KR100422094B1 - A composition having an effect of curing and preventing cancer by containing a methylenechloride extract of Rumex acetosa L. - Google Patents

Abstract

The present invention relates to a pharmaceutical composition capable of treating and preventing cancer by containing methylene chloride extract of swimming ( Rumex acetosa L. ) as an active ingredient.

Since the methylene chloride extract of swimming according to the present invention exhibits an excellent anticancer effect and at the same time has an effect of inhibiting mutations that cause cancer, it can be effectively used as a cancer treatment and a cancer prevention agent.

Description

A composition having an effect of curing and preventing cancer by containing a methylenechloride extract of Rumex acetosa L.}

The present invention relates to a pharmaceutical composition for treating and preventing cancer containing methylene chloride extract of swimming ( Rumex acetosa L. ) as an active ingredient. More specifically, the methylene chloride extract of swimming has an excellent anti-cancer effect. It is confirmed that the effect of inhibiting mutation, which is one of the causes of cancer, is excellent, and relates to providing a pharmaceutical composition for treating and preventing cancer containing the same as an active ingredient.

Cancer is one of the diseases that is increasing with the development of civilization, and treatment methods include surgical surgery, radiation therapy, and chemotherapy by chemotherapy.

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.

Therefore, there is a need for the development of anticancer drugs that can increase the anticancer rate while reducing side effects.

For this reason, attempts have been made to develop anticancer agents from natural products, in particular, herbal medicines, and specific examples are as follows.

In other words, 18 terpene compounds isolated from the methylene chloride fraction of Salvia miltiorrhiza showed anti-cancer effects, and according to cancer cells, ED ₅₀ values of about 20-500 ㎍ / mL were reported (Planta Medica). 63, 1997, Georg Thieme Verlag, Germany, SY Ryu, CO Lee, SU Choi, In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza, pp. 339-342), 6, isolated from the ethyl acetate fraction of Magnolia obovata Compounds with various structures of species also have anti-cancer effects and have been reported to have ED ₅₀ values of about 12.5-20.2 ㎍ / ml depending on the cancer cells (Planta Medica 65, 1999, Georg Thieme Verlag, Germany, YK Kim, SY Ryu, Cytotoxic components from stem bark of Magnolia obovata, pp. 291-292).

In addition, eight different compounds isolated from methylene chloride fractions of Artemisia princeps showed anti-cancer effects, and reported that they had ED ₅₀ values of about 28.5 to 45.8 μg / ml depending on cancer cells. (Planta Medica 63, 1997, Georg Thieme Verlag, Germany, SY Ryu, JO Kim, SU Choi, Cytotoxiccomponents of Artemisia princeps, pp. 384-385).

However, the conventional herbal extracts such as the anticancer drugs were able to reduce the side effects because they are herbal extracts, but the anticancer effect was weak and not effective in the treatment of cancer. Moreover, in the case of such conventional herbal extract anticancer drugs, no research has been conducted on whether the cancer-causing mutations can be suppressed, and thus, it was not possible to confirm whether they could prevent cancer.

Therefore, the present inventors conducted a study to find a herbal ingredient that has superior anticancer effect and antimutagenic effect than conventional herbal extract anticancer agents, and methylene chloride extract of swimming ( Rumex acetosa L. ) has excellent anticancer effect and antimutagenicity. It has been found to have an effect and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition capable of treating cancer and at the same time preventing cancer.

1 is a graph showing the antimutagenic effect of each swimming extract on mutagenic MNNG.

In order to achieve the above object, the pharmaceutical composition according to the present invention is characterized by containing methylene chloride extract of swimming ( Rumex acetosa L. ) in an amount effective to treat and prevent cancer.

Hereinafter, the present invention will be described in detail.

Swimmer (scientific name: Rumex acetosa ) is a perennial plant belonging to the polygonaceae that grows in wild fields in Korea. The root leaves are dense, the leaves are revived, and the petioles are long lanceolate. Male and female multicolored plant that has a torus, light green or greenish purple in a cone. A male flower has 6 calyxes, 6 stamens, and a female flower has 6 calyxes, 3 pistils. Fruits are achenes, triangular oval, black brown, yellowish white, with three ridges, and wings. The flowering period is from May to June, and the fruiting season is from August to September. 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 New Herbal Medicine, p. 880, Dongcheon Publishing Co., 1999), fever, diuresis, acupuncture points, jigal, earth blood, blood transfusion, sogal It is known to be effective.

The present invention finds that the extract obtained by extracting swimming having the above efficacy with methylene chloride has an anticancer effect and can suppress mutations that cause cancer, and provides a pharmaceutical composition having the same as an active ingredient.

The methylene chloride extract of swimming used in the present invention comprises drying the collected swimming in the shade sufficiently, and then pulverizing to powder; Adding a mixture of methanol and water to the swimming 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; And methylene chloride layer is concentrated with a vacuum condenser to remove methylene chloride and then dried to obtain a methylene chloride extract of swimming.

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 Swimming

After collecting from Ulsan area, the confirmed swimming was confirmed by Professor Kang Byung-su of the Department of Herbal Medicine, College of Oriental Medicine, Dongguk University, and dried to remove moisture. According to the Korean Pharmacopoeia Test Method, a conventional grinder is used to pulverize swimming outposts (including roots, leaves, and stems) into powder, and then 200 g of swimming powder is mixed with 2000 ml of 80% methanol (400 ml of water and 1600 ml of methanol). ) Was added and extracted by heating with a water bath for 3 hours. Methanol was removed by the vacuum concentrator and 300 ml of water extract was obtained.

Reference Example 2 Preparation of Hexane Extract of Swimming

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 leaving for 1 hour to give two layers, the upper hexane layer was separated. This was concentrated with a vacuum condenser to remove hexane, and then dried with a freeze dryer to obtain 11.2 g of hexane extract.

Example 1 Preparation of methylene chloride extract of swimming

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 give 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 extract.

Reference Example 3 Preparation of Ethyl Acetate Extract of Swimming

4.3 g of ethyl acetate extract was obtained in the same manner as in Example 1, except that ethyl acetate was added to the aqueous layer instead of methylene chloride.

Reference Example 4 Preparation of Butanol Extract of Swimming

11.5 g of butanol extract was obtained in the same manner as in Example 1 except that butanol was added to the aqueous layer instead of methylene chloride.

Experimental Example 1 Anticancer Effect Measurement

The cancer cell line was used as a cell line in culture at the Korea Research Institute of Chemical Technology, and the cell culture was maintained at 37 ° C., 100% humidity, and 10% CO ₂ concentration. D-MEM containing 10% of serum was added to each well in a 24-well plate on the day before sample administration, and the cells in culture were trypsinized and harvested, and the cell concentration was calculated by a cell counter.

Diluted by concentration with D-MEM and plated in each well with different cell numbers. After 18 hours of cell plating, the medium was aspirated, and then 1.0 mL of D-MEM containing no serum was added to each well, and the sample was diluted by concentration with D-MEM containing no serum and administered to each well. After 4 hours of administration, all of the medium was removed, and then 2.0 ml of D-MEM containing 10% of serum was added thereto per well. After 6 days of sample administration, all of the medium was removed, stained with crystal violet for 3 minutes and dried, and observed by an anatomical microscope, and the survival rate of cancer cells was calculated by comparing the number of colonies with the plated cells. The results are shown in Table 1.

Cancer cell line ED ₅₀ (μg / ml) Methanol extract Reference Example 2 (hexane Extract) Reference Example 3 (Ethyl Acetate Extract) Reference Example 4 (butanol extract) Example 1 (methylene chloride extract) A549 (lung cancer) 200 29.14 36.51 184.62 13.17 SK-OV-3 (ovarian cancer) 200 24.66 35.49 56.22 13.46 SK-MEL-2 (melanoma) 200 34.28 35.11 143.90 18.73 XF498 (Central Nerve Cancer) 200 47.07 43.64 193.67 18.35 HCT15 (Rectal Cancer) 200 38.15 40.16 140.03 17.62 * ED ₅₀ means a concentration that inhibits the proliferation of cancer cells by 50%.

From Table 1, it can be seen that the methylene chloride extract of the present invention has an excellent anticancer effect on human cancer cells. That is, the methylene chloride fraction of Salvia miltiorrhiza is as A549 (lung cancer cell line) ED ₅₀ medicine.Percocet.Where'd 30㎍ / ㎖ was, Arte ED ₅₀ is 28.5 ~ 45.8 ㎍ / ㎖ accordance with the methylene chloride fraction on a cyano US peurinsep's cancer cells is for the present invention All anticancer effects were weaker than the composition. In the case of Il Magnolia, ED ₅₀ is reported to be different for each type of cancer: lung cancer 18.5 µg / ml, ovarian cancer 20.2 µg / ml, melanoma 17.5 µg / ml, nerve cancer 12.5 µg / ml, and rectal cancer 17.5 µg / ml. However, when considering the error range of the results of Table 1 above, the swimming methylene chloride extract of the present invention was superior to the anti-cancer effect in the lung cancer and ovarian cancer than Il Magnolia, almost similar anticancer effect in melanoma and rectal cancer And, in the central nervous system cancer showed a slight anti-cancer effect.

Experimental Example 2 Anti-mutation Effect Measurement

2-1. Ames test

Histidine-required mutants Salmonella typhimurium TA100 and TA98 (substituted from the Korea Collection for Type Cultures of the Institute of Biotechnology, Korea Advanced Institute of Science and Technology) were tested. DMSO, a solvent in which the sample was dissolved, was used as a negative control, and the mutation rate was compared with the positive control group NPD (4-nitro- o- phenylenediamine) and NaN ₃ (sodium azide).

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 methylene chloride of Example 1 Extract 1 mg / 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 Table 2 and Table 3.

Antimutagenic Effects of Each Extract on Mutant NPD in Salmonella typhimurium TA98 sample Return mutation / plate % Inhibition Natural variation 6 ± 1 NPD 640 ± 24 NPD + Methanol Extract 391 ± 37.5 39.35 NPD + Hexane Extract (Reference Example 2) 110 ± 107.5 83.67 NPD + Ethyl Acetate Extract (Reference Example 3) 590 ± 11.5 7.96 NPD + Butanol Extract (Reference Example 4) 528 ± 14 17.66 NPD + Methylene Chloride Extract (Example 1) 31 ± 22 96.06

From Table 2, it can be seen that the hexane extract and the methylene chloride extract showed 83.7% and 96.1% of antimutagenic activity, respectively, for NPD, which is a direct mutagenesis source.

Antimutagenic Effects of Each Extract on Mutant NaN ₃ in Salmonella typhimurium TA100 sample Return mutation / plate % Inhibition Natural variation 150 ± 12.5 NaN ₃ 287 ± 3.5 NaN ₃ + Methanol Extract 234 ± 8.5 38.69 NaN ₃ + hexane extract (Reference Example 2) 244 ± 1.5 31.39 NaN ₃ + ethyl acetate extract (Reference Example 3) 237 ± 1.5 36.50 NaN ₃ + butanol extract (Reference Example 4) 275 ± 3 8.39 NaN ₃ + methylene chloride extract (Example 1) 154 ± 11 96.72

From Table 3, it can be seen that the methylene chloride extract showed a high antimutagenic activity of 96.7%, whereas the hexane extract was low as 31.4%, for the other mutant cause NaN ₃ .

2-2. SOS Chromotest

In order to more reliably confirm the antimutagenic effect of each swimming extract, SOS chromotest was carried out by MNNG (N-methyl-N'-nitrosoguadine) as a mutagen.

Cultured 0.5 ml of E. coli PQ37 (passage cultured in the laboratory) in 50 ml Liria Bertani medium (composition: tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1.0L, pH 7.0) Inoculation was incubated for 4 hours at 37 ℃ (OD = 0.4 ~ 0.8). 600 µl of this culture solution, 10 µl of mutagenesis (MNNG) and 10 µl of methylene chloride extract were put into a tube to induce SOS reaction. After standing at 37 ° C. for 2 hours, alkaline phosphatase enzyme activity 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. After reacting by adding 1.4 ml of 2.0 M Na ₂ CO ₃ solution, the absorbance at 420 nm was measured.

Alkaline phosphatase activity was measured using P buffer (composition: tris-base 121 g, SDS 1.0 g, distilled water 1,000 mL, pH 8.8) instead of B buffer and p-nitrophenyl-phosphogalactoxidase activity instead of ONGP solution. It was. 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 FIG. From Figure 1, it can be seen that the methylene chloride extract shows the lowest enzyme activity ratio value as in the Ames test.

That is, since methylene chloride extract of swimming effectively inhibits mutations, it can be seen from this that the methylene chloride extract of swimming according to the present invention has excellent cancer prevention effect.

The present invention provides a pharmaceutical composition containing the above methylene chloride extract of swimming in an amount effective for the treatment of cancer and the prevention of cancer.

The effective amount of methylene chloride extract of swimming in the pharmaceutical composition of the present invention can be determined within a wide range depending on the route of administration, the formulation, the purpose of use, the degree of disease of the patient, and the like. In general, however, extracts may be formulated to be administered at a concentration of 0.1-0.3 mg / kg / day, preferably 0.2 mg / kg / day.

Preparations for administering the methylene chloride extract of swimming in the above range may have a conventional form, for example, may be used in the form of pills, drinks, medicines or dietary supplements. 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 of ordinary skill in the art, and various excipients, carriers, Or a diluent or the like.

As described above, the methylene chloride extract of swimming ( Rumex acetosa L. ) can treat cancer and at the same time can prevent cancer by inhibiting mutations, so it can be effectively used as an anticancer agent and cancer prevention agent.

Claims (3)

A pharmaceutical composition capable of treating and preventing cancer by containing methylene chloride extract of swimming ( Rumex acetosa L. ) as an active ingredient. The pharmaceutical composition of claim 1, wherein the cancer is lung cancer, ovarian cancer, melanoma, central nerve cancer, or rectal cancer. The pharmaceutical composition of claim 1, wherein the prevention of cancer is by inhibition of mutation.