KR20050020202A - Method for isolating and purifying Xanthium strumarium L. extract having antimicrobial and antitumor activity, and the use of the extract - Google Patents

Abstract

PURPOSE: A method for isolating and purifying an extract of Xanthium strumarium L., and use thereof are provided, which method stably and effectively isolating the extract of Xanthium strumarium L. having antimicrobial and antitumor activity without loss of its stability and activity. CONSTITUTION: The method for isolating and purifying an extract of Xanthium strumarium L. comprises the steps of: repeatitively extracting the hot water extract of Xanthium strumarium L. using ether under neutral condition; treating the ether neutral extract with anhydrous Na2SO4, and drying and concentrating extract; dissolving the concentrate in methanol, and subjecting the solution to TLC using CH2Cl2 and ethylacetate and silica gel column chromatography, HPLC using methanol and water, and GC/MS, sequentially.

Description

Method for isolating and purifying Xanthium strumarium L. extract having antimicrobial and antitumor activity, and the use of the extract}

The present invention relates to a method for the separation and purification of Korean extracts from the extract of anti-cancer and anti-cancer activity in Korea. More specifically, the present invention provides a stable and efficient novel separation and purification method of the Korean docomari extract with antibacterial and anticancer activity, and relates to the use of the extract as an antibacterial and anticancer agent.

Plants not only provide humans with very useful substances, they also serve as excellent chemical factories, and over 20,000 new and unique bioactive substances derived from them have been identified. These useful substances, which are mostly secondary metabolites, are used in a variety of applications, including medicines, spices, fragrances, pigments, pesticides and high-purity chemicals, accounting for 25% of the global pharmaceutical market, with more than $ 9 billion in sales in the United States alone and 1,600 annually. More than one new plant-derived substance has been found. The material of this study, Xanthium strumarium L. belonging to the family Asteraceae ( Compositae ), has hairs all over, its stem is straight, and its height is about 1.5 m. Flowers bloom from August to September at the tip of stem with yellowish-brown head flowers. The fruit is similar to jujube seed, and has thick hairs in the shape of pericarp. It is an annual herb that grows mainly in fields or roadsides, and is widely distributed and grown in Northeast Asia and Europe including Korea. The study on the larvae is based on the Chinese antler ( Xanthium sibiricum Patr. Ex Widd), which has excellent effects on fever, sweating, pain, pain, breeze, ulceration, skin disease, neuralgia and malignant tumors. It is known that there is a significant inhibitory effect on Staphylococcus aureus as well as Typhoid bacteria and dysentery. Chang-i-leaf has various effects such as bloating, analgesic, antipyretic, insecticide, etc. The components contained in Chang-za are xanthinin, carotenoid, alkaloid, saponine with γ-lactone structure. ), Xanthostrumarin, xanthostrunarin and oleic acid are known. As a result, Docomari with these ingredients has been established as a traditional folk remedy and widely used for the treatment of skin diseases such as eczema, as well as having various pharmacological effects, and thus antimicrobial and anticancer substances are expected. Recently, research on natural physiologically active substances with new functions from herbal medicines, edible plants and various cultivated useful microorganisms (VBNC) has been actively conducted in East and West. Research is active. In particular, components such as ginnalin extracted from maple, asterisic acid extracted from sulphurous lake, beberin extracted from yellowish white or rhubarb, and hesperidin contained in citrus skin It has been reported that it has antibacterial effects, such as herbal medicines and spices having anti-proliferative effect on tooth decay, Streptococcus mutans , anticancer effect of Chopi extract, and anticancer effect of phenolic compounds secreted from turmeric. Among the components of medicinal plants, the antibacterial and anticancer effects of various components such as flavonoids, alkaloids, glycosides, polyphenols, and the like are known. In addition, a number of studies have been conducted on the effects of antibacterial, anticancer and various physiological functions by separating components contained in foods including green tea (You, YS et al. (1993), Kor. J. Appl.Microbiol.Biotechnol . , 21, 187-191 .; Kim, SH et al. (1993), Kor. J. Appl. Microbiol.Biotechnol ., 21, 628-634; Nagabhushan, M. and SV Bhide (1992), J. Am. Coll. Nutr ., 11, 192-198; Dkai, Y. et al. (1993), J. Ferment. Bioeng ., 76, 367-370; Mizuno, M. et al. (1989), Agric. Biol. Chem ., 53 , 959-964; Senji, S., M. et al. (1989), Agric. Biol. Chem ., 53, 2307-2311).

In view of this, the present inventors have a wide range of antimicrobial effects against bacteria and fungi as a component extracted from each part of the dog as a part for the exploration and application of new substances with various physiologically active functions, such as new antibacterial and anticancer effects, from Korean dogs. In addition, the results of the separation and purification of two antimicrobial substances having molecular weights of 386 and 230 have been reported (Kim, HS et al. (1997), Kor. J. Appl. Microbiol. Biotechnol. , 25, 183- 188.7). Subsequent studies have shown that these antimicrobial agents have potent growth inhibitory effects on rectal cancer cell lines. However, antimicrobial effects on various cancer cell lines, antimutagenic effects, and other biologically active functions are expected to be identified. Previous studies have raised problems with the stability of isolated antimicrobials. Therefore, the present inventors have established a new method for the stable and efficient purification of all components present in Korean oxtail, and investigated and confirmed the antimicrobial and physiological effects such as mutagenicity and antimutagenicity, and searched for excellent antibacterial and anticancer substances to provide high added value. The present invention has been completed to develop as a new drug.

Accordingly, an object of the present invention is to provide a stable and efficient method for separating and purifying an active substance having antimicrobial and anticancer activity from Korean dogs.

Another object of the present invention is to provide a use as an antimicrobial and anticancer agent of the Korean extract of Docomari.

The object of the present invention was achieved by extracting bioactive substances in neutral, acidic and basic conditions using ether and ethyl acetate in hot water extracts of Korean oxtail, and examining the antimicrobial and anticancer activity of each extract.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of invention is demonstrated concretely.

The present invention is composed of the separation and purification step of the biologically active substance in the Korean dog, and the antimicrobial and anticancer effect of the biologically active substance obtained from the step.

In the present invention, the separation and purification method of the biologically active substance derived from Xanthium strumarium L. of Korea is fractionally extracted with ether under neutral conditions using ether and neutral anhydrous Na ₂ SO ₄ The mixture was dried, concentrated and dissolved in methanol, and then subjected to TLC analysis and silica gel column chromatography using CH ₂ Cl ₂ and ethyl acetate (mixed in a ratio of 9: 1) as a developing solvent, followed by elution. HPLC was performed using methanol and water (mixed in a ratio of 6: 4) as a solvent, and then characterized by analysis through GC / MS.

Separation and purification method of the biologically active substance derived from the Korean oxtail ( Xanthium strumarium L.) of the present invention is characterized in that it further comprises the step of fractional extraction of the hydrothermal extract of the Korean oxtail under acidic conditions.

The present invention is characterized in that the separated purification method of the biologically active substance derived from the Korean Doco-mari ( Xanthium strumarium L.) further comprises the step of fractional extraction of the hydrothermal extract of the Korean Doco-marigold under basic conditions.

The present invention is characterized in that the separation and purification method of the biologically active substance derived from the Korean Dokomari ( Xanthium strumarium L.) further comprises the step of fractional extraction of ethyl acetate from the hot water extract of the Korean Dokomari under neutral conditions.

The antimicrobial effect of the extract of Korean Docomari in the present invention is Mycobacterium phlei KCTC 1932, Staphylococcus aureus KCTC 1927, Serratia marcescens KCCM 11809 ^T , Streptococcus equii KCCM 40306,, Streptococcus zooepidermicus KCTC 3318, Bacillus megaterium KCCM 11492, Bacillus subtilis PCI 219, Bacillus subtilis ( Bacillus thermoglucosis ), 9 Gram-positive bacteria of Bacillus licheniformis IFO 12197, Escherichia coli K-12, Escherichia coli KCTC 1923, Pseudomonas ae Logis labor (Pseudomonas aeroginosa) KCTC 1930, Pseudomonas fluorescein sense (Pseudomonas fluorescens) KCTC 1645, Enterobacter Oh Eroge Ness (Enterobacter aerogenes) KCTC 2190, Salmonella typhimurium (Salmonella typhi) KCTC 2425, Proteus vulgaris (Proteus vulgaris) KCTC 2512 Gram-negative bacteria and Cryptococcus neoformans KCCM 50564, Hansenul Three fungi, Hansenulla anomala B-7 and Aspergillus fumigatus IFO 5840, were selected and investigated.

Growth medium of the test strain to investigate the antimicrobial effect, LB agar (tryptone 10 g, NaCl 10 g, yeast extract 5 g / L) for bacteria, yeasts Y agar (10 g yeast extract, peptone 20 g, glucose 20 g / L) and fungi are characterized by the use of YM agar (3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g / L glucose, pH 5.5).

The anticancer effect of physiologically active substances derived from Korean docomari in the present invention is gastric cancer cell line (SNU 668), lung squamous cell carcinoma cell line (NCI H1703), lung adenocarcinoma cell line (NCI H 522), prostate cancer cell line (LN CAP clone), melanoma Cell line (Clone M3), colon cancer cell line (HT29), uterine cancer cell line HeLa cell, hepatic cancer cell line HepG2 cell, osteosarcoma cell line Saos2 cell and human normal skin fibroblast (Human Normal Skin Fibroblast) is characterized in that the irradiation.

As a growth medium of the test cell line to investigate the anticancer effect, gastric cancer cell line (SNU 668), lung squamous cell cancer cell line (NCI H1703), lung adenocarcinoma cell line (NCI H 522), prostate cancer cell line (LN CAP clone), black The species cell line (Clone M3) and colorectal cancer cell line (HT29) used RPMI-1640 medium containing 10% fat calf serum, 2 mM glutamine, HeLa cell, a uterine cancer cell line, and HepG2 cell, a liver cancer cell line. Silver 10% Fatal Calf Serum, F10 medium containing 2 mM Glutamine was used, and osteosarcoma cell lines Saos2 cells and Human Normal Skin Fibroblast were DMEM containing 10% Fatal Calf Serum, 2 mM Glutamine. It is characterized by culturing in 36 ℃, 5% CO ₂ incubator using high glucose media.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1: Preparation of Korean Dokomari Extract

In order to isolate and purify the physiologically active substance derived from the Korean Docomari, the Xanthium strumarium L. (Korean name: Docomari ), which is native to Sanya in Gyeongnam and Gyeongbuk, was collected in October 2000, and then dried and dried . Used. Figure 1 shows the process of separating and purifying the bioactive material from the leaves and berries of the dog.

First, 300 liters of the leaves and berries of the lizard were extracted with hot water three to five times with 3-5 L of water for about 5 hours, and then 1 L of the extract was concentrated using an evaporator (EYELA Co. Japan) using 0.1 N HCl and 0.1 N NaOH. 6 fractions were obtained by fractional extraction using ether and ethyl acetate as organic solvents under neutral, acidic and basic conditions.

Example 2: Identification of the Antimicrobial Activity of the Organic Solvent Extract of Korea

Antimicrobial activity of the six fractions of the Korean extract of the present invention obtained in Example 1 was investigated using the Agar diffusion method.

In order to investigate the antimicrobial activity according to the Agar diffusion method, each test bacterium that was incubated for 18 to 24 hours ( A. fumigatus was used for 7 days in a spore solution obtained after incubation in a slope medium) was included. A dry paper disk (φ: 6 mm, Whatman Co.) to which 20 µl of the various samples obtained in Example 1 was added to the solid medium was prepared, and at 24 ° C (yeast, mold) and 37 ° C (bacteria) for 24 hours. Incubated. The antimicrobial test was confirmed by the formation of clear zones and the antimicrobial effects of 50, 100, 150 and 200 ㎍ were examined. Identification of the antimicrobial substance was carried out on the agar plate containing the test bacteria, left for 30 minutes, the TLC plate was removed, and when the agar plate was incubated for 24 hours at 37 ° C. ) It was confirmed by the presence or absence of formation.

The antimicrobial activity of E. coli and B. subtilis was examined in the neutral and acidic extracts of ether and ethyl acetate (not shown). Therefore, the antimicrobial activity was examined by diluting these extracts to their respective concentrations.

              Test strain sample (㎍) E. coli K-12 B. subtilis PCI 219 Inhibitory zone (φ, mm) XE-N 50100150200 15161717 13172123 XE-A 50100150200 8101111.5 9111315 XEA-N 50100150200 -78.510.5 －-1011 XEA-A 50100150200 －－－－ -689

 -: Do not suppress

As shown in Table 1, the antibacterial effect of the neutral extract of ether was better than the neutral extract of ethyl acetate at the concentration of 50 g.

From the above results, the antimicrobial spectra of 9 Gram-positive bacteria, 7 Gram-negative bacteria, and 3 fungi were examined for 10 g of the ether neutral extract (XE-N) having the highest antibacterial effect among the extracts. The growth medium of the test strain was LB agar (tryptone 10 g, NaCl 10 g, yeast extract 5 g / L) for bacteria, and yeasts were Y agar (10 g yeast extract, 20 g peptone, 20 g / glucose). L) and fungi were YM agar (3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g / L glucose, pH 5.5).

Sample strain XE-N (10 μg) Gram-positive bacteria Inhibitory zone (φ, mm) Mycobacterium phlei KCTC 1932 Staphylococcus aureus KCTC 1927 Serratia marcescens KCCM 11809 T Streptococcus equii KCCM 40306 Streptococcus zooepidermicus KCTC 3318 Bacillus megaterium KCCM 11492 Bacillus subtilis PCI 219 Bacillus thermoglucosis Bacillus licheniformis IFO 12197 12.510.57.5137.587.5-7.5 Gram-negative bacteria Escherichia coli K-12 Escherichia coli KCTC 1923 Pseudomonas aeroginosa KCTC 1930 Pseudomonas fluorescens KCTC 1645 Enterobacter aerogenes KCTC 2190 Salmonella typhi KCTC 2425 Proteus vulgaris KCTC 2512 10.5-91310.57.59 leaven Cryptococcus neoformans KCCM 50564 Hansenulla anomala B-7 7.5  mold Aspergillus fumigatus IFO 5840 －

As shown in Table 2, as a result of investigating the antimicrobial spectrum of the ether neutral extract (XE-N) extracted from the Korean Dokomari in the present invention, Cryptococcus neopoman is a pathogenic yeast, including 8 Gram-positive bacteria, 6 Gram-negative bacteria Cryptococcus neoformans also showed antimicrobial effect.

Comparative Example 1: Investigation of the antimicrobial effect of the Korean extract of the present invention Dokmari Maritime according to FDA (fluorescein diacetate) method

The Agar diffusion method may be somewhat inaccurate depending on the sample due to the diffusion of the sample, the components in the medium, and the lighting required for a long time. Therefore, the fluorescein diacetate (FDA) method was used to quantitatively analyze the antimicrobial effect of the liquid culture.

First, the antimicrobial effect of XE-N (neutral extract of ether) and XEA-N (neutral acetate of ethyl acetate) was analyzed to establish the FDA analytical conditions on the extracts with proven antimicrobial effect. FDA measurements used a modified method of Chand et al. (Chand, S. et al., (1994), J. Antibiotics , 47, 1295-1304). That is, 175 μl of test log of the logarithmic sampler and 20 μl of sample (3 points for each sample) were put in a 96-well microplate, incubated at 30 ° C. for 40 minutes, and 5 μl of 0.2% FDA (dissolved in acetone) was added. Incubation was continued for 90 minutes. The antimicrobial effect was determined by using a microplate reader (Bio-rad Co. Model 550) to increase the production of fluorescein, an FDA degradation product of the esterase produced by the test strain, at absorbance of 490 nm and the growth of bacteria. Figure was measured at 655 nm, the antimicrobial effect was confirmed by the difference of the addition of the sample.

Wavelength 490 nm (FDA) 655 nm E P S C E P S C Control 1.101 1.485 0.715 0.539 0.171 0.171 0.294 0.371 XE-N   30 ng 0.943 1.634 0.666 0.507 0.201 0.372 0.291 0.339   50 ng 1.017 1.624 0.667 0.514 0.206 0.352 0.269 0.349  100 ng 0.964 1.622 0.700 0.479 0.207 0.348 0.286 0.317 XEA-N   30 ng 0.907 1.474 0.608 0.477 0.190 0.398 0.281 0.325   50 ng 0.917 1.568 0.667 0.536 0.183 0.366 0.302 0.384  100 ng 0.910 1.525 0.683 0.526 0.192 0.350 0.289 0.366 Control 1.391 0.543 0.578 0.559 0.175 0.180 0.245 0.371 XE-N   1 μg 1.237 0.641 0.653 0.494 0.197 0.195 0.258 0.319   5 μg 0.935 0.693 0.550 0.482 0.221 0.282 0.215 0.292  10 μg 0.816 0.738 0.556 0.516 0.228 0.362 0.233 0.321 XEA-N   1 μg 1.110 0.730 0.501 0.475 0.209 0.246 0.238 0.310   5 μg 0.970 0.616 0.494 0.497 0.234 0.272 0.213 0.323  10 μg 0.873 0.718 0.506 0.501 0.283 0.334 0.187 0.310

Note: E , Escherichia coli ; P , Pseudomonas aeruginosa ; S , Staphylococcus aureus ;

C , Candida albicans

As shown in Table 3, bacterium E. coli , P. aeroginosa , Staphylococcus aureus , and yeast Candida albicans ( B. C. albicans ) showed a significant inhibitory effect at 30 ng / mL against all three strains except P. aeroginosa .

Example 3: Purification of ether neutral extract with excellent antimicrobial activity

From the results of Examples 1 and 2 it was purified the ether neutral extracts of Korean dogs were found to have excellent antibacterial activity.

First, the ether neutral extract fraction (XE-N) extracted from Korean docomari was concentrated and the concentrate was dissolved in a small amount of methanol and analyzed by TLC (Silica gel 60 F254). CH ₂ Cl ₂ and ethyl acetate were mixed and used as a developing solvent in a ratio of 9: 1, respectively. The short wavelength UV lamp (254 nm) confirmed that the Rf values were 0.71 (S1), 0.59 (S2) and 0.39 (S3). Three signals were detected as). Signals S1, S2, and S3 having excellent antimicrobial activity were separated using a silica gel column through TLC plate analysis. The separated S1 was analyzed by HPLC (Shimadzu LC-10AD, Shimpak column C ₁₈ , MeOH: H ₂ O = 1: 1), and a peak appeared at retention time 20.664 min but a small amount of mixture coexisted. Second purification was performed by HPLC. A preparative Metachem nucleosil (C ₁₈ ) column was used to fractionate the detected peak at absorbance 254 nm as solvent of MeOH: H ₂ O = 6: 4.

As shown in Figure 2, it was detected as a single peak at 22.477 minutes, and analyzed by chromatography pack (chromatopak, Shimadzu Co.), it can be seen that the purity was purified to 100%.

Scanning purified XE-N-S1 at 200-900 nm using UV / Visible spectrophotometer (Parmacia Co. Biochrom 4060) showed maximum absorbance at 215 nm, as shown in Figure 3, GC / MS As a result of the analysis, as shown in Figure 4, it was confirmed as a single substance having a molecular weight of 248.

Experimental Example 1 Purification of S2 and S3 in Ether Neutral Extract

Among the ether neutral extracts, to separate the mixed S2 and S3 other than the above-mentioned S1, XE-N-S1 was first separated, and then, a small amount of MeOH was added to a concentrate containing Rf values of 0.59 (S2) and 0.39 (S3). Was dissolved and then analyzed using TLC. As a developing solvent, CH ₂ Cl ₂ and ethyl acetate were mixed at a ratio of 8.5: 1.5, respectively, and separated using a silica gel column under the same solvent conditions with reference to the TLC analysis result. The isolated XE-N-S3 was analyzed by HPLC (Shimadzu LC-10AD, Metachem nucleosil C ₁₈ , MeOH: H ₂ O = 6: 4), and the main peak appeared at retention time 10.735 minutes. 91%, with a peak of 8.979 minutes mixed (not shown). In order to isolate these compounds, Second-HPLC (Shimadzu LC-10AD, Methachem nucleosil C ₁₈ , MeOH: H ₂ O = 1: 1) was purified second, and confirmed that the retention time was a single peak of 20.275 minutes. As a result of measuring by GC / MS to investigate the molecular weight of the isolated compound, it was identified as a single substance having a molecular weight of 262, but was determined to be considerably unstable because it was decomposed within a few days after purification.

Experimental Example 2 Purification of Ether Acid Extract with Excellent Antimicrobial Activity

Separation and purification of physiologically active substances from the ether acid extract (XE-A) proved antibacterial effect from Example 2 was used the same method as the purification process of ether neutral extract (XE-N). The extract concentrate was first dissolved in a small amount of MeOH and then analyzed by TLC. CH ₂ Cl ₂ and ethyl acetate were mixed and used as a developing solvent in a ratio of 9: 1, respectively. The short wavelength UV lamp (254 nm) confirmed that the Rf values were 0.74 (S1), 0.63 (S2), and 0.55 (S3). ) And 0.38 (S4), four types of signals were detected. Antimicrobial activity for these signals was confirmed by TLC plate analysis, and showed three antimicrobial activities, respectively, on three signals, S1, S2, and S3. Therefore, to separate them, based on the TLC analysis, the concentrate was filled in a silica gel 60 column, and then eluted using the same solvent as above. As a result, XE-A-S3, which is easily separated among four signals, was isolated and analyzed using HPLC (Shimadzu LC-10AD, Metachem nucleosil C ₁₈ , MeOH: H ₂ O = 6: 4). The main peak appeared at 11.222 minutes, the purity was 92%, and the peak at 8.930 minutes was mixed slightly. To isolate these materials, the solvents were purified by HPLC (Shimadzu LC-10AD, Methachem nucleosil C ₁₈ , MeOH: H ₂ O = 1: 1) with different solvent conditions (not shown), but similar to XE-N-S3 Decomposition within the material suggests that this material is also quite unstable.

Experimental Example 3: Purification of Ethyl Acetate Neutral Extract with Excellent Antimicrobial Activity

From the results of Examples 1 and 2 were purified ethyl acetate neutral extract with excellent antimicrobial activity in the Korean Dokomari, was carried out in the same manner as the purification process of ether neutral extract.

First, the ethyl acetate extract was dissolved in a small amount of MeOH, and analyzed by TLC (Silica gel 60 F254). CH ₂ Cl ₂ and ethyl acetate were mixed and used as a developing solvent in a ratio of 9: 1, respectively. The short wavelength UV lamp (254 nm) confirmed that the Rf values were 0.73 (S1), 0.61 (S2) and 0.46 (S3). ), Three kinds of substances were detected. Based on the TLC analysis, the concentrate was filled in a silica gel 60 column, and then eluted using the same solvent as above. As a result, XEA-N-S2 was isolated and subjected to second purification using HPLC (Shimadzu LC-10AD, Metachem nucleosil C ₁₈ , MeOH: H ₂ O = 6: 4), resulting in a single peak of 14.691 minutes retention time. Although it was confirmed that this material also decomposed quickly.

Example 4: Investigation of the anticancer effect of the biologically active substance isolated and purified from Korean extract

The anticancer effect of the physiologically active substance separated and purified from the present invention Korean docomari extract was identified according to Examples 1 to 3 above.

To this end, the bioactive material separated and purified in the above example was used by dissolving in DMSO and EtOH, and XE-A and XE-B and XEA-A and XEA-B were not well dissolved.

In addition, gastric cancer cell line (SNU 668), lung squamous cell carcinoma cell line (NCI H1703), lung adenocarcinoma cell line (NCI H 522), prostate cancer cell line (LN CAP clone), melanoma cell line among the cell lines used to investigate anticancer effects (Clone M3) and colorectal cancer cell line (HT29) used RPMI-1640 medium containing 10% fatal calf serum and 2 mM glutamine, while HeLa cell, uterine cancer cell line, and HepG2 cell, liver cancer cell line, 10% fatal calf serum. F10 medium containing 2 mM glutamine was used, and osteosarcoma cell lines Saos2 cells and human Normal Skin Fibroblasts were treated with DMEM high glucose media containing 10% fat cap serum and 2 mM glutamine. C was incubated in a 5% CO ₂ incubator.

When the gastric cancer cell line, lung cancer cell line, uterine cancer cell line, liver cancer cell line, colon cancer cell line, osteosarcoma cell line, prostate cancer cell line, melanoma cell line and human normal skin fibroblast cell line were reached, and then reached the logarithmic growth stage, the extracts were diluted in stages. Samples were inoculated into each cultured cell and cultured for a predetermined time, and then examined for antitumor effects. Anti-tumor effects were analyzed by 3- (4,5-dimethyl-2-tetrazolyl) -2 and 5-diphenyl-2H tetrazolium bromide (MTT) assay. That is, 1 × 10 ⁴ cells in the logarithmic phase were suspended in 180 μl of culture solution and then dispensed into 96-well plates, incubated in 37 ° C., 5% CO ₂ incubator for one day, and then 10 μl of the diluted extracts were added. Time incubation. After incubation, 10 μl of MTT at a concentration of 5 mg / mL was added and incubated for 4 hours, 180 μl of the supernatant was removed, and 150 μl of DMSO was added and shaken in a rocker for 20 minutes. The anticancer effect was determined by measuring OD at 570 nm wavelength using an ELISA reader (Dynatech Co.), and a solvent in which the anticancer agent was dissolved was used as a control. In addition, etoposide, cisplatin, 5-fluorouracil (5-FU), and the like were used to compare and compare anticancer effects with commercially available anticancer agents.

In addition, the number of viable cells was mixed with 50 μl of trypsin-treated cells in 50 μl of 0.4% trypan blue, mixed in a hemocytometer, and counted after 1 minute.

sample MICs of human-derived cancer cell line ^a (µg / mL) SUN668 HeLa HepG2 HT29 Saos2 XE-N 228.8 110.9 110.9 55.4 13.8 XE-A > 1625.0 > 1625.0 > 1625.0 > 1625.0 NT XE-B 1250.0 625.0 625.0 312.5 NT XE-N-S1 109.3 27.3 27.3 54.7 <13.6 XE-N-S3 365.0 46.8 46.8 93.7 NT XE-A-S3 650.0 162.5 81.2 325.0 NT XEA-N 425.0 425.0 425.0 106.2 106.2 XEA-A > 1800.0 > 1800.0 > 1800.0 > 1800.0 NT XEA-B 1700.0 1700.0 1700.0 1700.0 1700.0 Etoposide 125.0 250.0 125.0 125.0 250.0 Cisplatin > 75.0 50.0 > 75.0 > 75.0 75.0 5-FU 7500.0 5000.0 2500.0 > 7500.0 5000.0

a: SNU668, gastric cancer cell line; HeLa, cervical cancer cell line; HepG2, liver cancer cell line;

   HT29, colorectal cancer cell line; Saos2, osteosarcoma cell line; NT, no test

sample MICs of human cell line ^a (μg / mL) NCI H522 NCI H1703 LN CAP Clone M3 HSF XE-N NT NT NT NT NT XE-A NT > 1625.0 203.1 NT > 1625.0 XE-B NT 625.0 78.1 NT 625.0 XE-N-S1 <6.8 <6.8 27.3 13.6 54.7 XE-N-S3 NT 46.8 <5.8 NT 46.8 XE-A-S3 NT 81.2 20.3 NT 162.5 XEA-N NT NT NT NT NT XEA-A NT > 1800.0 > 900.0 NT > 1800.0 XEA-B NT 1700.0 850.0 NT > 1700.0 Etoposide 250.0 125.0 125.0 125.0 250.0 Cisplatin 50.0 50.0 50.0 > 75.0 75.0 5-FU 5000.0 5000.0 5000.0 2500.0 5000.0

a: H522, lung adenocarcinoma cell line; NCI H1703, lung squamous cell carcinoma cell line;

   LN CAP, prostate cancer cell line; Clone M3, melanoma cell line;

   HSF, human normal skin fibroblasts; NT, not tested.

As shown in Tables 4 and 5, the cancer cell suppression concentration (MIC) for the various samples, the anticancer effect on HeLa cervical cancer cells was the best XE-N-S1, XE-N-S3 also showed an excellent effect. Two types of anti-cancer effects on HepG2 liver cancer cells, XE-N-S1 and XE-N-S3, and anti-cancer effects on HT29 colon cancer cells were superior to XE-N extract and XE-N-S1. XE-N-S1 was the best anticancer effect on osteosarcoma cancer cells, but XE-N extract was also excellent. In addition, XE-N-S1 had the best anticancer effect against NCI H522 lung adenocarcinoma cells, and XE-N-S1 had the best anticancer effect against NCI H1703 lung squamous cell carcinoma cells. It showed an excellent effect. XE-N-S3 had the best anticancer effect against LN CAP prostate cancer cells, and XE-A-S3 and XE-N-S1 also showed excellent effects. The anticancer effect on Clone M3 melanoma cancer cells was XE-N-S1. Since the extracts used in the experiments and the XE-A, XE-B, XEA-A and XEA-B extracts in the tablets were not well dissolved in the solvent, the anticancer effects on them were higher than those shown in Tables 4 and 5. It is believed to have. In addition, in order to investigate the cytotoxicity of various extracts and tablets on HSF human normal dermal fibroblasts, the commercially available anticancer drugs etoposide and cisplatin were purchased and compared, and as a result, XE-A, XEA-A and XEA-B extracts were compared. The lowest toxicity, XE-B extract was also relatively low compared to etoposide. Meanwhile, XE-N-S1 and XE-N-S3 were more toxic than etoposide, an anticancer drug. XE-A-S3 was more toxic than etoposide, but lower than cisplatin.

As described through the above Examples and Experimental Examples, the present invention relates to a method for the separation and purification of Korean extracts with antimicrobial and anti-cancer activity and its use, the novel stable and effective separation and purification of biologically active substances in Korean extracts There is an outstanding effect of providing one method. In addition, the present invention has an excellent effect of providing a biologically active substance having antimicrobial and anticancer activity purified from Korean extracts. Therefore, the present invention is a very useful invention in the material separation industry because it provides a novel method for separating and purifying Korean extracts.

Figure 1 illustrates the separation and purification of bioactive substances having antimicrobial and anticancer effects in the Korean dog.

Figure 2 shows the secondary HPLC results of XE-N-S1 in the ether neutral extract of the present invention Korean Docomari.

Figure 3 shows the results of observing the XE-N-S1 in the ether neutral extract of the present invention Korean Dogweed Mari under UV / Vis spectrophotometer.

Figure 4 shows the GC / MS results of XE-N-S1 in the ether neutral extract of the Korean Dokomari of the present invention.

Claims (7)

In the method of extracting the bioactive substance from the The hydrothermal extracts of Korean dokomari samples were fractionally extracted using ether under neutral conditions, Treated with ether neutral extract obtained from anhydrous Na ₂ SO ₄ and dried and concentrated, The concentrated solution was dissolved in methanol, subjected to TLC analysis and silica gel column chromatography using CH ₂ Cl ₂ and ethyl acetate (mixed in a ratio of 9: 1) as a developing solvent, and then methanol and water (6: 4) a method of separating and purifying a bioactive substance derived from Korean docomari ( Xanthium strumarium L.), which is obtained by analyzing by GC / MS after HPLC. The method of claim 1, Korean Xanthium strumarium, characterized in that the Korean Xanthium strumarium (Xanthium strumarium L.) separation and purification method of the physiologically active substance derived from further comprises a step of extracting sensible ether to the hot-water extract of Korean Xanthium strumarium under acidic conditions (Xanthium strumarium L.) Separation and purification method of derived biologically active substance. The method of claim 1, The Korean Xanthium strumarium (Xanthium strumarium L.) separation and purification method of the physiologically active substance derived from the Korean Xanthium strumarium further comprising the step of extracting sensible ether to the hot-water extract of Korean Xanthium strumarium under basic conditions (Xanthium strumarium L.) derived from Separation and purification of physiologically active substances. The method of claim 1, Separation and purification method of the biologically active substance derived from the Korean Dokomari ( Xanthium strumarium L.) further comprises the step of fractional extraction of ethyl acetate under neutral conditions of the hot water extract of the Korean Docomari ( Xanthium strumarium L.) Separation and purification method of derived biologically active substance. Extract of Xanthium strumarium L. from Korea with molecular weight 284 and antibacterial and anticancer activity. Antimicrobial composition, characterized in that containing the Korean extract of Docomari as an active ingredient. Anticancer composition, characterized in that containing the Korean extract of Dokomari as an active ingredient.