KR20020017675A - Chemopreventive agent of cancer - Google Patents

Abstract

PURPOSE: The titled inhibitor containing a Scutellaria baicalensis George extract as a main component is provided which has an effect on suppressing carcinogenesis and can be used for prevention of cancer generated in an organ in the body such as liver cancer, stomach cancer, esophageal cancer, lung cancer, breast cancer or the like. CONSTITUTION: This carcinogenesis inhibitor is obtained by extracting a Scutellaria baicalensis George and can suppress carcinogenesis by expression of glutathiones S-transferase(GST) Yc1/2 to transfer carcinogenic substances to nontoxicity before DNA of a cell is damaged. The agent can formulate to be administered orally or parenterally at a dose of 11 to 500mg/kg/day.

Description

Carcinogenic inhibitors {Chemopreventive agent of cancer}

The present invention relates to a carcinogenic inhibitor mainly composed of the extract of the root of Scutellaria baicalensis Georgi, more specifically, to the carcinogenic inhibitor of gold.

If the anticancer agent inhibits the treatment or metastasis of a cancer that has already occurred, or the agent containing the same, the carcinogenic inhibitor refers to a compound or an agent containing the same that exhibits an effect of inhibiting or delaying the cancer itself. Carcinogenic inhibitors used within the specification are in accordance with the above definitions.

BACKGROUND ART [0002] Studies on carcinogenic inhibitors that attempt to prevent cancer from being promoted by environmental pollutants using ingredients or compounds present in vegetables, fruits, and drug plants have been conducted.

In the US, the National Cancer Institute has been conducting research on over 400 cancer prevention candidates and conducting 60 clinical trials. A typical example is DNA that induces GST expression. A study on carcinogenic inhibitors that inhibit the development of cancer by removing epoxide metabolites that damage the

That is, it is a group of enzymes that exist in animals and plants and conjugates reduced glutathione to the electrophilic sites of hydrophobic substances (Chasseaud, 1979) and dimers, and about 100 species have been identified (Buetler and Eaton, Glutathione S-transferase (hereinafter referred to as 'GST') is a procarcinogen, aflatoxin B ₁ (hereinafter referred to as 'AFB ₁ '), benzo (a Metabolize benzo (a) pyrene, 7,12-dimethylbenz (a) anthracene, etc. In other words, these pro-carcinogens are converted into highly reactive epoxide derivatives, ultimate carcinogens, by cytochrome P450 enzymes, which form covalent adducts with DNA. It converts normal cells into cancer cells (Cai et al., 1997; Ewaskiewicz et al., 1991), and GST converts epoxides into nontoxic substances by binding glutathione to epoxide derivatives before these epoxide derivatives damage DNA. Conversion to the substance causing extracorporeal excretion. Therefore, it is intended to suppress the production of cancer by inducing the expression of GST.

Specific examples of carcinogenic inhibitors by this mechanism include aged garlic extract, ethoxyquin, tert-butyl hydroxyanisole (BHA), oltipraz, phenobarbital, and the like. There is this.

In other words, garlic extract has been shown to increase the GST activity in rat liver and breast tissues, thereby inhibiting the DNA damage caused by 7,12-dimethylbenz (a) anthracene and thus preventing the occurrence of breast cancer (Liu et al., 1992). . In addition, ethoxyquine, t-butyl hydroxyanisole, Oltipraz (Buetler et al., 1995), and phenobarbital (Langouet et al., 1996), etc. increase the expression of several GSTs, including GST Yc, including AFB ₁ . It has been reported to effectively eliminate the toxic effects of various substances. In particular, since oltifras increases the expression of several types of GST, including GST Yc2 (Kensler, 1997), Kensler and colleagues at Johns Hopkins University in the United States continue to contaminate AFB ₁ through food, leading to liver cancer incidence. In the high Qidong area of China, oltipraz was administered and the effects of hepatocellular carcinoma were investigated, and the expression of GST Yc lowered the incidence of liver cancer (Jacobson et al., 1997; Kensler et al., 1998; Wang et al., 1999).

Therefore, the present inventors also conducted a study on carcinogenic inhibitors that can inhibit the development of cancer by inducing GST expression, and the gold extract promotes the expression of GST Yc1 / 2 and inhibits DNA damage, thereby causing cancer. It has been found that the present invention can be suppressed to complete the present invention.

Accordingly, it is an object of the present invention to provide a carcinogenic inhibitor which can suppress the occurrence of cancer by promoting the expression of GST, which induces the intoxication of carcinogens in various organs.

1a to 1c are photographs of the effect of the golden extract on the expression level of various glutathione-S-transferases (GST) in vivo by Western blotting.

<Description of drawing symbol>

Lanes 1 to 3; 3 different controls with physiological saline

Lanes 4-8; 5 different rats receiving golden extract

2 is a graph analyzing the effect of the gold extract on the activity of GST Yc1 / 2 in vivo.

Figure 3 is a graph showing the effect of the golden extract on the viability of hepatocytes in the primary hepatocyte culture of rats.

Figures 4a and 4b is a photograph of the effect of the golden extract on the expression of GST Ya and GST Yc1 / 2 in primary hepatocyte culture of rats measured by Western blot.

Lane 1: control group (hepatocytes not treated with golden extract)

Lane 2: hepatocytes treated with 12.5 μg / ml of golden extract

Lane 3: hepatocytes treated with 25.0 μg / ml golden extract

Lane 4: hepatocytes treated with 50.0 μg / ml golden extract

Lane 5: hepatocytes treated with 100 μg / ml of golden extract

Figure 5 is a graph showing the effect of the gold extract in the primary hepatocyte culture of rats on the reaction of AFB ₁ to form a shared adduct with the DNA of hepatocytes.

6 is a graph showing the effect of the extracts on the golden AFB ₁ -8,9- GST Yc1 / 2 activity of rat cytosol to break up the epoxide formed by the tissue between the cytochrome P450 3A4 in human.

In order to achieve the above object, the carcinogenic inhibitor according to the present invention is characterized by containing the extract of gold as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Gold has been used to treat anti-inflammatory, anti-allergic, antibacterial, arteriosclerosis, hangover prevention, diarrhea, asthma, hyperlipidemia. Recently, some flavonoids contained in gold have been reported to have antimutagenic effects on Salmonella typhimurium (Lee et al., 2000) and to remove free radicals from animal cells (Gao et al., 1999).

In the present invention, the gold having the above-mentioned effect has been traditionally passed down from water by using water or alcohol, or extracted by a conventional extraction method described in Chinese medicine or textbook (hereinafter referred to as 'golden extract'), which is referred to as GST Yc1. / 2 to increase the expression of found that quickly AFB ₁ -8,9- epoxide was converted to the conjugate of glutathione ^{(glutathione conjugate), [3 H} ] FIG effectively prevent DNA damage by -AFB _1, and this It is to provide a carcinogenic inhibitor as an active ingredient.

The effective amount of the golden extract, which is an active ingredient in the carcinogenic inhibitor of the present invention, may be appropriately selected depending on the administration method, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease. In general, however, the golden extract may be formulated to be administered at a concentration of 10-500 mg / kg / day. Of course, the dose of the active ingredient may be an amount outside the above range.

Preparations for administering the golden extract within the above range may have a conventional form, for example, may be used in the form of pills, capsules or drinks, pharmaceuticals and the like. They can be administered orally or via various parenteral routes of administration for the inhibition and delay of the occurrence of cancer and are suitable for the dosage form and are already well known to those skilled in the art and readily selectable by those skilled in the art, Carrier or diluent and the like.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited only to these examples.

Experimental Method

1. Sample

William`s E medium, collagenase was purchased from Gibco (Grand Island, NY, USA) and AFB ₁ was purchased from Sigma (St. Louis, MO, USA). [ ³ H] AFB ₁ was purchased from Moravek Biochemical, Inc. (Brea, CA, USA), and anti-GST Ya-antibody, anti-GST Yc1 / 2-antibody and anti-GST Yp-antibody were from Biotrin (Dublin, From Ireland).

2. Golden Extract

Gold was purchased from Bohwadang Oriental Medicine Hospital in Iksan, Jeonbuk, and was tested by Korean medical doctor Kwon Kang-bum. After finely crushing 100 g of dried golden gold, a sufficient amount of water was poured, followed by a water bath at 54 ° C. for 3 hours or 90 minutes at 100 ° C., and freeze-dried to obtain 23 g of extract.

3. Rat Administration

The golden extract was administered orally 1 g / kg / day for 4 days in SD male rats 6 weeks old.

4. Cytosol Isolation of Liver Tissues

Liver tissue was isolated from the rats, and then homogenized with a 4-fold volume of cold 100 mM Tris-HCl (pH 7.4) buffer containing 100 mM KCl, 1.0 mM EDTA, and 20 ?? M BHT. Then, the homogenized was centrifuged at 9,000 × g for 20 minutes at 4 ° C., and then the supernatant was centrifuged at 105,000 × g for 1 hour at 4 ° C. to use as a cytosol solution.

Test Example 1 Effect of Golden Extract on the Expression of GST in Vivo

Rats were treated with physiological saline and golden extract (2 g / kg / day, 6 days), the cytosol of liver tissue was isolated, and Western blot of the isolated cytosol solution was used to measure the expression level of GST. Is shown in FIG.

In the test method, the cytosol solution was subjected to electrophoresis on a 16% SDS-polyacrylamide gel, and then transferred to a nitrocellulose filter and nonspecific binding for 1 hour in 3% BSA-Tris buffered saline (pH 7.5). Blocked. Add 15 ml of anti-GST Ya-antibody, anti-GST Yc1 / 2-antibody or anti-GST Yp-antibody (1/800 dilution at 1/400), store at 4 ° C. overnight, and then Washed three times with buffered Tween Salt. Next, the goat anti-mouse IgG-alkaline phosphatase conjugate was incubated at room temperature for 1 hour with a secondary antibody diluted 1: 1,000-fold and washed three times. Coloration uses nitroblo terzolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates (Carlson and Billings, 1996).

In Fig. 1, lanes 1 to 3 are cytosol treated with physiological saline, and lanes 4 to 8 were cytosol treated with golden extract, and expression levels of GST Ya and GST Yp were similar. However, the amount of GST Yc1 / 2 was 2-4 times higher in all cytosols treated with golden extract than the control. On the other hand, the administration of the gold extract in this test was not conducted by investigating the optimal dose, it will be possible to determine the concentration of gold to further increase the expression level of GST Yc1 / 2.

Test Example 2 Effect of Golden Extract on the Activity of GST in Vivo

AFB ₁ is cytochrome (microsomal cytochrome) AFB ₁ -8,9- epoxide (AFBO) by a P450 enzyme, AFQ ₁ and will switch to AFM _1, AFB ₁ -8,9- epoxide (AFBO) formed a glutathione GST Yc1 / 2 activity was measured in the cytosol of rats (2 g / kg / day, 6 days) treated with cytosol of mice, cytosol of rats and golden extract.

The method for measuring the activity of GST Yc1 / 2 is as follows.

The reaction mixture is 1 ml in total and includes 100 pmol of rat or human liver microsomes, 50 mM calcium phosphate buffer (pH 7.5), 2 mM glutathione, 50 μM AFB ₁ , and a NADPH generating system. As the GST activator, mice and rat cytosol were added, respectively. The mixture was reacted at 37 ° C. for 10 minutes and AFB ₁ metabolites were analyzed using Beckman's HPLC (Model 142). The column uses an Ultrasphere ODS C-18 reversed-phase column (Shiseido, 5 ?? m, 4.6 × 250mm) and the eluting solvent is 20 mM ammonium acetate (adjusted pH to 4.0 with acetic acid) and acetonitrile: Solvent B mixed with methanol: water in a ratio of 4.5: 4.5: 1.0 is used. The elution rate was 1.0 ml / min. After equilibrating the column with a solvent in which the solvent A and the solvent B were mixed at a ratio of 9: 1, 20 µl of the sample was injected and 30 minutes over a 30 minute continuous gradient. Samples are eluted with varying solvents at% A-70% B (Monroe and Eaton, 1987; Kim et al., 1997). The result is shown in FIG.

From FIG. 2, the synthesis rates of low toxicity AFQ ₁ and AFM ₁ were independent of the added cytosol. In contrast, the rate of formation of AFB ₁ -glutathione conjugate was 306, 32, 75 pmol / min in mice, rat control, and golden extract treated rats, respectively. This means that the activity of GST Yc1 / 2 in the cytosol of mice is higher than that of rat cytosol, and the administration of the golden extract increased the activity of GST Yc1 / 2 in the cytosol of rats by 2.3 times. the extract -8,9- AFB AFB _{1 1} a non-toxic to the epoxide at a faster rate produced by the cytochrome P450 enzyme can be seen that a transition sikindaneun glutathione conjugate.

Therefore, since gold extract-treated rats to promote the GST Yc1 / 2 expression and increases the GST Yc1 / 2 is efficiently remove the AFB ₁ -8,9- epoxide found that the cause of liver cancer, gold extract GST It is possible to protect the liver and other organs from various carcinogens and toxic substances, including AFB ₁ , which are converted to poisonous substances by Yc1 / 2.

Test Example 3 Effect of Golden Extract on Survival of Hepatocytes in Primary Hepatocyte Culture

The rats were anesthetized by injection of urethane (1 g / kg body weight) into the abdominal cavity, and then the incision was made and the portal vein was connected with an 18G catheter, followed by Hanks' balanced salt solution (hereinafter referred to as' HBSS '). Inferior vena cava was immediately cut while flowing at 15-20 ml / min. Then, after flowing HBSS for 5 minutes, the circulation was connected by connecting a 14G catheter to the superior vena cava, and then tied the inferior vena cava. After collagenase was added to HBSS solution for perfusion for 15-20 minutes, hepatocytes were separated by small sections of liver tissue, and washed twice with HBSS and Williams' E medium.

The viability of the isolated hepatocytes was analyzed by staining with 0.4% trypan blue solution (Patterson, 1979). Only cells with a viability of 95% or more were used. Hepatocytes were 5.00 × 10 ⁶ cells in a Petri dish 100 mm in diameter coated with collagen, incubated at a temperature of 37 ° C. in a CO ₂ incubator (Carlson and Billings, 1996), followed by fresh Williams' E medium after 4 hours. Exchanged. Then, 12.5, 25.0, 50.0, 100.0 and 150.0 μg / ml of the golden extract were administered and incubated for 48 hours to measure the survival rate of hepatocytes, and the results are shown in FIG. 3. The control group is hepatocytes without the golden extract.

From FIG. 3, when the golden extract was administered up to 50.0 μg / ml, the viability of hepatocytes was hardly affected, but significantly decreased from 100.0 μg / ml to 51% at 150.0 μg / ml.

Test Example 4 Effect of Gold Extract on the Expression of GST in Primary Hepatocyte Culture

After treating the golden extract to the concentrations of 12.5, 25.0, 50.0 and 100.0 μg / ml in the primary hepatocytes cultured in Test Example 3 and incubating for 48 hours, the expression level of GST Ya and GST Yc1 / 2 was tested. Western blot analysis of 1, the results are shown in FIG.

From FIG. 4, the expression of GST Ya was independent of the throughput of the golden extract, whereas the expression of GST Yc1 / 2 was 6, 10 and 21 times higher than the control at the concentrations of 12.5, 25.0 and 50.0 μg / ml. .

On the other hand, when the concentration of the gold extract is 100.0 ㎍ / ㎖, the expression of GST Yc1 / 2 was significantly reduced than 50.0 ㎍ / ㎖, as can be seen in Test Example 3, since the viability of hepatocytes is reduced, It can be surmised that expression of GST Yc1 / 2 is suppressed. Nevertheless, the results obtained by administering gold at 50.0 μg / ml are far better than those of ethoxyquine, t-butyl hydroxyanisole, Oltipraz (Buetler et al., 1995; Kensler, 1997), which are known to express GST Yc. 4 times better.

In addition, this result is 5 to 10 times better than the in vivo results of Test Example 1 suggests that the amount of gold extract administered in Test Example 1 is not optimal.

[Test Example 5] Effect of Golden Extract on [ ³ H] -AFB ₁ and DNA Adducts in Primary Hepatocytes of Rats

The first hepatocytes of rats cultured in the same manner as in Example 3 were administered with gold extract at concentrations of 12.5, 25, 50 and 100 μg / ml, and cultured for 48 hours to fully express GST Yc1 / 2 in cytosol. And medium was exchanged.

Each cell was dosed with [ ³ H] -AFB ₁ (11 Ci / mmol, 1 μCi / ml) and incubated for 12 hours, after which the DNA of hepatocytes was isolated. DNA separation in hepatocytes was performed by Sharma et al. (1994). That is, the cells were washed twice with cold phosphate-buffered saline, and then reacted with 500 μl of proteinase K (100 μg / ml) in 200 mM Tris and 100 mM EDTA (pH 8.5) at 37 ° C. for 10 minutes to harvest the cells. Then, 50 μl of 10% SDS solution was added and left on ice for 30 minutes, followed by centrifugation at 13,000 × g for 15 minutes. Two times the volume of cold ethanol was administered to the supernatant, stored at -20 ° C. overnight, the DNA was precipitated, and the supernatant was discarded by centrifugation at 13,000 × g for 20 minutes. DNA pellet was washed with 70% EtOH and resuspended with 500 μl of 10 mM Tris and 1 mM EDTA buffer (pH 8.0). RNA was removed by treating 5 μl of RNase (10 mg / ml) and 5 μl of RNase T1 (5 unit / ml) at 37 ° C. for 1 hour, and only DNA was isolated.

The amount of separated DNA and the amount of radiation in the DNA were measured to determine the amount of DNA-AFB ₁ adduct (Wang et al., 1991), and the results are shown in FIG. 5.

From Fig. 5, it can be seen that when the golden extract is administered at concentrations of 12.5, 25.0, 50.0 and 100.0 μg / ml, only 36.5, 22.1, 11.5 and 14.8% of adducts are formed as compared to the control group. It can be seen that inhibits the binding of DNA and [ ³ H] -AFB ₁ . In other words, it can be seen that the golden extract is effective in effectively blocking DNA damage caused by AFB ₁ in hepatocytes.

[Test Example 6] Effect of gold extracts on human tissues GST Yc1 / 2 activity to remove AFB ₁ -8,9- epoxide (AFBO) formed by the activity of cytochrome P450 3A4 in liver

In male SD rats (6 weeks old), golden extracts were orally administered 0.0, 0.5, 1.0, and 2.0 g / kg / day for 6 days (5 per group), and cytosol of liver tissue was isolated, and then cytosol GST Yc1 / 2 activity was measured at the rate at which AFBO formed by human liver tissue was converted to AFB ₁ -glutathione, and the results are shown in FIG. 6. Human liver tissue was used for HL110 microsomes. As reported by Kim et al. (1997), only AFQ ₁ and AFBO were synthesized in AFB ₁ .

As shown in FIG. 6, the activity of GST Yc1 / 2 in rat cytosol was increased by 2-4 times as compared to the control (group without the golden extract) by administration of the golden extract, but increased at the dose of 2 g / kg / day. The trend was slightly reduced. In any case, the expression of GST Yc1 / 2 was expressed depending on the dose of the treated golden extract, and the carcinogen (AFBO) formed by human liver tissue was also rapidly treated as a non-toxic material. Switched. From this, the expression of GST Yc1 / 2, which degrades AFBO, which was found to cause liver cancer, was increased by the golden extract, which resulted in efficient inhibition of DNA damage (formation of AFB1-DNA adducts). It can be seen that the occurrence of cancer can be suppressed by preventing the reaction of converting into an advanced cancer.

As described above, the gold extract can inhibit the occurrence of cancer by promoting the expression of GST Yc1 / 2 and converting carcinogens into nontoxic substances before the DNA of the cells is damaged. It can prevent cancers of various internal organs such as liver cancer, stomach cancer, esophageal cancer, lung cancer and breast cancer.

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Claims (1)

Carcinogenic inhibitor, characterized in that it contains an extract of golden (root of Scutellaria baicalensis Georgi) as an active ingredient.