KR20140108796A - Drug composition containing extract of Juncus Effusus L. for anti-oxidative and anti-tumor activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a Juncus Effusus extract as an active ingredient for anti-oxidation and preventing or treating cancer, wherein the Juncus effuses extract contains dehydroeffusol. The pharmaceutical composition according to the present invention has excellent anti-oxidative and anti-cancer effects.

Description

[0001] The present invention relates to a pharmaceutical composition having an antioxidant and anticancer effect,

The present invention relates to a pharmaceutical composition having an antioxidant and anticancer effect using an extract of Codonopsis lanceolata as an active ingredient.

Reactive oxygen species (ROS) generated by the promotion of oxidation in the body due to westernized diet, environmental pollution and excessive stress can be classified into potential causes such as cell membrane destruction, enzyme inactivation, lipid oxidation, DNA denaturation and cell senescence Causing cell damage and leading to serious pathological disorders such as atherosclerosis including cancer, autoimmune disease, and the like.

There are antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase, which protects cells and tissues from damage in the body. Enzymes gradually decrease at the time of adult, and it is necessary to consume in vitro. In addition, as the theory that aging and diseases of adult diseases are caused by active oxygen has been recognized, researches on the development of antioxidants, which are known as substances capable of controlling them, are being actively carried out.

Recently, in the field of food industry, there has been a growing interest in eating antioxidants that delay aging or effectively inhibit the production of active oxygen in the body, and side effects such as synthetic antioxidants and anticancer drugs are serious, Research on natural products having physiological activity is actively underway.

The present invention is a technique related to a pharmaceutical composition with a rush, rush (Juncus Effusus L.) has been reported to have pharmacological effects such as anti-inflammation, fever, diuretic, and hemostasis in the oyster area of the raspberry (soft rind removed from the hard shell of the raspberry). Rhesus has long been known as a medicinal herb used in the civilian roots and grapefruit herbs as herbal medicine, herbal medicine registered in Korean and Chinese herbal medicine catalogs, and having anti-inflammatory, antipyretic, diuretic and hemostatic effects. Also, the ground part of the rush is used to make mats, cushions and tatami mats. In the 1970s, domestic ryegrass was cultivated for export purposes. However, due to the labor cost problem, cultivated farmers have disappeared and grow wild in national rivers, wetlands and paddy fields. The present invention And the physiological activities of the compounds were investigated and further their value as a new natural material.

On the other hand, referring to related patents, U.S. Patent Application Publication No. 2010-0104518 discloses a technique relating to chewing gum including rosemary extract, and US Patent Application Publication No. 2008-0138491 proposes a technique relating to juice powder containing rosemary extract have.

U.S. Patent Application Publication No. 2010-0104518 United States Patent Application Publication No. 2008-0138491

It is an object of the present invention to provide a pharmaceutical composition having an antioxidant effect and an anticancer effect by using an extract of Codonopsis lanceolata.

The present invention provides a pharmaceutical composition for antioxidant and anticancer therapy or prevention comprising an extract of Juncus Effusus L. as an active ingredient.

Particularly, it is preferable that the rumen extract comprises dehydroeffusol of the following structural formula (1).

[Structural formula 1]

Particularly, it is preferable that the rumen extract comprises effusol of the following structural formula (2).

[Structural formula 2]

Particularly, it is preferable that the above-mentioned Seaweed extract is obtained by fractionating an extract obtained by extracting with methanol or ethanol again with ethyl acetate.

In particular, the anti-cancer effect is preferably an anti-cancer effect against lung cancer.

In particular, the pharmaceutical composition may be any one selected from tablets, hard or soft capsules, granules, solutions, suspensions, injectable solutions or suspensions.

In particular, the pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier.

In the experiment of the present invention, the ethanol extract of Jucus effusus was fractionated by solvent (Water, EtOAc, n-BuOH), and the compound 1 (dehydroeffusol) and 2 (effusol) were measured for antioxidant activity and anticancer effect. Compounds 1 and 2 exhibited more than 90% DPPH free radical and ABTS free radical scavenging activity at 500 μM and showed antioxidative activities similar to those of the control group (AsA; ascorbic acid, vitamin C). As a result of examining the anticancer effect by measuring growth inhibitory effect on human gastric cancer cell line (AGS) and lung cancer cell line (A549), both of compounds 1 and 2 showed similar or higher inhibitory effects on cancer cell growth than those of anti-cancer drug (CPA, cyclophosphamide) . In particular, Compound 1 was found to have an excellent effect on inhibition of A549 growth, and thus it was confirmed that Compound 1 was useful as a natural anticancer drug targeting lung cancer cells.

FIGS. 1A and 1B show the results of experiments on the anticancer effect on gastric cancer cells and liver cancer cells according to concentrations of compound 1 (dehydroeffusol) and compound 2 (effusol) and CPA (cyclophosphamide) as a control group, respectively.
FIG. 2 is a comparison chart showing the anticancer effect of gastric cancer cells and liver cancer cells measured at 40 μM concentrations of Compound 1 and Compound 2, respectively.

Hereinafter, the present invention will be described through experiments.

Example  : rush  Preparation of extract

It is preferable that the extract is firstly extracted with an alcohol solvent such as methanol or ethanol, and then the extract is suspended in water and extracted with ethyl acetate or n-butanol. In the following experiments, ethanol was used for extraction.

The jujube ( Juncus Effusus L. ) used in the experiment of the present invention was purchased and used in Kyungdong market. In order to facilitate the extraction, 70% ethanol (EtOH) was added to 10 kg of dried and ground sirloin and extracted at room temperature for 24 hours. The extract was filtered, and the residue was extracted twice with the extraction solvent (EtOH) by the same method. The obtained filtrate was combined and concentrated under reduced pressure to obtain an ethanol extract. The ethanol extract was suspended in water, partitioned with ethyl acetate (EtOAc), and the H ₂ O layer was partitioned and extracted with n-butyl alcohol (n-BuOH). Each layer was concentrated under reduced pressure to obtain an ethyl acetate fraction (26.05 g), an n-butyl alcohol fraction (10.18 g) and an H ₂ O fraction.

Silica gel column chromatography fraction from ethyl acetate (26.05g) Photography (cc) (φ 5 × 15cm , CHCl 3 - MeOH = 10: 1 → 7: 1 → 5: 1, CHCl 3 - MeOH-H 2 O = 65: 35 : 10 → 6: 4: 1) to obtain 16 fractions (JEE1 to JEE16). Among these, the fraction of JEE-8 (2.2 g) was fractionated by silica gel chromatography (φ5 × 14 cm, CHCl ₃ -MeOH = 45: 1 → 40: 1 → 30: 1 → 20: 1 → 5: 1 → 2: And 12 fractions were obtained. JEE8-4 (668.9 mg) was further purified by silica gel column chromatography (5 x 12 cm, n-hexane-EtOAc = 6: 1 -> 4: 1 -> 2: 1) Eight fractions were obtained. The JEE8-4-2 (227.8mg) through a sephadex LH-20 ^® compound 1 (20mg, SiO2 TLC Rf 0.5 , n-hexane: EtOAc = 1: 2)] and a compound 2 (6.3mg, SiO2 TLC Rf 0.5 , n-hexane: EtOAc = 1: 1).

Compound 1 (Dehydroeffusol): powder, mp 228-229 ° C, IR v _max 3270, 1660, 1540, 1420, 1405, 1350, 1270, 1160 cm ^-1 ; ^{1 H-NMR (400 MHz,} CD 3 OD): 8.48 (1H, d, J = 8.8 Hz, H-4), 7.85 (1H, d, J = 9.0 Hz, H-10), 7.57 (1H, d , J = 9.0 Hz, H- 9), 7.47 (1H, dd, J = 10.4, 17.6 Hz, H-12), 7.15 (2H, br.s, H-6, 8), 7.08 (1H, d, J = 8.8 Hz, H-3), 5.74 (1H, d, J = 1.2, 17.6 Hz, H-13 '), 5.42 (1H, dd, J = 1.2, 10.4 Hz, H- , s, H-11). ^{13 C-NMR (100 MHz,} CD 3 OD); (C-7), 153.45 (C-2), 143.43 (C-12), 139.87 (C-5), 134.75 (C-4), 126.33 (C-4a), 124.62 (C-5a), 124.29 (C-10), 119.44 (C-6), 118.77 C-3), 114.45 (C-13), 112.82 (C-8), 11.44 (C-11).

Compound 1 ¹ H-NMR spectrum of 6 aromatic hydrogen _{(δ H 8.48, d, J} = 8.8 Hz, H-4; 7.08, d, J = 8.8 Hz, H-3) In, (δ _H 7.85, d, J = 9.0 Hz, H-10 ; 7.57, d, J = 9.0 Hz, H-9), (δ H, 7.15, br.s, H-6, 8) have been observed, and these coupling constants (coupling contant ) Values were used to identify ortho and meta-coupling systems. The exomethylene signal was observed at δ _H 5.74 (1H, d, J = 1.2, 17.6 Hz, H-13 '), 5.42 (1H, dd, J = 1.2, 10.4 Hz, H- . Seventeen carbons were observed in the ¹³ C-NMR spectrum and two hydroxylated aromatic carbon signals at δ _c 155.57 (C-7) and 153.45 (C-2) and six methine carbons And methylene carbon was observed at δ _c 114.44. Based on these results, Compound 1 was found to be a phenanthrene compound, dehydroeffusol.

[Structural formula 1]

Compound 2 (Effusol): mp. 210-211 C, IR v _max 3250, 1605, 915 cm ^-1 ; ^{1 H-NMR (400MHz, CD} 3 OD): 7.19 (1H, d, J = 8.0Hz, H-4), 6.94 (1H, dd, J = 10.0,17.6Hz, H-12, 6.86 (1H, d , J = 1.5Hz, H-6 ), 6.66 (1H, d, J = 8.0Hz, H-3), 6.65 (1H, d, J = 1.5Hz, H-8), 5.65 (1H, dd, J = 2.0, 17.6 Hz, H-13 '), 5.21 (1H, dd, J = 2.0,10.0 Hz, H-13), 2.70 (2H, br.s, H-9), 2.64 , H-10) and 2.22 (3H, s, H-11).

^{The results of 13} C-NMR (100 MHz, CD ₃ OD) were 156.54 (C-7), 155.19 (C-2), 141.80 (C-5), 140.61 (C-1a), 128.51 (C-4), 127.91 (C-4a), 127.18 (C-5a), 122.14 (C-1), 115.13 C-3), 112.47 (C-8), 31.65 (C-9), 26.80 (C-10) and 11.90 (C-11).

Compound 2 showed a very similar pattern as compound _{1, δ H 7.19 (1H,} d, J = 8.0 Hz, H-4) and 6.66 (1H, d, J = 8.0 Hz, H-3) o-coupled in a ring And hydrogen was meta-coupled at δ _H 6.86 (1H, d, J = 1.5 Hz, H-6), 6.65 (1H, d, J = 1.5 Hz, H-8). Exo-methylene coupling was also observed at? _H 5.65 (1H, dd, J = 2.0, 17.6 Hz, H-13 '), 5.21 (1H, dd, J = 2.0, 10.0 Hz, H-13). ^{In the 13} C-NMR spectrum, all 17 carbons were observed, and methylene carbon was observed at δ _c 31.65 and 26.80, indicating that Compound 2 was effusol.

[Structural formula 2]

Experimental Example

Experimental Example  1: Antioxidant activity measurement result

The antioxidant activity of sulphate was evaluated on the basis of DPPH free radical scavenging ability and ABTS free radical scavenging ability. The most characteristic mechanism of antioxidants is to react with free radicals. Free radical scavenging activity is used as a measure to inhibit the antioxidant effect and the aging in the human body by donating electrons to the active radicals. Representative methods widely used to measure free radical scavenging activity of natural products are DPPH and ABTS systems. The DPPH free radical scavenging activity was measured by dilution with 100, 250 and 500 ug / mL to determine the antioxidative activity of the fractions (EtOAc, n-BuOH, H ₂ O) (Data not attached). The antioxidant activity of DPPH and ABTS free radical scavenging activity of the compound (Compound 1: Dehydroeffusol, Compound 2: Effusol), which had the highest antioxidant activity, was determined in Tables 1 and 2.

DPPH (1,1-diphenyl-2-picrydrazyl) free radical scavenging activity was measured by modifying the method of Blois et al. To measure DPPH free radical scavenging activity. That is, 0.1 mL of 2 × 10 ^-4 M DPPH solution was added to 0.1 mL of each concentration, and the mixture was incubated for 10 seconds. Then, the mixture was incubated at room temperature for 60 minutes, and the absorbance was measured at 525 nm using a UV / VIS spectrophotometer . The DPPH free radical scavenging activity was expressed as a percentage difference between the absorbance of the sample solution and that of the sample solution.

[Equation 1]

free radical scavenging activity (%) = {1- (Atest-ABlank / Bcontrol)} 100

Here, "Atest" is the absorbance of the sample, "ABlank" is the absorbance of the blank, and "Bcontrol" is the absorbance of the sample without added sample.

Positive control used AsA (Ascorbic acid), a natural antioxidant, which has been proven to have excellent antioxidant activity. DPPH free radical scavenging activity was measured. As shown in Table 1, compounds 1 and 2 exhibited excellent scavenging activity of 79.1% and 83.7% at a low concentration of 50 μM and 90% activity at 100 μM, respectively . It was confirmed that the two compounds isolated from the fractions of ethyl acetate fraction were found to have DPPH free radical scavenging ability close to the control group AsA.

In addition, the antioxidant activity of the sample was measured using ABTS according to a method such as Re. ABTS (2,2'-azinobis- (3-ethylbenzothiazoline-6-sulphonic acid)) was dissolved in distilled water at a concentration of 7 mM and then 2.4 mM potassium persulfate was added to generate ABTS free radicals. For 12 ~ 16 hours. The radical-generated ABTS solution was diluted with 99% ethanol solution and adjusted to an absorbance of 0.70 ± 0.02 at 700 nm. The removal efficiency was determined by mixing 100 μL of the ABTS solution and 100 μL of the sample, reacting for 6 minutes, and then measuring the absorbance at 700 nm.

As shown in Table 2 above, the ABTS free radical scavenging activity was measured, and it was confirmed that the scavenging activity of the compounds 1 and 2 increased in a concentration-dependent manner ( p <0.05). Compound 1 showed a low scavenging activity of 18.0% at 50 μM, but showed over 90% scavenging activity at 500 μM concentration. Compound 2 exhibited a relatively high scavenging activity at 35.9% as compared with Compound 1 at a low concentration of 50 μM and a scavenging activity similar to AsA from 500 μM to 99.2%, indicating a high antioxidative activity.

Experimental Example  2: Cancer cell growth inhibitory effect

We investigated the effects of compounds 1 and 2 isolated from beef stalk using tumor cells (AGS) and lung cancer cells (A549), which are typical for Korean people, on cancer cell growth.

The antioxidant activity of each compound was confirmed by the antioxidant activity. In order to confirm the effect of inhibiting the growth of cancer cells, the concentration was reduced to 1, 10 and 40 μM. As a result, it was confirmed that each compound effectively inhibited the growth of AGS and A549 (see Figs. 1A and 1B).

The human cancer cell line (AGS) and the lung cancer cell line (A549) were purchased from the Korean Cell Line Bank (KCLB). Each cell was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin G and 50 μg / mL streptomycin (Gibco Co., Grand Island, NY, USA) The cells were cultured in a 5% CO ₂ and 37 ° C incubator. When the cell density increased, trypsin-EDTA treatment was performed to subculture the cells.

(3 - [4,5-dime thythizol-2-yl] -2,5-diphenyl tetrazolium bromide, Lonza) assay according to the method of Young et al. The cancer cells were prepared at a concentration of 1 x 10 ⁵ cells / ml, and 100 μl of each microplate was dispensed uniformly into each well of a 96-well microplate. The cells were incubated for 48 hours in a cell culture incubator. Each compound was dissolved in DMSO (dimethylsulphoxide) and diluted with RPMI 1640 medium. Each of the samples from which the impurities had been removed was prepared at 1, 10, and 40 μM concentration, and 100 μl was added to each sample. In the control group, 100 μl of RPMI 1640 medium was added in place of the sample solution and incubated again for 24 hours. Then, 10 μl of MTT (1 mg / ml PBS) was added to each well and incubated again. After 4 hours, the absorbance was measured at 540 nm using an ELISA reader.

Referring to FIG. 1A, in the case of AGS, Compound 1 showed a relatively low growth inhibition effect at 1 μM to 8.5%, and showed the highest inhibitory effect at 20 μM at 40 μM, but there was no significant difference according to the concentration. Compound 2 showed an inhibitory effect on AGS growth of 20-28% at a concentration of 1-40 μM and did not show a significant difference according to the concentration as in Compound 1. Compounds 1 and 2 showed similar antitumor activity as CPA (cyclophosphamide) used as an anticancer agent.

Referring to FIG. 1B, the effects of Compound 1 (10.5-20.7%) and Compound 2 (5.1-7.3%) on the growth of A549 were similar to those of AGS growth inhibition at concentrations of 1 and 10 μM , There was no significant difference between the compounds 1 and 2. On the other hand, at 40 μM, compound 1 and compound 2 inhibited A549 growth by 58.8% and 44.9%, respectively. Compared to CPA at the same concentration, compound 1 and compound 2 were 1.5 times and 2 times more than control (CPA) High growth inhibition effect.

Comparing the cell growth inhibitory effect of Compound 1 and Compound 2 against two cancer cells (AGS, A549), it was confirmed that they effectively inhibited the growth of A549 than AGS (p <0.05) (see FIG. 2). In particular, Compound 1 is expected to be useful as a novel functional material targeting lung cancer cells (A549).

Claims (7)

(Juncus Effusus L.) extract as an active ingredient.
2. The pharmaceutical composition according to claim 1, wherein the extract of mustard extract comprises dehydroeffusol of the following structural formula (1).
[Structural formula 1]

2. The pharmaceutical composition according to claim 1, wherein the extract of mustard extract contains effusol of the following structural formula (2).
[Structural formula 2]

[Claim 2] The pharmaceutical composition according to claim 1, wherein the fragrant extract is a fraction obtained by fractionating an extract obtained by extracting with methanol or ethanol with ethyl acetate.
The pharmaceutical composition according to claim 1, wherein the anti-cancer effect is an anti-cancer effect on lung cancer.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is any one selected from tablets, hard or soft capsules, granules, solutions, suspensions, injectable solutions or suspensions. .
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

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