KR100623436B1 - Pharmaceutical composition containing emodin for preventing and treating cancer from Rumex acetosa L - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for cancer prevention and treatment comprising emodin derived from maternal muscle as an active ingredient.

Emodine according to the present invention has an anti-mutagenic effect that can prevent cancer with an excellent cancer treatment effect at the same time can be used as a cancer treatment and a cancer prevention agent, in particular, the effect of reducing the toxicity of the existing anti-cancer agent in combination with the existing anti-cancer agent There is.

Emodin, ovarian cancer treatment effect, antimutagenic effect, reduction of toxicity

Description

Pharmaceutical composition containing emodin for preventing and treating cancer from Rumex acetosa L}

1 is a graph showing the change in body weight of the drug-unadministered group and the emodin pretreatment group,

Figure 2 is a graph showing the change in body weight of the group treated with emodin alone, Taxol alone and combination of Emodine and Taxol after cancer,

Figure 3 is a photograph showing that the tumor was enlarged in the non-drug group (A: before the experiment, B: after completion of the experiment),

4 is a photograph showing that the size of the ovarian tumor in the emodine alone administration group (A: before administration, B: after administration),

Figure 5 is a photograph showing that the ovarian tumor size is reduced in the combined administration of emodin and taxol (A: before administration, B: after administration),

6 is a picture of cancer cells (A) not treated with emodine and cancer cells (B) necrotic treated with emodine, and the arrows show necrotic cancer cell portions,

Figure 7 is a bar graph showing the antimutagenic effect of each component on the mutagenic NPD in Salmonella typhimurium TA98, S is blank, 1 is chrysopanol, 2 is pion, 3 is emodine, 4 is emodine -8- O -D-glucopyranoside,

8 is a bar graph showing the antimutagenic effect of each component on the mutant NaN ₃ in Salmonella typhimurium TA100,

9 is a bar graph showing the antimutagenic effect of each component on the mutagenic MNNG in E. coli PQ37,

10 is a bar graph showing the antimutagenic effect of each component on mutant NQO in E. coli PQ37.

The present invention is an acid hair roots to emodine in Isolated from (Rumex acetosa L.) It relates to cancer prevention and treatment of pharmaceutical composition containing as an active ingredient, more particularly, emodine the acid follicle-derived anticancer effect and cancer The present invention relates to a pharmaceutical composition for preventing and treating cancer, which has excellent effect of inhibiting mutation, which is one of the causes, and reduces the toxicity of the existing anticancer agent when used in combination with the existing anticancer agent. .

Cancer is one of the diseases caused by the accumulation of mutations in the genome due to various causes, and the treatment methods include surgical surgery, radiation therapy, and chemotherapy by the administration of anticancer substances.

However, most of these treatments are limited to the treatment of early cancer patients or specific cancers, and death due to cancer is steadily increasing. In particular, radiation therapy or chemotherapy causes many side effects.

In Korea, for example, ovarian cancer accounts for 2.9% of all female cancers (Ministry of Health and Welfare, 1993), and the incidence of about 4% in the United States (1994, National Institutes of Health). Since radiation therapy is not effective, postoperative, chemotherapeutic agents are mainly used, and therefore, the development of new anticancer drugs that can increase the anticancer rate while reducing side effects is required.

In order to solve such a problem, the research to develop an anticancer agent from natural products, especially plant herbal medicine, is made in recent years.

Among them, Taxol (Paxol; paclitaxel) isolated from yew tree is known as an anticancer agent, and is mainly used as an ovarian cancer treatment agent.

Taxol combines with microtubules involved in the transport of various substances including chromosomes and maintains the skeleton of cells within cancer cells, and has an active mechanism that kills cancer cells by interfering with chromosomal movement of cancer cells. Because it also acts on other normal cells, it can cause other diseases, and the poor solubility in water has been pointed out as a serious toxicity and side effects.

On the other hand, the ovarian cancer treatment effect of the emodin achieved in the present invention has been confirmed through animal experiments that caused ovarian cancer, and there are no previous studies on this, and in the case of the antimutagenic effect, there are some reports, but the research is very insufficient. .

Specific examples of studies on emodin are as follows.

Reaction of emodin with ovarian cancer cells with carboplatin, a therapeutic agent for ovarian cancer, did not enhance the inhibitory effect of cancer cells, but it was reported that the drug was enhanced with etoposide or novabiosin (Cancer Research 59). , 1999, Jan G. Hengstler et al., Contribution of c-erbB-2 and topoisomerase II to chemoresistance in ovarian cancer, pp. 3206-3214). It is known (Cancer Research 55, 1995, Lisha Zhang et al., Supressed transformation and induced differentiation of HER-2 / neu-overexpressing breast cancer cells by emodin, pp. 3890-3896). In addition, emodin is a potent inhibitor of casein protein kinase II and is known to have a growth inhibitory effect on uterine cancer cells (Korean Patent Registration No. 0221762).

In addition, emodin has a weak leukemia cancer cell inhibitory effect and has been reported to search for substances that have a strong blood cancer inhibitory effect among the synthetic derivatives (Journal of Medicinal Chemistry 31, 1988, M. Koyama et al., Novel type of potential anticancer agents derived from chrysophanol and emodin.Some structure-activity relationship studies, pp. 283-284; 32, 1989, M. Koyama et al., Intercalating agents with covalent bond forming capability.A novel type of potential anticancer agents. 2. Derivatives of chrysophanol and emodin, pp. 1594-1599).

On the other hand, studies related to the antimutagenic effect of emodin showed mutation activity when using the S-9 mixture among TA 1573 strains among several Salmonella TA strains. No activity was reported (Applied and Environmental Microbiology 37, 1979, F. Wehner et al., Mutagenicity of the mycotoxin emodin in the Salmonella / microsome system, pp. 658-660).

However, it has not been determined whether the emodin has antimutagenic activity in the presence of various mutagens, and the results of the previous studies on the emodin could not confirm whether the emodine is effective in treating and preventing ovarian cancer.

Accordingly, the object of the present invention is to find a herbal ingredient that has superior anticancer effect and antimutagenic effect than conventionally known anticancer agents, and as a result, excellent anticancer effect and anticancer effect from methylene chloride extract of Mame root ( Rumex acetosa L.) By separating the components with mutagenic effects and characterizing the structure, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition containing a herbal ingredient capable of treating cancer and simultaneously preventing it.

Still another object of the present invention is to provide a pharmaceutical composition containing herbal ingredients that can treat ovarian cancer while simultaneously preventing it.

Still another object of the present invention is to provide a pharmaceutical composition containing a herbal ingredient that can treat lung cancer, melanoma, central nerve cancer and rectal cancer while simultaneously preventing it.

Still another object of the present invention is to provide a method of inhibiting cancer cell death or proliferation which can maintain anticancer effect while greatly reducing the toxicity of an existing anticancer agent by administering in combination with an existing ovarian cancer treatment agent.

Still another object of the present invention is to provide a method for separating herbal ingredients that can simultaneously and prevent cancer from maternal muscle.

In order to achieve the above object, the pharmaceutical composition according to the present invention is characterized by containing the emodine represented by the following formula (1) in an amount useful for preventing and treating cancer.

[Formula 1]

In addition, to achieve another object, the present invention provides a method for inhibiting cancer cell death or proliferation using the pharmaceutical composition.

In addition, in order to achieve another object, the present invention provides a method for producing emodine from maternal muscle.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

Maternal root (scientific name: Rumex acetosa ) refers to the roots of perennial herb, belonging to the polygonaceae, which grows in wild fields in Korea.

Young leaves and stems have been used for food, and roots have been used for medicinal purposes. Swim, fever, jigger, blood donation, convergence, bleeding, skin disease, hematopoiesis, bleeding, scabies, ringworm, dysentery (above Asian medicinal map, p. 149, cutlery, Kyungwon publisher, 1997), postpartum, neuralgia, arthritis ( New Herbal Medicine, p. 880, Dongcheon Publishing Co., 1999), fever, diuresis, acupuncture points, jigal, earth blood, blood transfusion, Sogal (Supplemental natural drug metabolism, Haekwon, p. 125, Kim Jae Gil, Namsandang, 1984) It is known to be effective.

The present invention finds that the extract obtained by extracting the mother root having the above efficacy with methylene chloride has an anticancer effect and can suppress mutations that cause cancer, and isolates four active ingredients therefrom It is to provide a pharmaceutical composition containing a strong component of emodine.

In the present invention, the pharmaceutical composition containing emodin as an active ingredient can prevent and treat ovarian cancer, lung cancer, melanoma, central nerve cancer and rectal cancer.

And the composition according to the present invention has the property of inhibiting mutations.

In addition, when used in combination with taxol or taxol derivatives, the emodine according to the present invention has the characteristics that the anticancer effect is maintained while significantly reducing the side effects of the existing taxol.

At this time, the taxol derivative is in the group consisting of known 10-deacetyl baccatin III, bactin III, 10-deacetyl taxol, cepharomannin and 7-epi-10-deacetyl taxol. The selected taxol derivative may be used, and any derivative that exhibits an effective effect of taxol may be used.

And, the mother root-derived emodine used in the present invention, after drying the mother root in the shade sufficiently, pulverized by pulverizing; Adding a mixture of methanol and water to the maternal root powder, followed by heat extraction to obtain a water extract; Adding water and hexane again to the water extract and shaking to separate the aqueous layer; Adding methylene chloride to the aqueous layer and shaking to separate the methylene chloride layer; Concentrating the methylene chloride layer with a vacuum concentrator to remove methylene chloride, and then drying to obtain a methylene chloride fraction; And methylene chloride fractions are subjected to silica gel chromatography using a 9 to 1 mixed solvent of methylene chloride and methanol as a separation solvent.

On the other hand, in the pharmaceutical composition of the present invention, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and liquid preparations for oral use include suspensions, contents, emulsions, syrups, and the like. This includes a variety of excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to the commonly used simple diluents, water and liquid paraffin.

In addition, the effective amount of emodine in the pharmaceutical composition of the present invention can be determined within a wide range depending on the route of administration, the dosage form, the purpose of use, and the degree of disease of the patient. However, it is generally formulated to be administered at a concentration of 22-26 mg / kg / week, preferably 24 mg / kg / week.

At this time, the formulation for administering the emidein in the above range may have a conventional form, for example, it can be used in the form of a pill or medicine or dietary supplement.

In addition, they may be administered for the prevention and treatment of cancer via oral or various parenteral routes of administration, are suitable for the dosage form and are already well known to those skilled in the art and can be easily selected by those skilled in the art, Carrier, diluent, and the like.

In addition, when co-administering Taxol or a derivative of Taxol in the pharmaceutical composition of the present invention, it is preferable that the combined effective amount of Taxol follows a conventional dosage, but since the actual dosage should be determined in view of various related factors, The use range is not limited.

Hereinafter, although an Example and an experimental example are given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Reference Example 1 Preparation of Water Extract of Maternal Muscle

After collecting from Ulsan area, the mother root was confirmed and confirmed by Professor Kang Byung-su of Dongguk University's primary school and dried to remove moisture. According to the Korean Pharmacopoeia test method, maternal roots are pulverized using a conventional grinder to make powder, and then, 200 g of powder is added with 2,000 ml of 80% methanol (400 ml of water and 1600 ml of methanol) and extracted by heating with a water bath for 3 hours. It was. Then, methanol was removed by a vacuum concentrator and 300 ml of water extract was obtained.

Reference Example 2 Preparation of Methylene Chloride Fraction of Maternal Muscle

300 ml of water was added to 300 ml of the water extract of Reference Example 1, and the mixture was placed in a separatory funnel, and 500 ml of hexane was mixed and vigorously shaken 10 times. After standing for 1 hour to form two layers, the lower aqueous layer was separated. The aqueous layer was placed in a separatory funnel again, and 500 ml of methylene chloride was added thereto, followed by strong shaking 10 times. After leaving for 1 hour to form two layers, the lower methylene chloride layer was separated. The methylene chloride layer was concentrated with a vacuum condenser to remove methylene chloride, and then dried with a freeze dryer to obtain 13.1 g of methylene chloride fraction.

Example 1 Separation of Active Ingredients

The methylene chloride fractions were subjected to component separation by silica gel column chromatography. First, a mixture of components 1 and 2 was separated using methylene chloride as a separation solvent. Next, components 3 and 4 were separated using a solvent having a component ratio of 9: 1 to methylene chloride and methanol as a separation solvent. In the mixture of components 1 and 2, two components were purely separated using a mixed solvent of petroleum ether and methylene chloride (petroleum ether: methylene chloride = 1: 1). Instrumental analysis was performed to identify the structure of the four isolated components.

Example 2 Structural Analysis of Active Ingredients

Nuclear Magnetic Resonance Spectroscopy (Gemini-200, Varian, USA), Mass Spectrometry (MAT95, Finnigan, Germany), Infrared Spectroscopy (MB100-10, Bomem) , Canada), ultraviolet spectroscopic analysis (UV-2001S, Schimadzu, Japan) and the like were analyzed and the melting point was measured. Analysis of the instrument analysis shows that component 1 is Chrysophanol, component 2 is Physcion, component 3 is Emodin and component 4 is Emodine-8- O -D-glucopyrano It was identified as the seed (Emodin-8- O- D-glucopyranoside), and the analysis result of component 3 (emodin) having the strongest activity among them is shown below. Emodine crystals were scarlet in red, and their melting point was measured at 262-263 ° C.

Molecular Formula: C ₁₅ H ₁₀ O ₅

Nuclear magnetic resonance spectroscopy: δ H (CDCl ₃ + DMSO-d ₆ , 200 MHz) (ppm) = 6.97 (H-2), 7.46 (H-4), 7.18 (H-5), 6.57 (H-7) , 2.40 (CH ₃ ), 12.10-12.13 (OH-1, OH-6, OH-8).

Nuclear Magnetic Resonance Spectroscopy: δC (CDCl ₃ + DMSO-d ₆ , 50 MHz) (ppm) = 162.4 (C-1), 121.1 (C-2), 148.2 (C-3), 124.4 (C-4) , 109.5 (C-5), 166.1 (C-6), 108.7 (C-7), 165.5 (C-8), 190.4 (C-9), 182.0 (C-10), 133.4 (C-4a), 110.0 (C-8a), 113.8 (C-9a), 135.4 (C-10a), 22.3 (CH ₃ ).

Melting Point (mp): 262∼263 ℃

Infrared spectroscopy ν max (nujol): 3435 (OH), 1680 (CO), 1628cm ^-1 (CO)

UV spectrometry λ max (logε) (CH ₂ Cl ₂ ): 438 (3.07), 288 (3.24), 266 (3.24), 230 nm (3.25)

Mass spectrometry (m / z) (rel. Int.): 270 (M ^{+ ·} , 100%), 242 (M-CO, 18%), 241 (M-CHO, 24)

Experimental Example 1 Anticancer Effect Measurement

Human cancer cell line was used five kinds of cell lines in culture at Korea Research Institute of Chemistry, and the inhibition of cancer cell proliferation was SRB method (Sulfrhodamine B method) (Skehan, P) which is the most widely used method to measure the primary anticancer effect of drugs at the National Cancer Center. Et al., 1990: New colorimetric cytotoxicity assay for anticancer drug screening. J. Natl. Cancer. Inst. 82, 1107-1112). The cancer cell proliferation rate (% cell growth) of the sample group was [(T-Tz) / (C-Tz)] × 100 when Tz> T, and [(T-Tz) / Tz] × when Tz <T. Calculated by the formula of 100. In this case, C is the number of cells in the control group, Tz is the number of cells in zero time, and T is the number of cells in the sample group. Based on the cancer cell proliferation rate of the sample groups measured at each concentration, the data regression of the Lotus program was used to calculate the concentration at which the sample inhibited the growth of the corresponding cancer cells (IC ₅₀ ).

The results are shown in Table 1 below.

Cancer cell line IC ₅₀ (μg / ml) Reference Example 1 (Methylene Chloride Fraction) Example 2 (Crysopanol) Example 2 (Pcion) Example 2 (Emodine) Example 2 (Emodine-8-O-β-D-Glucopyranoside) A549 (lung cancer) 13.17 34.76 25.16 3.32 > 30.0 SK-OV-3 (ovarian cancer) 13.46 7.28 22.41 2.94 > 30.0 SK-MEL-2 (melanoma) 18.73 5.83 26.07 3.64 > 30.0 XF498 (Central Nerve Cancer) 18.35 > 30.0 26.40 2.98 > 30.0 HCT15 (Rectal Cancer) 17.62 > 30.0 36.88 3.10 > 30.0 * IC ₅₀ means a concentration that inhibits the proliferation of cancer cells by 50%.

From Table 1, it can be seen that the emodine of the present invention has an excellent anticancer effect among human cancer cells, in particular, ovarian cancer cells. That is, the emodine had an IC ₅₀ of 2.94 μg / ml for the ovarian cancer cell line.

Experimental Example 2 Effect of Inhibiting Ovarian Cancer in Rats

Emodine of the present invention was orally administered 2 mg (10 mg / kg) once a week to a lett (Korean experimental animal development) having an average body weight of about 205 g for 5 months, and compared the effect with that of the control group, weight change, ovary Cancer incidence, gross and histological differences in ovarian cancer, and the extent of prevention and treatment of ovarian cancer were analyzed.

(1) Prevention of Ovarian Cancer According to Weight Change in Pretreatment

Emodine was administered at the same time as ovarian cancer was induced, and the effect of inhibiting the oncogenesis of emodin was observed as a change in body weight. At the beginning of the experiment, the average body weight was 205.5g. Over time, the weight of the emodine-treated group increased but the weight of the emodine-free group decreased.

Thus, it has been shown to inhibit weight loss by carcinogens during the period of emodin pretreatment.

(2) Ovarian cancer incidence during pretreatment

When ovarian cancer was induced with emodin, 2 of 10 animals died and 4 of 8 survivors developed cancer and atypical linear proliferation was observed. The control group survived only 5 animals, 4 of them developed cancer, and the other one showed atypical linear proliferation. The incidence of cancer was 50% in the experimental group (emodine-administered group), whereas the control group was 80%.

(3) Visual and histological aspects of cancer produced during pretreatment

The mean size of ovarian cancer was 2.3 ± 1.3cm in the control group and 1.7 ± 0.9cm in the experimental group. Histologic type of tumor was 3 cases of adenocarcinoma and 1 case of adenocarcinoma and squamous cell carcinoma in both experimental and control groups.

Experimental Example 4 Effect of Emodine on Ovarian Cancer Treatment

DMBA (9,10-dimethyl-1,2-benzanthracene, Sigma Chemical Co, Mo., USA) was inserted directly into the left ovary to induce tumors in the ovary for 4 months, and then the tumors were generated in the present invention. The effect of emodin was analyzed.

At this time, 20 of 40 rats died as test animals, and 20 surviving animals were tested.

Emodine was orally administered 3mg (12mg / kg) twice a week for 8 weeks in the group treated with the emodin alone and the group treated with the emodin and taxol, and the taxol 2mg (for the group treated with the emodine and taxol alone and taxol alone). 8 mg / kg) twice a week for 8 weeks intraperitoneally to compare the anticancer effect.

(1) Weight change during the experiment

Body weight change during the entire experimental period after cancer is shown in FIG. 2. In the non-drug group, the mean weight before the start of the experiment was about 275g, and increased to 287.5g after 8 weeks of the experiment. In addition, the average weight of the emodine alone group was about 262 g before the start of drug administration, but increased to 270 g at 8 weeks. However, body weight was reduced in the combination of emodin and taxol and the taxol alone group, which is considered to be a side effect of taxol.

(2) Comparison of therapeutic effect between single administration and combination administration

After the onset of cancer, the mean size of the cancer was 1.2 ± 0.7 cm. Drug administration was carried out on 20 tumors, and the experimental results are shown in Table 2 below. In the non-drug group, the mean tumor size was 1.5 ± 1.1 cm before drug administration but increased 2.5 cm to 4.0 ± 2.1 cm after drug administration (see FIG. 3). In addition, in the case of the emodine-only group (see FIG. 4) and the combined administration group (see FIG. 5), the average increase was 0.1 cm and 0.4 cm, respectively. The size increased or decreased, and the non-drug group increased significantly in size compared to the three experimental groups (p <0.05).

However, there was no statistically significant difference in size change between the other Taxol alone, Emodine-only and co-administration groups (p> 0.05).

(3) Comparison of mortality rate between single dose and combination dose

Comparing the survival rate of the drug-nonadministered group and the Taxol alone group, the emodine-only group and the combination-administered group, as shown in Table 2, the non-drug group was 100% surviving for 8 weeks during the experimental period, and also in the case of the emodine-only group. Compared to 100%, survival rates of Taxol and Co-administered groups were 0% and 50%, respectively.

The mortality rate of the taxol-administered group was higher than that of the emodine-only group, demonstrating that the taxol had higher anticancer effect but higher toxicity and side effects.

Tumor size before administration (cm) Tumor size after administration (cm) Tumor size change (cm) death rate(%) Drug non-administration group 1.5 ± 1.1 4.0 ± 2.1 2.5 ^* 0 Taxol alone group 1.0 ± 0.6 0.5 ± 0.1 -0.5 ^* 100 Emodine + Taxol combination group 1.1 ± 0.8 1.2 ± 0.8 0.1 ^* 50 Emodine-only group 1.1 ± 0.7 1.5 ± 2.0 0.4 ^* 0

(Kruskal-Wallis test or Chi-square test * p <0.05)

Therefore, the emodine of the present invention was confirmed to suppress ovarian cancer while maintaining normal activity when inducing ovarian cancer, and when applied to humans, the emodine is 22 to 26 mg / kg per week on a per person basis It is expected that it may be formulated to be administered orally, preferably at a concentration of 24 mg / kg.

In addition, when taxin or a taxol derivative showing the effect of taxol is used together with the emodin of the present invention, it has been found that the anticancer effect is increased while the side effect of taxol is significantly reduced.

(4) Effect of Emodine on Cultured Ovarian Cancer Cells

As a result of culturing ovarian cancer cells treated with emodine and untreated ovarian cancer cells, necrosis occurred in ovarian cancer cells when treated with emodine (see FIG. 6B).

Experimental Example 4 Anti-mutation Effect Measurement

4 -1. Ames test

Histidine-required mutants, Salmonella typhimurium TA100 and TA98 (generated from the Gene Collection Bank of Korea Institute of Science and Technology; Korea Collection for Type Cultures) were tested. As a negative control, DMSO (dimethylsulfoxide), a solvent in which a sample was dissolved, was used. As a positive control, a mutation rate was determined using NPD (4-nitro- o- phenylene-diamine) and NaN ₃ (sodium azide). Comparison was made with each other.

Meanwhile, before the experiment, the strains were used as test strains after checking genotypes such as histidine-required deep rough character, UV sensitivity, and R factor.

The medium for anti-mutation experiments and preparation of the necessary reagents were performed according to the method of Maron and Ames.

That is, 0.1 ml of strain cultured overnight (1-2 × 10 ⁹ cells / ml), 0.1 ml of mutagen (containing 10 µg of NPD, 1 µg of NaN ₃ ), 0.5 ml of sodium phosphate buffer, and each component of Example 2 100 μg / plate was added to the test tube, followed by gentle vortexing and preincubation at 37 ° C. for 20 minutes. 2 ml of each top agar stored at 45 ° C. was poured into tubes, vortexed for 3 seconds, plated on minimal glucose agar plates, incubated at 37 ° C. for 48 hours, and returned. The number of mutations was counted. The results are shown in FIGS. 7 and 8.

From Fig. 7, it can be seen that, for NPD which is a direct mutagenesis agent, emodine (component 3) exhibits strong antimutagenic activity by reducing the number of return mutations by NPD by 71.6%. In addition, it can be seen from FIG. 8 that emodine (component 3) also reduces the number of back mutations caused by NaN ₃ by 53.2% with respect to another mutagen, NaN ₃ , to show good antimutagenic activity.

4 -2. SOS Chromotest

To more reliably confirm the antimutagenic effects of the components isolated from maternal muscle, SOS chromotest was performed using N-methyl-N'-nitrosoguadine (MNNG) and 4-nitroquinoline-1-oxide (NQO) as mutagens. It was carried out in the same manner.

Incubated 0.5 ml of E. coli PQ37 (passage of bacteria stored in the laboratory) was inoculated in 50 ml of Liria Bertani medium (composition: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1.0 l of distilled water, pH 7.0) for 4 hours at 37 ° C. The culture was shaken for a while (OD = 0.4-0.8). 600 µl of this culture solution, 20 µl each of mutant MNNG or NQO (1 µg / assay) and 10 µg of each component were put in a test tube to induce an SOS reaction. After standing at 37 ° C. for 2 hours, the enzymatic activity of β-galactoxytase and alkaline phosphatase was measured.

β-galactoxydase activity was determined by 1.8 ml of B buffer (composition: Na ₂ HPO ₄ 5.5 g, KCl 0.75 g, MgSO ₄ 0.25 g, SDS 1.0 g, β-mercaptoethanol 2.7 ml, distilled water 1,000 ml, pH 7.0 ), The mixture was allowed to stand at 37 DEG C for 5 minutes to uniform the temperature, and then 0.4 ml of o -nitrophenyl-β-galactoxytase (ONGP) solution was added to maintain color development for 10 minutes. And, the absorbance was measured at 420nm after the reaction by addition of 2.0M Na ₂ CO ₃ solution of 1.4㎖.

Alkaline phosphatase activity was measured in the above method for measuring activity of β-galactoxytase, instead of B buffer, P buffer (composition: tris-base 121 g, SDS 1.0 g, distilled water 1,000 ml, pH 8.8), instead of ONGP solution. The activity was measured by adding p-nitrophenyl-phosphogalactoxydase solution. To stop the enzymatic reaction, 0.7 ml of 2.5M HCl was added and then the reaction was stopped for 5 minutes, and 0.7 ml of 2.0M Tris-HCl buffer was added.

The activity of each enzyme was measured using the formula of 1000 × A ₄₂₀ / t (wherein A ₄₂₀ represents the absorbance at ₄₂₀ nm and t represents the reaction time in minutes). Was calculated by dividing β-galactoxytase activity by alkaline phosphatase activity. The results are shown in FIGS. 9 and 10.

9 and 10 show that emodine (component 3) shows the lowest enzyme activity ratio value.

That is, since the emodin, which is a component of the maternal muscle, effectively inhibits mutations, it was found that the emodine according to the present invention is excellent in preventing cancer.

As described above, emodine, which is an active ingredient of the mother root ( Rumex acetosa L.), can treat cancer and at the same time can prevent cancer by suppressing mutations, and thus can be effectively used as an anticancer agent and a cancer prevention agent.

In addition, when administered in combination with the existing anticancer drugs to reduce the toxicity of the existing anticancer drugs and the anticancer effect is maintained.

Claims (6)

delete A pharmaceutical composition for the prevention and treatment of ovarian cancer comprising an emodine represented by the following formula (1) as an active ingredient. [Formula 1]

delete delete delete (a) drying the maternal muscles sufficiently in the shade and then pulverizing to powder; (b) adding a mixture of methanol and water to the maternal root powder, followed by heat extraction to obtain a water extract; (c) adding water and hexane again to the water extract and shaking to separate the aqueous layer; (d) adding methylene chloride to the separated aqueous layer and then shaking to separate the methylene chloride layer; (e) concentrating the separated methylene chloride layer in a vacuum condenser to remove methylene chloride and then drying to obtain a methylene chloride fraction; And (F) a method for producing emodine represented by the formula (1), characterized in that the methylene chloride fraction comprises silica gel chromatography using a 9 to 1 mixed solvent of methylene chloride and methanol as a separation solvent.

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