Properties

Absorbance: 200 nm, 430 nm

Color: yellow

Example 4

Oxime derivatives

10 mg of DC (29.4 μmol) was dissolved in 1 ml of methanol, benzyl hydroxylamine (10.81 mg, 67.75 μmol) and triethylamine (9.4 μl, 67.75 μmol) were added, followed by stirring under heating for 12 hours. I was. After the reaction, 3 mg of DC oxime derivative was isolated by TLC and HPLC analysis.

The molecular weight and molecular structure of the DC oxime derivative were confirmed by ESI MS and ¹ H-NMR. The molecular weights and physicochemical properties were as follows:

Molecular Weight: 547

Chemical Formula: C ₃₄ H ₃₁ O ₅ N ₂

. Physicochemical Properties

Maximum Absorbance: 200nm, 325nm

Color: Light Yellow

Example 5

Hydrazine derivatives

5 mg of DC (14.7 μmol) was dissolved in 1 ml of methanol, hydrazine (4.7 mg, 146 μmol) was added, and then 50 μl of acetic acid was added and reacted with stirring for 24 hours. After the reaction, 2 mg of the DC hydrazine derivative was isolated by TLC and HPLC analysis.

The molecular weight and molecular structure of this DC hydrazine derivative were confirmed by ESI MS and ¹ H-NMR. The molecular weight and physicochemical properties were as follows:

Molecular Weight: 333

Chemical Formula: C ₂₀ H ₁₇ O ₃ N ₂

. Physicochemical Properties

Maximum Absorbance: 337nm

Color: Light Yellow

In Examples 6-11 below, various angiogenesis inhibitory experimental methods and results of DC and various derivatives thereof extracted and purified by the method of the present invention will be described.

Example 6

Toxicity to vascular endothelial cells

Growth inhibitory activity experiments were conducted to determine whether DC extracted and purified by the method of the present invention is toxic to vascular endothelial cells.

Vascular endothelial cells were incubated in a 5% CO ₂ cell culture medium at 37 ° C. for 3 hours with 1 ml of M199 (containing 20% serum) in a 96-well plate to 5 × 10 ³ cells per well. After the cells have grown to a density of 80%, the wells are washed with 1 ml of PBS, the culture medium is replaced with fresh M199 (containing 20% serum), and the purified DC is administered so that the final concentration is 2.5 to 25 µg / ml. It was. After 72 hours, MTT reagent was added, DMSO was administered, and then measured by ELISA. The growth rate of vascular endothelial cells was calculated according to the following Equation 1.

As shown in FIG. 3, DC extracted and purified by the method of the present invention did not show toxicity to vascular endothelial cells at a treatment concentration of 10 μg / ml, and mild toxicity before and after 10% at a concentration of 20 μg / ml. Indicated.

Therefore, the neovascularization inhibitory activity carried out afterwards was performed at 5 μg / ml, which is a concentration at which DC is not toxic to vascular endothelial cells and exhibits sufficient growth inhibition to vascular endothelial cells.

Example 7

Growth Inhibitory Activity on Vascular Endothelial Cells

The fact that DC inhibits the proliferation of vascular endothelial cells by stimulation of angiogenesis means that angiogenesis can be suppressed, and thus the effect of DC on vascular endothelial cell growth was confirmed as follows.

Vascular endothelial cells cultured in a serum-free medium for 6 to 12 hours were seeded in 96-well plates to a cell count of 5 × 10 ³ cells / well and then cultured in a 5% CO ₂ incubator at 37 ° C. for 3 hours. . When the cells were attached to the bottom, the wells were washed with 1 ml of PBS, and 100 µl of the culture solution as 1 ml of serum-free M199 medium and bFGF and DC were administered by concentration. After 72 hours, MTT reagent was added and DMSO was administered, followed by ELISA, and then the proliferation rate of vascular endothelial cells was calculated according to the following equation.

As shown in FIG. 4, the DC extracted and purified by the present invention effectively inhibited the proliferation of vascular endothelial cells at a concentration of 2.5 μg / ml, and 50% growth inhibition at a concentration of 5 μg / ml. The effect of (IC ₅₀ ) is shown.

Example 8

Angiogenesis inhibitory activity

To confirm that DC substantially inhibits vascular endothelial angiogenesis, vascular endothelial cells were divided into gelatin-coated 48-well multiplates at a density of 1 × 10 ⁴ cells / well and 1 ml of medium was added to 5 μg of DC. Treat / ml. At 37 ° C., the presence of blood vessels is photographed by time while incubating in a 5% CO ₂ cell incubator (see FIG. 5).

Example 9

Inhibitory Activity on Chemotaxis of Vascular Cells

As an angiogenic feature of vascular endothelial cells, these vascular endothelial cells have the property of exhibiting motility in response to vascular generating factors derived from primary cancer cells. The following experiment was performed to determine the inhibitory activity against chemotaxis of vascular cells of DC. An insert chamber with a polycarbonate filter having a pore size of 8.0 μm was used. The bottom of this filter is covered with gelatin. Into the main chamber (24-well multiplate), 600 μl of medium containing blood vessel generating factor, 6 μl of BSA (100 μg / ml), 1.8 μl of bFGF (10 μg / ml), and the like are inserted into the insert chamber. Add 400 μl of medium containing 1 × 10 ⁵ cells / well), then insert the insert chamber into the main chamber. After incubating for 4 hours in a 37 ° C. incubator, the polycarbonate filter in the insert chamber was removed, fixed, and stained with hematoxylin-eosin to calculate the number of cells that passed through the membrane by chemotaxis. The results are expressed as the number of vascular endothelial cells that have passed through the membrane per well, and the chemotaxis of vascular endothelial cells by the vascular generating factor is calculated by the following equation.

As shown in FIG. 6, DC (5 μg / ml) extracted and purified by the method of the present invention inhibits chemotaxis induced by angiogenesis factor (bFGF).

Example 10

Inhibition of Basal Tissue Infiltration Activity of Vascular Cells

Vascular endothelial cells not only chemotactic, but also penetrate the tissue surrounding the tumor, which is a major process of angiogenesis. In order to confirm the effect of DC on the basal tissue penetration activity of vascular cells, the following experiment was performed.

An insert chamber equipped with a polycarbonate filter having a pore size of 8.0 μm is used. The bottom of the filter is coated with gelatin and the top of the filter is coated with Matrigel. To the main chamber (24-well multiplate) add 600 μl of medium containing blood vessel generating factor, 6 μl of BSA (100 μl / ml), 6 μl of each sample according to the concentration, and 1.8 μl of bFGF (10 μg / ml). Into the insert chamber, 400 μl of medium containing vascular endothelial cells (1 × 10 ⁵ cells / well) is added, and then the insert chamber is placed in the main chamber. After incubating for 16 to 20 hours in a 37 ° C. incubator, the polycarbonate filter in the insert chamber was removed, fixed and stained with hematoxylin-eosin to decompose the matrigel and measure the number of cells that passed through the filter. The results are expressed as the number of vascular endothelial cells passing through the filter per well, and the basal tissue penetration of vascular endothelial cells is calculated according to the following equation.

As shown in FIG. 7, DC (5 μg / ml) extracted and purified by the method of the present invention was found to effectively inhibit basal tissue penetration induction by angiogenesis factor (bFGF).

Example 11

Inhibitory activity of yolk sac angiogenesis

In vitro test for inhibiting angiogenesis of vascular endothelial cells of DC and further inhibiting angiogenesis of vascular endothelial cells of vascular endothelial cells of the present invention in vivo. Activity was measured. Hatching eggs are incubated for 3 days in the incubator at 37 ℃. After three days, egg albumin is removed by subcutaneous saliva to remove CAM and yolk sac from the shell membrane. When embryonic after 4 to 5 days, DC is treated on thetheroxox coverslip, followed by drying to place on the CAM surface. After 2 days, 2 ml of 10% fat emulsion is injected into the CAM and the CAM is observed under a microscope. After DC treatment, if avascular zone is observed in CAM, it is measured positively. Each experiment was performed three times and 20 eggs were used per experiment. As can be seen in Table 4, DC exhibits a neovascularization inhibitory activity almost similar to the control retinoic acid, and thus it was confirmed that it is a new angiogenesis inhibitor.

Inhibitory activity of egg yolk neovascularization Volume / disk control(%) Control 2 μl methanol 15 Retinoic acid 20 μg 89 DC 20 μg 86 Dimethylated DC 20 μg 85 Oxime derivatives 20 μg 80 Hydrazine derivatives 20 μg 94

In the above, the present invention has been described with reference to the accompanying drawings, but these configurations and embodiments are illustrative of the present invention and should not be construed as limiting the present invention. It will be appreciated that various modifications and changes are possible within the scope of. Accordingly, the following claims should be construed as including both such modifications and changes.

The present invention provides a method for synthesizing hydrazine demethoxycurcumin from demethoxycurcumin and hydrazine demethoxycurcumin. Hydrazine demethoxycurcumin has excellent angiogenesis inhibitory activity, and thus can be used in anticancer functional agents containing it, especially in beverages.

Claims (4)

Hydrazine demethoxycurcumin of Formula 4: Formula 4 A method for synthesizing hydrazine demethoxycurcumin of the formula (4) comprising the step of reacting demethoxycurcumin of the formula (1) with methyl iodide and K ₂ CO ₃ in acetone: Formula 1 Formula 4 An anticancer functional agent that inhibits neovascularization comprising hydrazine demethoxycurcumin of Formula 4 Formula 4 The anticancer functional agent according to claim 3, wherein the agent is a beverage further comprising Bifidobacteria or Lactobacillus as lactic acid bacteria.