KR20040021183A - Anticancer composition comprising an extract and a compound isolated from Rhodiola sachalinensis - Google Patents

Abstract

PURPOSE: An anticancer composition containing a Rhodiola sachalinensis extract exhibiting anticancer activity for suppressing proliferation of cancer cells is provided. It inhibits a topoisomerase enzyme and thus is expected to have inhibiting activity against cancer. CONSTITUTION: The pharmaceutical composition contains 0.01 to 80% by weight of a Rhodiola sachalinensis extract, based on the total weight of the composition. The Rhodiola sachalinensis extract is prepared by extracting Rhodiola sachalinensis in a polar solvent with a single reflux cooler, concentrating under reduced pressure, fractioning with a nonpolar solvent such as chloroform, ethylacetate and ether and then carrying out silica gel column chromatography or TLC. The purified fraction has Rf values of 0.50 to 0.60 on TLC. The polar solvent is water, methanol, ethanol or a mixture thereof.

Description

Anticancer composition comprising an extract of Rhodiola saline and a compound isolated therefrom {Anticancer composition comprising an extract and a compound isolated from Rhodiola sachalinensis}

The present invention relates to a composition containing a herbal extract having anticancer activity and a compound isolated therefrom.

DNA Topoisomerase is an enzyme that regulates and modifies the morphological state of DNA by simultaneously catalyzing the cleavage and recombination of DNA strands during DNA replication, repair, gene recombination and transcription (D'Arpa). et al., Biochim. Biophys.Acta. , 989 , p163, 1989). Topoisomerase II enzymes modify the morphological state of DNA by cleaving the double strand of DNA. Many agents that block cleavage / recombination of DNA topoisomerase are known to effectively kill cells when converting these enzymes to complete DNA cleavage enzymes (LF Liu, Adv. In Pharmacol. , 28B, 1994; LK Wang et al., Chem. Res. Toxicol. , 6 , p813, 1993).

Thus mammalian topoisomerase II is an effective pharmacological target for developing chemical cancer therapeutics (AY Chen et al., Annu. Rev. Pharmacol. Toxicol. , 34 , p191, 1994). Clinical agents recognized and in use as topoisomerase II inhibitors include etoposide (VP-16), teniposide (VM-26), mitoxantrone, m-AMSA, adriamycin (doxorubicin), ellipsine and There is daunomycin.

Compared to these topoisomerase II inhibitors, topoisomerase I inhibitors are relatively little known. For example, camptotaxin, known for its anticancer activity, is the most widely studied mammalian topoisomerase 1 inhibitor (RC Gallo et al., J. Natl. Cancer Inst. , 46 , p789, 1971; BC Giovanella et. al., Cancer Res. , 51 , p3052, 1991), the compounds block the cleavage and recombination reactions of the DNA of topoisomerase I, resulting in the accumulation of covalent intermediates, resulting in reversible DNA cleavage of topoisomerase I. State, "cleavable complex" (YH Hsiang et al., J. Biol. Chem. , 260 , p14873, 1985; SE Porter et al., Nucl.Acids Res. , 17 , p8521, 1989; C Jaxel et al., J. Biol. Chem. , 266 , p20418, 1991), causing the cells to die.

SV40 DNA replication system applied in the present invention is an excellent model system for studying the DNA replication mechanism of eukaryotic cells. This is because the SV40 virus uses a host eukaryotic replication system for its own replication, except for the SV40 T-Ag. The SV40 viral gene is a 5.2 kb circular duplex minichromosome with a nucleoprotein structure similar to the intracellular chromatin, and DNA is bidirectionally linked by T-Ag binding to the origin of replication. Replication starts. And elongation of the SV40 DNA chain is caused by host replication proteins, similar to the method that occurs in chromosomal replication forks. Three factors in the SV40 DNA replication system, SV40 T-Ag, Replication proteinA (RPA), and the DNA pol α-primase complex, are essential factors in the initiation process. In the presence of topoisomerase, SV40 T-Ag continuously releases highly overlapping DNA (Borowiec JA, et al.,Cell,60,pp181-184, 1990). The role of these three factors and topoisomerase in DNA synthesis is very important (Wold, M.S. and Kelly, T.J.,Proc. Natl. Acad. Sci. USA.,85, pp 2523-2527, 1988).

During replication, RPA is involved in the unwinding of DNA containing the SV40 origin of replication in the presence of SV40 T-Ag and topoisomerase I. RPA also interacts with the SV40 T-Ag and the DNA kinase alpha-primase complex, which is essential for the initiation of SV40 DNA replication (Murakami Y, Eki T, Hurwitz J., Proc. Natl. Acad. Sci. USA , 89 , pp 952-956, 1992). Topoisomerase I transiently causes single-stranded breaks in DNA and acts as a DNA strand transferase, which plays an important role in transcription and recombination as well as DNA replication. (Wang TA, Li JJ., Curr. Opin. Cell. Biol. , 7, pp414-420, 1995).

On the other hand, Honggyeongcheon, which is called a true stone,Rhodiola sachalinensis A. Bor) Is a Rhdiola plant of Crassulasceae (景天 科). There are three kinds of honggyeongcheon genus plants distributed in Korea.Rhodiola angustaNakai), Branch Stone Flower, Fine Stone Flower (R. ramosaNakai) and stone flowers, rock flowers, true stone flowers (R. roseaL. (R. tachiroei Nakai; R. elongata (Ledeb.) Fisch. Et C. A. Meyer; R. sachalinensis A. Bor). Honggyeongcheon has fleshy rhizome and important medicinal parts are roots and stems, and there are 96 kinds of plants belonging to the cactus flower in the world. There are about 70 species in China. Occupy. Honggyeongcheon has the special adaptability that other plants do not have in low temperature, dryness, oxygen deficiency, strong ultraviolet rays, and day and night temperature difference 2,000 ~ 5,000m above sea level. (中國 本草 圖鑑, 驪 江 出版Co., p314, 1994; 高 庚 式, 金 潤 植 .Derivatives of the National Academy of Medicines, Academic Books, p277, 1977; 鄭 台 鉉.

Hong Kyungcheon is a kind of health medicinal plant recently discovered after ginseng and thorny ginseng, and has the nickname of "highland ginseng" because it can rejuvenate, overcome disease and poison, and have a long life. The main effects and indications are: refreshment, long life, oxygen deficiency, cold, fatigue, radiation of microwaves, increased attention, business efficiency, body decline inhibition, geriatric disease, physical strength, intelligence, business ability improvement, blood pressure normal recovery, memory, various nerves It is known to be effective for hypersensitivity, coronary artery disease, work sickness, diabetes, bleeding, hemorrhage, pneumonia, women's bags, bruises and burns. Clinical clinical studies have shown that chalcedony flowers are not only effective in overcoming oxygen deficiency, cold, fatigue, and microwave radiation, but also enhance attention, slow down the body, and prevent geriatric diseases. It is used as a sedative, antipyretic and astringent in civilian countries, and is widely used as a drink and tea in China (Zang ZH, et al .;Chin. J. Chin. Mater. Med.,14, p687, 1989).

The phytochemical studies of the rhododendron plants have been actively conducted around the world. In 1925, Bridel isolated Salidroside. In 1864, Thieme isolated and rescued saliroside from the rhododendron plants (Bridel CR .;Hebd. Seances Acad. Sci.,183p231, 1926; Thieme H .;Naturwissenschaften,451p360, 1964), and from 1974 to 1879, Zapeschnaya, etc.R. algida)Alginin, Rhodalgin and Rhodalin were isolated from (Zapeschnaya GG. Et al .;Khim. Prir. soedin.,19,p23, 1983), and in 1979, Krasnov, et al.R. algida, R. krylovii)From Gerolin, Gelidolin, Litvinolin, Rhodalgiside, Rhodalgisin, and Zapeschnaya from 1983 to 1985 This same plant (R. rosea)Rhodionin, Rhodiosin, Rhodalidin and Rhodionidin were isolated from. From 1984 to 1985, Kurkin and other plantsR. rosea)Rhodiolgin, Rhodiolin, Rhodiogidin, and Rosiridin were isolated (Kurkin VA. Et al .;Khim. Prir. soedin.,20: p657, 1984).

Active research is in progress in Korea, and in 1996, Yoon Yeo-pyo reported anti-fatigue effect and platelet aggregation effect that significantly increase swimming time after oral administration of methanol extract of Rhodiola sachalinensis to mice for 7 days. Et al., Research on New Drug Development Using Natural Resources from China, Institute of Pharmaceutical Resources, College of Pharmacy, Chungbuk National University, 1996). Kim et al. Reported in 1997 that Rhodiola sachalinensis water extract inhibited the concentration of ethanol in blood by concentration-dependently inhibiting the absorption of ethanol in the gastrointestinal tract of rats treated with oral ethanol. (Kim MH and Park CK . ; Arch. Pharm. Res. , 20, pp432-437, 1997).

In 1998 ryugwangryeol etc., in vitro tests (in vitro) dongsok plant and the organic solvent extract of the antioxidant (R. sachalinensis A. Bor) honggyeongcheon from the oxidative stress induced by the drug in an in vivo test (in vivo) in It has been reported to restore significantly to the liver antioxidant system (Ryu KY. Et al .; Yakhak Hoeji , 42, pp312-318, 1998). Therefore, anti-aging effect and anti-toxic effect among the pharmacological effects of Hong Kyung-cheon are considered to be closely related to their antioxidant activities (Kim, Young-Ho et al .; Antioxidative activity of methanol extract of Rhodiola sachalinensis, Spring Association of Food Science Association), p450, 1999).

However, none of the above-mentioned prior art mentions or describes that red ginseng will be useful as an anticancer agent.

Accordingly, the present inventors have continued to study the honggyeongcheon as a result, it was found that the honggyeongcheon extract and compounds isolated therefrom effectively inhibit the topoisomerase enzyme to complete the present invention.

It is an object of the present invention to provide a composition comprising a herbal extract useful as an anticancer agent and compounds isolated therefrom.

1 is a view showing a manufacturing process for obtaining the honggyeongcheon extract of the present invention,

Figure 2a is _a diagram showing the TLC aspect of the P _a fraction, 2b is a diagram showing the TLC aspect of the P _b fraction, 2c is a diagram showing the TLC aspect of the P _b-1 fraction,

Figure 3a is _a diagram showing the GC spectrum results of the P _a fraction, 3b is _a diagram showing the MS spectral results of the P _a fraction,

Figure 4 is a diagram showing the experimental results of inhibiting the SV 40 DNA replication process of the Hong Kyungcheon extract

Figure 5 is a diagram showing the effect of the extract of honggyeongcheon on the activity of topoisomerase I,

FIG. 6 is a diagram showing the effect of Rhodiola sapa L. extracts on single-strand DNA binding capacity of RPA,

7 is a view showing the results of experiments confirming the effect of inhibiting the SV 40 DNA replication process of commercially available tyrosol.

In order to achieve the above object, the present invention is to provide a soluble extract of Rhodiola sachalinensis non-polar solvent having anticancer activity.

The nonpolar solvent is a solvent containing a nonpolar solvent such as chloroform, ethyl acetate, ether and the like.

In addition, the present invention is a non-polar solvent such as chloroform, ethyl acetate, ether and the like obtained by filtration and decompression concentration after extracting refrigerated refrigeration with a polar solvent such as water, methanol or ethanol or a mixed solvent by cutting the dried Honggyeongcheon Separation was carried out to obtain a soluble extract and residue of a nonpolar solvent, to provide a purified fraction having a R _f value of 0.70 to 0.80 on TLC obtained by performing silica gel column chromatography or TLC on the nonpolar solvent. After the residue is extracted with a polar solvent such as water or methanol, butanol, and then subjected to silica gel column chromatography or TLC, a purified fraction having an R _f value of 0.50 to 0.60 on TLC and a purified form having 0.70 to 0.80 are obtained. It is to provide a purified fraction of.

In addition, the present invention is to provide a pharmaceutical composition for the treatment and prevention of cancer containing the Rhodiola saline extract.

In addition, the present invention is to provide a pharmaceutical composition for the treatment and prevention of cancer containing a non-polar solvent soluble extract or polar solvent soluble extract.

The nonpolar solvent includes a solvent including a nonpolar solvent such as chloroform, ethyl acetate, ether, and the polar solvent includes water and a lower alcohol solvent such as methanol, butanol and the like.

The present invention also provides a pharmaceutical composition for the treatment and prevention of cancer containing the purified fraction.

In addition, the present invention is to provide a pharmaceutical composition for the treatment and prevention of cancer containing a tyrosol separated from the Hong Kyungcheon as an active ingredient.

Such cancers include solid cancers such as lung cancer, breast cancer, ovarian cancer, liver cancer and the like, as well as non-solid cancers such as blood cancer, lymph gland cancer and the like.

For example, the amount of extract of the present invention provides a pharmaceutical composition having a weight ratio of 0.01 to 80%, preferably 1 to 50% of the total composition.

Hereinafter, the present invention will be described in detail.

Referring to the process of obtaining each extract of Honggyeongcheon for treatment of various cancers of the present invention,

First, the crude extract of Honggyeongcheon is characterized in that it is extracted with water or an aqueous alcohol solution.

10 to 15 times the volume / weight of the rhodiola medicinal herb in dry state, lower alcohols such as methanol, ethanol and the like or a mixed solvent thereof, preferably 50 to 80% ethanol solvent, are added at a temperature of 4 to 6 ° C. Cold extraction for 8 hours, or extracted using reflux cooling extraction for 2 to 5 times for 1 to 24 hours, preferably 3 to 5 hours at an extraction temperature of 80 to 100 ℃ or more and filtered the extracted extract and concentrated under reduced pressure It can be prepared through the first step to obtain a crude extract,

The non-polar solvent soluble extract of Rhodiola sachaline is suspended by adding the crude extract to water and then adding a non-polar solvent such as chloroform, ethyl acetate, ether, etc., preferably ethyl acetate acetate, about 2 to 10 times the volume of the suspension, and separating the funnel. Three to ten aliquots were used to obtain extracts soluble in a nonpolar solvent, followed by column chromatography and TLC with a nonpolar solvent soluble extract, whereby the R _f value on the TLC was 0.70 to 0.80 (developing solvent condition, butanol: Fractions with ethanol: water = 5: 2: 1) can be prepared through a second step,

In addition, the polar solvent soluble extract of Honggyeongcheon is added to a lower alcohol solvent such as methanol, butanol, or the like, preferably butanol, by using a separatory funnel by adding about 2 to 10 times the volume of the remaining residue volume through the second step. 10 aliquots may be prepared by a third step of obtaining extracts soluble in a polar solvent,

Subsequently, three steps of the polar solvent soluble extract are performed by column chromatography and TLC, wherein the silica gel column has a chloroform: ethanol 1: 2 to 2: 1 (w / w) ratio, preferably 0.7: 1 to 1.5: 1 ratio. After developing a developing solvent having a column with a fraction, the purified fraction having an R _f value of 0.50 to 0.60 and the fraction having an R _f value of 0.70 to 0.80 were collected on a TLC to obtain a single fraction and dried to obtain a purified fraction of a purified form. It consists of a fourth step.

In another aspect, the present invention is to provide a health supplement for the prevention of various cancers using the Hong Kyungcheon crude extract as an active ingredient.

In addition, the present invention is to provide a health supplement for the prevention of various cancers using the Honggyeongcheon non-polar solvent soluble extract or polar solvent soluble extract as an active ingredient.

The health supplement food may include a health food for health, health drinks, additives and the like.

In order to confirm the prophylactic and therapeutic effects of the extracts against cancer, the extracts of the present invention were subjected to SV 40 DNA replication test, topoisomerase enzyme inhibition test, and single-strand DNA binding test. By showing the effect was found to be effective in suppressing cancer.

On the other hand, the compositions of the present invention can be prepared by extracting and extracting the extracts separated from the above methods, and then lyophilized to prepare a powder extract, and then mixed with the substrate used for the treatment.

As the base material used for oral administration, drinking water such as sterile water or physiological saline was used, and a drinkable alcohol such as shochu was added to the base material and used as an alcoholic aqueous solution.

The extract of the present invention may be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a composition for preventing and treating various cancers. The pharmaceutical composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, It may be formulated in unit dosage forms or in multiple dosage pharmaceutical formulations, such as liquid or parenteral formulations.

The pharmaceutical composition containing the extract of the present invention can be administered parenterally or orally according to the desired method, and the amount of 0.01 to 10g, preferably 1 to 5g per 1kg body weight as an active ingredient per day 1 to several times Can be administered in divided doses. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, and severity of disease.

The extract of the present invention may be added to food or beverage for the purpose of prevention of various cancers. At this time, the amount of the extract in the food or beverage can generally be added at 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight, the health beverage composition is 1 to 30g, preferably based on 100ml Preferably it can be added in 3-10 g of ratio.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of said natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Foods to which the extract of the present invention may be added for development for health foods include various foods, beverages, gums, vitamin complexes, health supplements, and the like.

The cancer treatment composition produced above is preferable because it can be used in all cancer patients.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1. Preparation of Honggyeongcheon extracts (see FIG. 1)

Dried Rhodiola sachalinensis A. Bor purchased from Kyungdong Market medicinal herb and dried 200 g of fine red Rhodiola saline in a beaker, and added 1 liter of ethanol until the sample was immersed. After extraction, the filtrate was filtered and concentrated under reduced pressure on a water bath equipped with a reduced pressure concentrator (R124, Buchi) and dried to obtain crude extract 100g.

In order to remove the fatty precipitate from the crude extract obtained above, the same amount of distilled water was added to the concentrate and filtered again. After concentrating the filtrate under reduced pressure, the solution was repeated five times with 1 L ethyl ether solvent to separate the dissolved ethyl ether layer as a fatty precipitate, and the remaining water layer was added with 1 L chloroform solvent to separate the supernatant. After continuously removing the phosphorus chloroform layer, 1 liter of ethyl acetate solvent was added to the remaining water layer, and the mixture was separated. The ethyl acetate layer as a supernatant was continuously concentrated under reduced pressure to obtain 200 ml of an ethyl acetate soluble extract, which was soluble in an ethyl acetate solvent. One extract of 380 mg was obtained. Thus, 0.38 g of P _a fraction having _a TLC pattern (developing solvent; butanol: ethanol: water = 5: 2: 1) having an R _f value of 0.76 in FIG. 2A was obtained, and the GC / MS spectrum (Auto-spec Ultima, As a result of analyzing the components by Micromass Co), it was confirmed that a large amount of tyrosol having a molecular weight of 138.

GC / MS (EI-MS): 138 (M +), 107, 77 (see FIGS. 3A and 3B)

800 mL of butanol solvent was added to the remaining water layer, and the obtained butanol solvent layer was concentrated under reduced pressure to obtain 20 mL of a butanol solvent soluble extract, which was then subjected to silica gel column chromatography (diameter: 5.2 cm, length: 54 cm, CHCl ₃ : EtOH, 6: 4,4: 6) was added to the flask by 20 ml to obtain 30 fractions. The 5th to 13th eluates were collected and concentrated under reduced pressure to obtain 0.117 _g of a P _b fraction having a TLC pattern with an R _f value of 0.54 as shown in FIG. 2b of 146 mg. The eluate was collected and concentrated under reduced pressure to obtain a concentration of 580 mg. 2.32 g of a P _b-1 fraction having a TLC pattern with an R _f value of 0.73 such as 2c was obtained and used as a sample.

Experimental Example 1. In vitro SV40 DNA replication inhibition test

In order to test the activity of the extracts obtained in Example 1 as an anticancer agent, an in vitro SV40 DNA replication inhibition test described in Kim et al. ( J. Biol. Chem. , 271, pp15124-15129, 1995) was performed. The application was carried out as follows.

40 μl of the reaction mixture contains 40 mM creatine phosphate / di-tris salt (pH 7.7), 1 μg creatine kinase, 7 mM magnesium chloride, 0.5 mM DTT, 4 mM ATP, 200 μM UTP, GTP, and CTP, 100 μM dTTP, dGTP, and dCTP, 20 μM [α- ³² P] dATP (specific activity 20,000 cpm / pmol), 0.8 μg SV40 T-Ag, 0.3 μg SV40 origin of origin (SV40 origin-containing DNA) And an appropriate amount of HeLa cell extract. 24 and 96 ㎍ of P _a , P _b and P _b-1 Hong Kyung _- cheon extracts of Example 1 were added to each reaction mixture to compare the degree of inhibition of SV40 DNA replication.

After the reaction mixture was reacted at 37 ° C. for 90 minutes, the reaction was stopped by 80 μl of a solution consisting of 20 mM EDTA, 1% SDS, and E. coli tRNA (0.5 mg / ml). The replication product in the reaction mixture was separated by 1.2% alkaline agarose gel (40 mM NaOH, 1 mM EDTA) at 2 V / cm for 12-14 hours, dried and exposed to x-ray film for analysis.

As shown in FIG. 4, all of the extracts of Hongkyungcheon inhibited the SV40 DNA replication process. Replication products include RFI and RFII, circular duplex DNA containing at least one single strand break, many topoisomers between them, and slower- Agarose-gel electrophoresis confirms the presence of migrating species. It was confirmed that SV40 DNA replication was further inhibited as each Rhodiola sap extract increased the concentration. P _a was more predominantly inhibited than P _b and P _{b-1 in} Rhodiola sachalinensis extract.

Experimental Example 2. Topoisomerase I Inhibition Experiment

In Experimental Example 1, it was found that the extracts of Honggyeongcheon inhibit DNA replication. Therefore, we investigated the effects of honggyeongcheon extracts on the activity of topoisomerase I in order to determine the possibility of inhibiting the origin-binding protein or other copying proteins required for replication forkification during initiation. For example, the experiment was conducted by applying the topoisomerase inhibition test described in Lin et al. (Lin LF, and Miller KG., Proc. Natc. Acad. Sci. USA , 78 : pp3487-3491, 1981).

The topoisomerase activity was measured to the extent that the superhelical plasmid DNA was relaxed, and the extent to which the activity of this enzyme was inhibited by the Rhodiola sachaline extract was observed. 20 μl of reaction mixture was prepared with 50 mM TrisHCl (pH 7.5), 120 mM potassium chloride, 10 mM magnesium chloride, 0.5 mM DTT, 0.5 mM EDTA, bovine serum albumin (30 μg / ml), pSA DNA ( the 20 ㎍ / ㎖) and topoisomerase as in example 1, the P _a, P _b and P _b-1 honggyeongcheon extract of 100s included. After 30 min reaction at 30 ° C., the reaction was stopped with 5 μl of a 25% (wt / vol) Ficoll 400 (Ficoll 400, Pharmacia) solution with 0.25 mg bromophenol blue per 5% SDS / ml. After the analysis, the results were analyzed by electrophoresis and UV photography. One unit of topoisomerase is the amount of topoisomerase that converts half of the superhelical pSA DNA into a relaxed form under the above reaction conditions.

As shown in FIG. 5, lane 1 represents a superhelical form of DNA, and in lane 2, supercoiled DNA is relaxed by topoisomerase. It shows the conversion to a relaxed form. Honggyeongcheon extract P _a 38 ㎍ addition group is its relaxed (relaxation) This was inhibited more than 90% (lane 3), P _b 146 ㎍ addition group was approximately _{70%, P b-1 145} ㎍ addition group was approximately 60% inhibition (Lanes 5 and 7). These results were consistent with the results of P _a inhibiting the DNA replication process much more than P _b and P _b-1 in the DNA replication experiment of Experimental Example 1. It was confirmed that Rhodiola sachalinensis extract inhibited DNA replication by inhibiting the activity of topoisomerase during DNA replication, thereby inhibiting proliferation in cancer cells.

Experimental Example 3. Single-stranded DNA binding measurement of RPA

In the process of replication, RPA (replication protein A) is involved in the unwinding of DNA containing the SV40 origin in the presence of SV40 T-Ag and topoisomerase I. In addition, RPA interacts with SV40 T-Ag and DNA pol α-primase complexes to produce complexes essential for the initiation of SV40 DNA replication. Therefore, the inhibitory effect of honggyeongcheon extract is likely to occur by interaction with RPA. In this study, based on these findings, Kim et al. (Kim DK, et al . ; J. Biol. Chem. , 271, pp15124-15129, 1995) to investigate the effects of Rhodiola sap extract on the single-strand DNA binding capacity of RPA . The experiment was performed as follows by applying the single-stranded DNA binding measurement test of RPA described in the following.

20 μl of the reaction mixture contains 50 mM Hepes-KOH (pH 7.5), 150 mM sodium chloride, 1 mM magnesium chloride, 0.5 mM DTT, 10% glycerol, 50 fmol 5′- ³² P-labeled oligo (dT) ₅₀ (2200 cpm / fmol), including the appropriate amount of RPA and various amounts of extract for the reaction. The reaction is reacted at 25 ° C. for 15 minutes. The DNA-protein complex was then separated with 5% polyacrylamide in 1 × TBE (89 mM Tris borate, 2 mM EDTA) buffer at 12 V / cm. The gel was dried at 80 ° C. for 1 hour and exposed to X-ray film to analyze the results.

As a result, as shown in Figure 6, it can be seen that as the amount of RPA increases the DNA-protein complex is gradually formed (lane 2, 3). Rhodiola sachaline extract P _a significantly inhibited the single-strand DNA binding of RPA (lanes 4 and 5), followed by high inhibition of P _b (lanes 6 and 7) and the least inhibitory effect of P _b-1 . (Lanes 8 and 9).

By inhibiting the single-stranded DNA binding capacity of RPA participating in the DNA replication process, it was confirmed that it may be effective in preventing proliferation in cancer cells by inhibiting the overall DNA replication initiation and progression.

Experimental Example 4. In vitro SV40 DNA replication inhibition test by tyrosol

According to the GC-MS data of Example 1, it was confirmed that the P _a fraction of the Honggyeongcheon extract contained a large amount of tyrosol. Therefore, the excellent DNA replication inhibition and replication protein activity inhibitory effect of P _a is thought to be caused by tyrosol. Thus, in order to reconfirm the experimental results, it is possible to purchase a commercially available tyrosol (TCI, H0720) on the DNA replication process. In order to measure, it experimented using the experiment method described in Experimental example 1.

In the replication reaction, SV40-derived DNA (pUC-ori +), SV40 T-Ag, HeLa cytoplasmic extract (100 μg), [ ³ H] dTTP and tyrosol were added, and the mixture was incubated at 37 ° C. for 2 hours. Acid-insoluble radioactivity of the reaction product was measured.

As shown in Figure 7, it was confirmed that the DNA replication process was inhibited quantitatively as the concentration of tyrosol was increased. Therefore, the inhibitory effect on DNA replication and replication protein activity by P _a fraction was confirmed to be due to tyrosol.

Extract of the present invention can be administered in the following formulations, the formulation examples below are merely to illustrate the invention, whereby the content of the invention is not limited.

Formulation Example 1 Preparation of Injection

100 mg of extract of Example 1

Sodium Metabisulfite3.0 mg

Methylparaben0.8 mg

Propylparaben0.1 mg

Sterile Distillation Amount for Injection

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

200 mg of extract of Example 1

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

100 mg of extract of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Liquid

1000 mg of extract of Example 1

20 g of sugar

Isomerized sugar 20 g

Lemon flavor

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

1000 mg of extract of Example 1

Vitamin mixture

Vitamin A Acetate70 μg

Vitamin E1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C10 mg

Biotin 10 ㎍

Nicotinic Acid Amide1.7 mg

Folic acid 50 ㎍

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate25.3 mg

Potassium phosphate monobasic 15 mg

Dibasic calcium phosphate55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride24.8 mg

Although the composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method, Granules may be prepared and used to prepare health food compositions according to conventional methods.

Formulation Example 6 Preparation of Healthy Drink

1000 mg of extract of Example 1

Citric Acid1000 mg

Oligosaccharides 100 g

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

The compositions provided by the present invention are expected to inhibit the topoisomerase enzyme and inhibit the cancer, and can be used as a medicine and health supplements composed of natural drugs that are not toxic.

Claims (20)

Soluble extract of Rhodiola sachalinensis non-polar solvent having anticancer activity. The extract of claim 1, wherein the nonpolar solvent is selected from chloroform, ethyl acetate, ether. The dried Honggyeongcheon was chopped and reflux-cooled with a polar solvent such as water, methanol or ethanol, or a mixed solvent thereof. The extract obtained by filtration and concentration under reduced pressure was separated by adding a nonpolar solvent such as chloroform, ethyl acetate, and ether. To obtain a non-polar solvent soluble extract, wherein the non-polar solvent soluble extract is purified by silica gel column chromatography or TLC to obtain a purified fraction having a R _f value of 0.70 to 0.80 on TLC. The dried Honggyeongcheon was chopped and reflux-cooled with a polar solvent such as water, methanol or ethanol, or a mixed solvent thereof. The extract obtained by filtration and concentration under reduced pressure was separated by adding a nonpolar solvent such as chloroform, ethyl acetate, and ether. To obtain a non-polar solvent soluble extract and residue, which was extracted with water or a polar solvent such as methanol and butanol, and then subjected to silica gel column chromatography or TLC to obtain an R _f value of 0.50 to 0.60 on TLC. Refined fractions in a modified form. The dried Honggyeongcheon was chopped and reflux-cooled with a polar solvent such as water, methanol or ethanol, or a mixed solvent thereof. The extract obtained by filtration and concentration under reduced pressure was separated by adding a nonpolar solvent such as chloroform, ethyl acetate, and ether. To obtain a non-polar solvent soluble extract and residue, which was extracted with water or a polar solvent such as methanol and butanol, and then subjected to silica gel column chromatography or TLC to obtain an R _f value of 0.70 to 0.80 on TLC. Refined fractions in a modified form. A pharmaceutical composition for the treatment and prevention of cancer containing the Hong Kyungcheon crude extract. A pharmaceutical composition for the treatment and prevention of cancer containing the Honggyeongcheon non-polar solvent soluble extract or polar solvent soluble extract. 8. The composition of claim 7, wherein the nonpolar solvent comprises a nonpolar solvent such as chloroform, ethyl acetate, ether. 8. The composition of claim 7, wherein the polar solvent comprises water and a lower alcohol solvent such as methanol, butanol and the like. A pharmaceutical composition for the treatment and prevention of cancer containing the purified fraction of any one of claims 3, 4 and 5. A pharmaceutical composition for the treatment and prevention of cancer containing tyrosol isolated from Hong Kyung-cheon as an active ingredient. The composition of claim 6, wherein the cancer is a solid cancer. The composition of claim 12, wherein the solid cancer is lung cancer, breast cancer, ovarian cancer, liver cancer. The composition of claim 6, wherein the cancer is a non-solid cancer. The composition of claim 14 which is hematological cancer, lymph gland cancer. The pharmaceutical composition according to claim 6, wherein the amount of extract has a weight ratio of 0.01 to 80% of the total composition. The pharmaceutical composition according to claim 6, wherein the pharmaceutical form is used in the form of a powder, a granule, a tablet, a capsule, a liquid, or an injection. Health supplement food for the prevention of various cancers using Hong Kyung-cheon crude extract as an active ingredient. Health supplement foods for the prevention of various cancers using a non-polar solvent soluble extract or polar solvent soluble extract as an active ingredient. 20. A dietary supplement according to claim 18 or 19, in the form of a dietary supplement, a beverage or an additive.