KR20130024563A

KR20130024563A - N-butanol substracts of ecklonia cava

Info

Publication number: KR20130024563A
Application number: KR1020110088089A
Authority: KR
Inventors: 서영완; 이진혁; 이정임
Original assignee: 한국해양대학교 산학협력단
Priority date: 2011-08-31
Filing date: 2011-08-31
Publication date: 2013-03-08

Abstract

PURPOSE: An n-butanol extract of Ecklonia cava is provided to suppress cancer metastasis. CONSTITUTION: An n-butanol extract of Ecklonia cava is prepared by drying and pulverizing Ecklonia cava, extracting with methanol, and extracting the crude extract with n-butanol. The n-butanol extract is prepared by heating Ecklonia cava powder in a methanol solvent, extracting, dissolving in methanol, concentrating, desalting to prepare the crude extract, and sequentially fractioning with n-hexane, 85% soluble methanol, n-butnaol, and water.

Description

N-Butanol substracts of Ecklonia cava}

The present invention relates to an extract of brown algae Ecklonia cava, and more specifically, to extract the brown algae epaulettes containing polyphenols through a novel extraction method for the anti-oxidation, anti-inflammatory and cancer metastasis N-butanol extract of brown algae breaches for use.

With the development of science and technology, the medical level of today has also grown rapidly. However, due to environmental pollution, increased stress caused by various causes, and changes in eating habits, modern people are more exposed to carcinogenic substances and stimuli. The increase in cancer incidence and deaths is a global trend, steadily increasing to 1.3% and 4.9% per year on average for men and women (National Cancer Information Center, 2010). Since the 20th century, as the interest in cancer has increased, the development of treatments has been studied at a high level. However, most anti-cancer therapies use radiation or synthetic drugs with high toxicity. It is a generalized fact that it causes serious abnormalities in function. Although studies on the development of natural anticancer drugs to reduce these side effects and selectively inhibit only cancer cells have been conducted, remarkable treatments are still unknown.

Cancer progresses in malignant tumor genes such as initiation stage, which is a process in which various stresses such as free radicals are transformed into cancer cells due to disturbance, damage, promotion stage, invasion, and metastasis. It is divided into three stages of progression stage. Metastasis refers to the metastasis of cancerous cells from early tumors, into blood vessels or lymphatic vessels, into other target tissues, where they multiply and form secondary tumors (Liotta, 1992). One important factor for this transition is to break down proteins in the cellular matrix (ECM) and basement membrane, which are the support structures of the cell. There are many studies on serine proteinase and metalloproteinase among these various proteinases, and matrix metalloproteinase (MMP), which breaks down the major components of ECM and basement membrane, plays an important role (Chambers et al. , 1997; Yoon et al., 2003). MMPs generally increase in the development of tumors and play an important role in tumor metastasis as well as neovascularization, apoptosis, proliferation and differentiation in tumor tissues (McCawley et al., 2000; Renckens et al., 2006 ). The MMP family consists of more than 20 protein groups, collagenase (MMP-1, 8, 13), which decomposes fibrillar collagen according to substrate specificity, gelatinase (MMP-2, 9), and proteoglycan, which decompose type IV collagen and gelatin. And stromelysin (MMP-3, 10, 11) and membrane-type MMP (MT-MMP), which break down glycoproteins. In particular, MMP-2 (type IV collagenase, gelativase A) and MMP-9 (type IV collagenase, gelativase B) are known to play the most important role in cancer invasion and metastasis (Moon et al., 2000). This is because the main component of the basement membrane is type IV collagen. Therefore, it is expected that if MMPs are expressed at high levels in cancer cells, tumor invasion and metastasis may be suppressed to control cancer progression and to seek a positive direction for anticancer treatment.

Research has been conducted to find natural product-derived MMPs inhibitors that act specifically on cancer cells while reducing side effects and improving immune function (Ohtsuki et al., 2008). Among these natural products, polyphenols, which are found in many plants, have recently attracted interest as food, beverage and health supplements as representative antioxidants as well as MMPs inhibitors (Sil et al., 2010). Antioxidants can be used to chelate metal ions, terminate superoxide dismutase activity, and radical reaction by enzyme-like activity in relation to oxidation of food or organic matter or aging of human body. Excellent ability in the prevention and treatment effect (Babizhayev et al., 1994).

The living body plays an important role in producing NO by nitiric oxide synthase (NOS) from L-arginine by stimulation such as infection and inflammation, killing tumor cells or protecting the living body from infection by microorganisms. Excessive production can result in depletion of intracellular NAD (Ev et al., 2007) or inhibition of DNA synthesis (Kwon et al., 1991). Or induction of mutations affect the like (Nguyem et al, 1992.) , Peroxynitrite (ONOO -) reacts with superoxide radical has been known to cause tissue damage by forming (Beckman et al, 1990.) . In this regard, polyphenols have been reported to have an excellent effect on the anti-inflammatory action of inhibiting NO production (Shanmugam et al., 2008).

Ecklonia cava used in the present invention is a plant of Laminariales seaweed (Alariaceae), which lives mainly in the coasts of Korea and Japan (Fig. 1.). The length is 1 ~ 2 m, the stem is cylindrical, and the base is the root shape of the plant. It lives in the depths of the luncheon and is an algae plant constituting the marine forest, serving as a food for abalone and conch, and plays an important role as a primary producer in the coastal ecosystem. Recently, polyphenols isolated from Ecklonia cava are used for various health functional drinks, toothpastes, shampoos, cosmetics, and the like.

Accordingly, the present invention has been made to solve the above problems, the present invention is effective in containing a polyphenol (polyphenol) compound which is a secondary metabolite contained in Ecklonia by purifying it more purely than Ecklonia itself in the present invention It is an object to provide a n-butanol extract of brown algae Ecklonia cava containing polyphenols for separating the components in high purity and for inhibiting cancer metastasis of antioxidants, anti-inflammatory agents and anticancer agents.

Another object of the present invention is to provide an extract for inhibiting cancer metastasis derived from brown algae Ecklonia cava, wherein the active extracts obtained by extracting the n-butanol extract of brown algae Ecklonia cava in more detail can be used for cancer metastasis inhibition. It is done.

In order to achieve the above object, n-butanol extract of brown algae Ecklonia cava according to one embodiment of the present invention is dried and crushed brown algae ecklonia (Ecklonia cava) to produce a powder, and then crude extract extracted from methanol again Secondly, extraction again from n-butanol is a technical subject matter. Preferably, the n-butanol extract is extracted by heating the Ecklonia cava powder to a boiling point of a methanol (methanol (MeOH)) solvent, and the resulting extract is dissolved in methanol (MeOH) at room temperature and concentrated and then desalting. The crude extract was partitioned sequentially according to the polarity of the solvent, n-hexane ( n -hexane), 85% aqueous methanol (85% aq. MeOH), n-butanol ( n -BuOH), water (H ₂ O) fractions N-butanol extract obtained.

The n-butanol extract has antioxidative activity with radical scavenging activity and hydrogen peroxide scavenging activity, anti-inflammatory activity with NO production inhibitory activity, or MMP-2 and MMP-9 at the cellular level. It is characterized by having a cancer metastasis inhibiting ability to suppress the expression of.

In addition, another feature of the present invention, n-butanol extract of brown algae erosion is divided into detailed fractions through silica column chromatography, and each fraction is dissolved in an appropriate concentration of MeOH in CHCl ₃ and then sephadex LH20 The active extract is separated through column chromatography. The separated active extract is any one of Triphlorethol-A, Eckol, Ecktolonol, Phlorofucoeckol or Dieckol. And

Each of the active extracts, n-butanol extract of brown algae Ecklonia cava was divided into detailed fractions by silica column chromatography, and 10% MeOH in CHCl ₃ fractions in each fraction were separated by sephadex LH20 column chromatography. Separating the Nocks (Eckstolonol), and combined the 20% MeOH in CHCl ₃ and 30% MeOH in CHCl ₃ subfraction of the remaining fractions again by silica column chromatography divided into 7 fractions, 15% MeOH in CHCl ₃ subfraction Triphlorethol-A was isolated by sephadex LH20 column chromatography, and sephadex LH20 column chromatography was also performed on 20% MeOH in CHCl ₃ fractions to carry out triphalorethol-A and Xtoro. Further separation of ckstolonol, new dieckol, and sephadex LH20 column chromatography and reverse-phased HPLC were performed sequentially for 25% MeOH in CHCl ₃ subfraction. It was carried out as a trichlorolore-A (Triphlorethol-A) is further separated and characterized in that the separation of Eckol (Eckol) and Florofucoeckol (Phlorofuroeckol). These active extracts are characterized by having cancer metastasis inhibiting ability to inhibit the expression of MMP-2 and MMP-9 at the cellular level.

The present invention can easily obtain a new novel material having no side effects by using an extract containing a polyphenol derived from the brown algae Ecklonia cava, thereby reducing the production cost.

In addition, the present invention has another effect of providing a new way to the treatment of cancer cells by obtaining substances that can be used for inhibiting cancer metastasis.

1 is a view showing the extraction process and the amount of extract by each solvent from brown algae Ecklonia cava.
2 shows the amount of total polyphenols in each solvent extract.
Figure 3 illustrates the extraction process of the active extracts 1 to 6 using n-butanol extract.
Figure 4 is a diagram showing the spectral data of ¹ H NMR and ¹³ C NMR of each separated active extract 2 to 6.
5a to 5e are graphs showing spectra of ¹ H NMR, ¹³ C NMR, gDQCOSY and TOCYS of each of the separated actives 2-6.
Figure 6 is a view showing the structure of each of the separated active substances 1-6.
7 shows cell viability for Raw 264.7 cells.
8 is a bar graph showing the results of ROS (total free radical measurement) scavenging activity of crude extracts and solvent fractions
9 is a view showing the results of ROS (total free radical measurement) scavenging activity of crude extracts and solvent fractions.
10 is a diagram showing the results of NO production inhibition experiment to determine the anti-inflammatory activity for Raw 264.7 cells.
Figure 11 shows the expression level of iNOS in Raw 264.7 cells.
12 is The results of cell viaability for the MTT assay effect of crude extract and solvent fraction on HT-1080 cells are shown.
Figure 13 is a gelatin zymography method for showing the MMP activity of the crude extract and solvent fractions MTT assay effect on HT-1080 cells.
14 is a diagram showing the expression effect of mRNA genes of MMP-2 and MMP-9 to confirm the effect on the HT-1080 cell invasion factor of a part of Ecklonia cava extract.
Figure 15 is a view showing the expression effect of mRNA genes of MMP-2 and MMP-9 to confirm the effect on the HT-1080 cell invasion factor of a part of Ecklonia cava extract.
Figure 16 shows the cell viability to determine the effect of MTT assay of the active compound (compounds) 2-6 of the five compounds isolated from the n- BuOH extract on HT-1080 cells.
Figure 17 shows the inhibitory effect of the active compound (compounds) 2-6 of five compounds isolated from n -BuOH extract on HT-1080 cells.
Figure 18 shows the effect of the expression of mRNA genes of MMP-2 and MMP-9 in order to confirm the effect on the HT-1080 cell invasion factor of five compounds 2-6 isolated from n-butanol extract of Ecklonia cava Shown.

Hereinafter, the n-butanol extract of brown algae roe containing polyphenol for use for inhibiting cancer metastasis according to the present invention will be described in detail.

Preparation for experiment

Selection of materials

May 2004 were used for the experiment to buy Ecklonia cava (Ecklonia cav a) one collected in Jeju Island aewoleup gwideokri was extracted by grinding in grinder and dried in the shade.

Reagents, Extraction, Fractionation and Separation

All solvents used for column chromatography were purchased from a first-class reagent. Sephadex LH-20 (bead size 25 ~ 100 ㎛, Sigma) was used for Sephadex column chromatography, and silica gel (YMC-Pack SIL, 12 nm, S-5 ㎛, 250 × 10 mm ID, YMC, USA), and high performing liquid chromatography (HPLC) was purified using a YMC-pack ODS-A column (250 × 10 mm, S-5 μm, 12 mm) and a guard column (7.5 × 4.6 mm, Alltech). Separated. The solvent used for NMR measurement was CD ₃ OD (Merck. Deuterium degree 99.95%).

activation

1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 3-morpholinsydnonimine (SIN-1), dihydrorhodamine 123 (DHR 123), penicillamine (DL-2-amino-3-mercapto-3-methyl butanoic acid was purchased from Sigma (St Louis, MO, USA). Peroxynitrite (ONOO ^-) was purchased from Cayman (Ann Arbor, MI, USA ). DMEM (Dulbecco's Modified Eagle's Medium) and RPMI-1640 for cell culture were purchased from Hyclone (Logan, Utah, USA). FBS (Fetal Bovine Serum), Trypsin and 100 units / mL Penicillin-Streptomycin were purchased from GIBCO (USA). The kit for MTT assay (MTT cell proliferation assay) was purchased from R & D systems (Minneapolis, MN, USA). DCFH-DA used for ROS measurement was Molecular Proves inc. It was purchased from (Eugene, OR, USA) and used. Lipopolysaccharide (LPS) and Griess reagent sulfanilamide and NED were purchased from sigma. iNOS (inducible nitric oxide synthase, forward 5'-AGA-GAG-ATC-CGG-TTC-ACA-3 ', reverse 5'-CAC-AGA-ACT-GAG-GGT- ACA-3') used for mRNA expression , GAPDH (Glyceraldehyde-3-phosphate dehydrogenase, forward 5'-CGG-AGT-CAA-CGG-ATT-TGG-TCG-TAT-3 ', reverse 5'-AGC-CTT-CTC-CAT-GGT- GGT-GAA -GAC-3 ') gene, primer and agar, EtBr (ethidium bromide) used for electrophoresis were purchased from Sigma. Phorbol myristate acetate (PMA), which was used to measure the expression of Metrix metalloproteinase (MMP), was purchased from Sigma and MMP-2 and MMP-9 were purchased from R & D systems Inc.

Device used

¹ H-NMR, ¹³ C-NMR and two-dimensional NMR experiments were performed using a Varian NMR 300 spectrometer. Compounds were purified using a Varian RI detector and high performance liquid chromatography (HPLC, Dionex p580). UV-Vis spectrophotometer (Thermo Spectronic, England) and Multi-detection microplate fluorescence spectrophotometer Synergy HT (Bio-TEK instruments, USA) were used for the measurement of antioxidant activity and MTT. Cultivation of cells is CO ₂ An incubator (Forma Scientific, Japan) was used, PCR (BIORAD, USA) was used for amplification of the gene, and Mupid-2plus (Takara, Japan) was used for electrophoresis. In addition, rotary evaporator (EYELA, JAPAN), vacuum pump, pH meter, water bath, pipet (JBM-pipet), filter and the like were used.

Extraction, fractionation and separation

Extraction and fractionation

Ecklonica cava purchased from Gukdeok , Jeju, Korea was stored at room temperature after drying and used to extract 850g by grinding with a grinder. Using a heating mantle, the methanol (MeOH) solvent was heated to a boiling point and extracted three times for 12 hours. The extract was extracted using a rotary vacuum evaporator (EYELA) in a 40 ° C water bath. JAPAN, NN series), the obtained extract was dissolved in methanol (MeOH) at room temperature again and concentrated to obtain a desalt (desalting) extract. As shown in FIG. 1, the original extract was 135.93 g (Crude extract: 135.92 g), using this to obtain 57.25 g of crude extract, which was sequentially partitioned according to the solvent polarity, and n-hexane ( n -hexane). , 85% aqueous methanol (85% aq. MeOH), n-butanol ( n- BuOH) and water (H ₂ O) fractions were obtained 8.60 g, 3.42 g, 45.20 g and 78.08 g, respectively.

Example 1 bracket Fraction gun Polyphenol Content measurement

The polyphenol content of each fraction was determined by a modification of the Folin-Denis method (Folin et al., 1912). 1 mg / mL (0.1% stock) of n- hexane, 85% aq. To 20 μL of each fraction of MeOH, n- BuOH, H ₂ O, 250 μL of H ₂ O and 250 μL Folin-Denize reagent were added and reacted at room temperature for 3 minutes. 500 μL of 35% Na ₂ CO ₃ dissolved in H ₂ O at 70 ° C. was added and reacted at room temperature for 20 minutes, and the absorbance was measured at 750 nm. Total polyphenol content was obtained from the standard curve prepared using tannic acid.

Content measurement result of polyphenol

The total polyphenol content in each fraction of Ecklonia cava extract was measured using tannic acid as a reference. As shown in Figure 2, the total polyphenol content is butanol ( n -BuOH), 85% aq. In the order of MeOH, H ₂ O and n- hexane fractions, 68.78, 22.89, 3.86 and 1.16 mg / ml, respectively, were found in the n- BuOH fraction with the highest total polyphenol content. It is expected to contain compounds with excellent physiological activity.

Example 2 . n- Butanol Isolation of Active Ingredients from Extracts

Polyphenols (polypheol) content measurement results and ¹ H NMR subjected to flash column chromatography NP (nfc) using a mixture of CHCl ₃ and MeOH for n -BuOH fractions based on the spectral analysis 100% CHCl ₃ (nf. 1), 5% MeOH in CHCl ₃ (nf. 2), 10% MeOH in CHCl ₃ (nf. 3), 20% MeOH in CHCl ₃ (nf. 4), 30% MeOH in CHCl ₃ (nf. 5), 40% MeOH in CHCl ₃ (nf. 6), 50% MeOH in CHCl ₃ (nf 7), 70% MeOH in CHCl ₃ (nf. 8), 100% MeOH (nf. 9) and 90% MeOH in H ₂ O (nf. 10) solvent fractions were obtained.

As shown in FIG. 3, each fraction obtained was subjected to nf. In consideration of ¹ H NMR spectroscopy data and the amount of sample. 3 fractions were separated using 100% MeOH solvent using Sephadex LH 20. A total of 13 subfractions were obtained, of which active extract 1 (compound 1) was obtained in subfraction 9-11 and active extract 4 (compound 4) in subfractions 3 and 4. In addition, nf. 4 and nf. Add 5 and run nfc again to get 100% CHCl ₃ (nf. 1), 10% MeOH in CHCl ₃ (nf. 2), 15% MeOH in CHCl ₃ (nf. 3), 20% MeOH in CHCl ₃ (nf. 4), 25% MeOH in CHCl ₃ (nf. 5), 30% MeOH in CHCl ₃ (nf. 6), and 100% MeOH (nf. 7) ) Solvent fractions were obtained. Nf. Sephadex LH 20 was used to separate 3 fractions with 30% MeOH in CHCl ₃ solvent. A total of 31 subfractions were obtained, of which active extract 2 (compound 2) was obtained from subfraction 27. nf. Similarly, the 4 fractions were separated using 30% MeOH in CHCl ₃ solvent using Sephadex LH 20, and a total of 75 subfractions were obtained. Among them, active extract 2 (compound 2) was obtained in subfraction 18-23, and active extracts 4 and 6 (compound 4 and 6) were obtained in subfraction 11-14 and subfraction 5-7, respectively. nf. The 5 fractions were first separated with 30% MeOH in CHCl ₃ solvent using Sephadex LH 20 to obtain a total of 32 subfractions. Among them, active extract 2 (compound 2) was separated from subfraction 10 and 11 and active extract 3 (compound 3) from subfraction 14-16, and subfraction 22-24 was added together to reversed-phase HPLC (ODS-A, 40). % aq.MeOH) was used to isolate the active extract 5 (compound 5).

Determination of Structure of Separated Active Materials

4 is a diagram showing spectral data of ¹ H NMR and ¹³ C NMR of each of the separated active extracts 2-6, and FIGS. 5A to 5E are ¹ H NMR, ¹³ C NMR, of each of the separated active extracts 2-6, It is a graph showing the spectrum of gDQCOSY and TOCYS, Figure 6 is a diagram showing the structure of each of the active extracts 2-6 separated. As shown,

The active extract 1 (Compound 1) was separated into white powder, and the molecular formula C ₆ H ₆ O ₃ was determined by mass spectrometry and ¹³ C NMR data. It has a symmetrical 1,3,5-tryhydroxybenzene structure, and the ¹ H NMR spectrum peak of the benzene ring was shifted upfield by the hydroxy group at δ 5.77 ppm. Two peaks at ¹³ C NMR were seen at δ 160.0 ppm shifted to the downfield and 95.5 ppm to the shifted upfield, indicating that oxygen is bound to the 1.3.5-position of benzene.

The active extract 2 (Compound 2) was isolated as a yellowish white powder, and the molecular formula C ₁₈ H ₁₄ O ₉ was determined by mass spectrometry and ¹³ C NMR data. Referring to the signal originating from the aromatic compound at δ 5.7-6.0 ppm of the ¹ H NMR spectrum, 14 peaks were observed in ¹³ C NMR, indicating that two of the three benzene rings are symmetrically present. This was exactly in agreement with the unsaturation value (U = 12), and these benzene rings could be expected from the combination of compound 1 monomers. ¹ H NMR ₂ system AB in the spectrum signal [δ 6.00 (1H, d , 2.2), δ 5.92 (1H, t, 2.2)] and AB system signal [δ 6.04 (1H , d, 2.8), δ 5.75 (1H, d, 2.8)] and A ₂ system [δ 5.89 (1H, s)]. In addition, it was confirmed that each benzene ring is connected by ether bond through ¹³ C NMR spectrum. Additional structures were determined using 2D NMR experiments such as ¹ H COSY, TOCSY, gHMQC, gHMBC. This is a well-known compound Triphlorethol-A, which is in good agreement with the NMR data of the literature (Fukuyama et al., 1985).

The active extract 3 (Compound 3) was separated into a light brown powder. The molecular formula C ₁₈ H ₁₂ O ₉ was determined by mass spectrometry and ¹³ C NMR data. ^{In the 13} C NMR spectrum, 12 signals were predicted to be aromatic carbons derived from oxygen around δ 160-100, and five peaks (δ 99.69, δ 99.20, δ 97.54, unsubstituted near δ 100-90). δ 95.61, δ 95.20) were confirmed. In addition, the characteristic signals of the three aromatic protons in the ¹ H NMR spectrum, AB _{2 of} The δ 5.92 (1H, s) peak attributable to the system, the δ 5.93 (1H, s) peak attributable to the AB system, and the δ 6.12 (1H, s) peak due to the singlet were observed. Through this, it can be expected that the active extract 3 is a compound having three phloroglucinol units. In addition, the active extract 3 ¹ H NMR and ¹³ C NMR spectra are active extracts two structures and eotneunde indicate a very similar pattern of when compared one aromatic hydrogen (H-5, δ 6.04) in the ¹ H NMR spectrum and ¹³ C The unsubstituted aromatic carbons (C-5, δ 94.8) shown in NMR disappear and new aromatic carbon signals (C-5a, 147.05) are substituted with oxygen, resulting in open- s in active extract 3 (compound 3). It was confirmed that the chain bond is connected to form an aryl-ether structure, and that the compound has a typical Dibenzo- p- dioxi skeleton. The overall structure of the active extract 3 (Compound 3) was determined using 2D NMR experiments such as ¹ H COSY, TOCSY, NOESY, gHMQC, gHMBC, and the results were compared well with the NMR data of the literature with Eckol, a known compound. (Herron et al., 1986; Fukuyama et al., 1989).

The active extract 4 (Compound 4) was isolated in the form of yellowish white powder. The molecular formula C ₁₈ H ₁₀ O ₉ was determined by mass spectrometry and ¹³ C NMR data. ^{In the} vicinity of δ 155-124 ppm of the ¹³ C NMR spectrum, 13 oxygen-bonded aromatic carbons were found. In the vicinity of δ 100-95 ppm of the ¹³ C NMR spectrum, five non-substituted carbon signals were observed. In addition, five peaks due to aromatic protons appeared in the vicinity of δ 6.2-5.9 ppm of the ¹ H NMR spectrum, which is similar to the active extract 3 (compound 3), and the active extract 4 (compound 4) is dibenzo- p. -dioxin has a basic skeleton. ^{In the 1} H NMR spectrum, one aromatic proton peak (H-2 ', δ 5.92) and one non-substituted aromatic carbon peak (C-2', δ 95.18) disappeared compared to active extract 3 (compound 3). In addition, a new aromatic carbon peak (C-8a, δ 124.73) was substituted with oxygen, indicating that the active extract 4 (compound 4) was an aryl-ether bond added between C-8a and C-7a. This is a well known compound, eckstolonol, which is in good agreement with the NMR data of the literature (Kang et al., 2003).

The active extract 5 (Compound 5) was separated into light brown powder, and the molecular formula C ₃₀ H ₁₈ O ₁₄ was determined by mass spectrometry and ¹³ C NMR data. ^{By 13} C NMR spectra, 19 carbon peaks and 2 quaternary carbon peaks derived from 9 methine, 2 quaternary carbon and oxygen among 30 aromatic carbons were identified. ^{In the 1} H NMR spectrum, two AB ₂ system peaks [δ 5.96 (2H, d, 2), δ 5.91 (1H, t, 2)] and [δ 5.93 (1H, t, 2.2), δ 5.87 (2H, d , 2.2)] and three singlets (δ 6.62, δ 6.39, δ6.25) were identified. Molecular weight and NMR spectral analysis showed that the compound was composed of five phloroglucinol units, and it was confirmed that the compound is very similar to the active extract 2 (compound 2) having a dibenzo- p- dioxin skeleton. ¹³ C NMR in by the aryl ether bond of carbon other than the peak due to the aryl ether bond appears in dibenzo- p -dioxin skeletal carbon peak of C-12a (δ 152.97) and C-11a (δ 150.96) appeared additionally. The chemical shifts of quaternary carbons, C-6 (δ 105.17) and C-7 (δ 105.22), suggest that C-6 and C-7 are linked through aryl-aryl bonds. Active extract 5 (Compound 5) Structural determination Ecklonia through additional 2D NMR experiments and literature of It was confirmed that it was phlorofucofuroeckol A isolated from Kurome , and it was consistent with the spectroscopic data (Fukuyama et al., 1990; Sugiura et al., 2006).

The active extract 6 (Compound 6) was also separated into light brown powder, and determined by mass spectrometry and ¹³ C NMR data as molecular formula C ₃₆ H ₂₂ O ₁₈ with an unsaturation of 26. It was found that the aspect of the ¹³ C NMR spectrum of the active extract 6 (Compound 6) each peak are shown in pairs on the spectrum as a very similar fashion to the active extract 3 (Compound 3), which may be combined with two eckol unit Guessed. ^{In 1} H NMR, the proton peak of H-4 '''paired with the H-4' (δ 5.92) peak of eckol disappeared, and C paired with C-4 '(δ 97.53) in the ¹³ C NMR spectrum. -4 '''(δ 126.26) showed a typical oxygen substituted aromatic carbon peak. In addition, the difference between C-7 '' (δ 154.28, -OR) and the chemical shift value of C-7 (δ 155.76, -OH) is marked, so that the bond between the two eckol groups is C-7 and C-4. It was confirmed that the aryl ether bond is connected between '''. In addition, the chemical shift values of C-1 '''(δ 157.57) and C-3''' (δ 152.16) are also upfield than C-1 '(δ 161.57) and C-3' (δ 159.81). Moved. In view of these points, compound 6 can be inferred as a dimer of Compound 2 , and H-2, the peak that appears in pairs with H-2 ', 6' (δ 5.92), a strong singlet peak in the ¹ H NMR spectrum. The chemical shift of the ''',6''' (δ 6.08) to the down filed further confirmed the dimer form of eckol. The overall structure was elucidated by 2D experiments and the literature review confirmed that the active compound 6 (compound 6) was a dieckol, a compound isolated from Ecklonia cava, and was consistent with the reported spectroscopic data (Lee et al., 2009).

As described above, by further dividing the n-butanol extract, it was possible to separate the active extracts of the distilled branches having different colors, and based on the analysis data of the separated active extracts, as shown in FIG. 6. I could see it.

Example 3 . Antioxidant activity experiment

DDPH radical How to measure scavenging activity

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) is a water-soluble substance with chemically stable free radicals that is dark purple in color and has a specific absorption band at a wavelength of 518 nm. When radicals in DPPH react with hydrogen-like phenols such as hydrogen or electron donors to become stable molecules, diphenylpicrylhydrazine loses its original purple color and becomes yellow, eliminating the strong absorption band at 518 nm. Since the color changes and the absorbance decreases in proportion to the reaction, the radical scavenging activity can be known by measuring the decrease in absorbance. DPPH radical scavenging activity is a widely used simple antioxidant screening method and is widely used to measure the antioxidant activity of phenol and aromatic amines (Blois, 1958).

Dissolve each sample (0.1-50 μg / mL) in MeOH. Prepare a DPPH dilution by mixing 1200 μL of DPPH stock solution prepared by dissolving 2 mg of DPPH reagent in 15 mL of EtOH in 500 μL of DMSO and 3000 μL of EtOH. Put the prepared DPPH diluent into the cuvette and measure the absorbance and adjust the concentration so that the absorbance is 0.94 ~ 0.97. 100 μL of the prepared sample and 900 μL of the DPPH dilution solution are mixed by vortex for 10-20 seconds, and measured after 10 minutes at a UV value of 518 nm. Free radical scavenging activity was expressed as a percentage compared to the negative control without the sample.

DPPH radical Scavenging activity

The Ecklonia cava was extracted with methanol solvent heated to boiling point, and experimented using fractions sequentially extracted in the order of n- hexane, 85% aq.MeOH, n- BuOH, H ₂ O according to polarity. Each of the obtained extract fractions was searched for DPPH radical scavenging activity at concentrations of 50, 10, 5, 1 and 0.1 μg / mL, respectively. L-ascorbic acid and BHT (butylated hydroxy toluene) were used as controls.

As a result, the control L-ascorbic acid showed 96.41% scavenging effect and BHT showed 78.67% scavenging effect at 50 μg / mL. In comparison, crude extract MeOH extract (54.05%) did not show a high effect, but some of the fractions obtained by fractionation of crude extract with organic solvent showed relatively high DPPH radical scavenging activity. In particular, n -BuOH fraction of the four solvent fractions showed the highest radical scavenging activity (91.69%), which was almost the same level as the control group L-ascorbic acid. Rather than using BHT, it showed a higher level of radical scavenging activity, and the experimental results are shown in Table 1 below.

The crude extract and solvent fraction samples used for the DPPH radical scavenging activity were unpurified, and considering the antioxidant activities of L-ascorbic acid and BHT used as controls, the antioxidant effect of n -BuOH extract fraction itself was very high. It is excellent, and it can be seen that there is a substance contributing to the antioxidant effect in the extract fraction.

Method for Measuring Peroxynitrite Scavenging Activity

ONOO ^- scavenging activity was detected by measuring the degree of oxidation of dihydrorhodamine 123 (DHR 123). DHR 123 (5 mM) was dissolved in dimethylformamide and the stock solution was purge with nitrogen and stored at -80 ° C. Dilutions of the DHR 123 (fc 5 μM) solution were prepared before use on ice in the dark. Buffer was prepared by mixing 90 mM sodium chloride, 50 mM sodium phosphate (pH 7.4), 5 mM potassium chloride, and 100 μM (fc) of diethylentriaminepenta acetic acid (DTPA), and refrigerated before use. After mixing the DHR 123 solution to the buffer solution, the sample and peroxynitrite were added and left at room temperature for 5 minutes, and measured by multi-detection microplate fluorescene spectrophotometer Synergy HT. In the case of adding SIN-1 instead of Authentic peroxynitrite, it was measured by adjusting the time allowed to stand at room temperature for 1 hour while performing the same method. Oxidation by DHR 123 by SIN-1 occurs gradually, while authentic peroxynitrite oxidizes very rapidly. Excitation wavelength was 485 nm, emission wavelength was 530 nm and measured at room temperature. And ONOO ^- based on the solution of (fc 10 μM) was used to 0.3N NaOH, experiments were line in triplicate, the results are the average values obtained by subtracting the blank was calculated as a percentage of the control group (Kooy et al, 1994. ).

Peroxynitrite Scavenging Activity

Since DHR 123 reacts with intracellular ONOO ^- to change to the fluorescent substance rhodamine 123, the absorbance of this product can be measured to examine the ONOO ^- scavenging activity of Ecklonia cava. ONOO ^- scavenging activity is expressed as a percentage (%) compared to the control without addition of the sample.

Crude extract and four fractions obtained by fractionating it were n- hexane, 85% aq. The scavenging activity of SIN-1 and authentic peroxynitrite was investigated at concentrations of 100, 50, 10, 5, 1 and 0.1 μg / mL for the MeOH, n- BuOH and H ₂ O fractions, respectively. L-ascorbic acid and penicillamine were used as controls.

As a result of SIN-1 treatment, control L-ascorbic acid showed 99.78%, 99.82%, 99.40%, 99.20%, 95.15%, 66.33% at 100, 50, 10, 5, 1, 0.1 μg / mL. Penicillamine was 103.99%, 101.41%, 99.95%, 92.39%, 64.88% and 27.78%. Crude extracts and fractions showed high scavenging activity at 100 μg / mL compared to the control, especially n- BuOH and 85% aq. MeOH and n- hexane fractions were 79.05%, 71.85%, and 76.56%, respectively, and showed higher activity than the control penicillamine. Among them, the n -BuOH fractions were lower than 0.1 μg / mL. Comparable high activity was shown, which is shown in Table 2.

As a result of scavenging activity against Authentic perxoynitirite, L-ascorbic acid and penicillamine used as controls showed 100% scavenging activity at concentrations of 100, 50, and 10 μg / mL, but 5 μg / From mL, the overall antioxidant activity of SIN-1 was found to be lower than that. As shown in Table 3, the scavenging activities of the crude extract and n- hexane, 85% aq.MeOH, n- BuOH, and H ₂ O fraction were measured, respectively, except for the H ₂ O fraction. In particular, n -BuOH fraction showed an excellent activity effect corresponding to penicillamine, which is concentration dependent. Based on these results, it is expected that a compound having high scavenging activity can be obtained by further separating the n- BuOH fraction.

Example 4. Activity at the cellular level

Cell culture

Raw macrophage cell lines, Raw 264.7 cells, human fibrosarcoma cell lines, HT-1080 cells, and human gastric cancer cell lines, AGS, were purchased from the Korean Cell Line Bank (KCLB). Raw 264.7 and HT-1080 were incubated in DMEM medium containing 100 unit / mL penicilin- streptomycin and 10% Fetal Bovine Serum (FBS), and AGS was 100 unit / mL penicilin- streptomycin and 10% Fetal Bovine Serum (FBS). It was incubated with the containing RPMI culture. All cells were incubated in a cell culture dish at 37 ° C. and 5% CO ₂ incubator. The cultured cells were exchanged 2-3 times a week and washed with PBS in 6-7 days, and then HT-1080 and AGS were subcultured by separating cells attached with 0.05% Trypsin-0.02% EDTA. 264.7 was subcultured with cell scraper.

How to measure cell viability

MTT assay can be used to determine the effect of secondary metabolites of Ecklonia cava on cell viability. The MTT assay is an indirect measure of cell proliferation and cell viability, and is widely used as a primary screening test for the sensitivity of anticancer drugs. In the case of cancer cells whose metabolism is intact, the water-soluble MTT formazan [3- (4,5-dimethylthiazol-2-yl) -2,5- which is a blue purple color of MTT tetrazolium, a yellow water-soluble substrate by the action of dehydrogenase of mitochondria. dipheyl-tetrazolium bromide] crystals. The absorbance of the resulting MTT formazan is maximal at a wavelength of 540 nm, and the absorbance measured at this wavelength reflects the concentration of live, metabolized cells.

Each cell of raw 264.7, HT-1080, and AGS cells was counted, and 100 μL of 1 × 10 ⁵ cells / mL was injected into 96 well micro-plate and incubated in 37 ° C. and CO ₂ incubator for 24 hours. The prepared samples were treated in each well and incubated in a CO ₂ incubator at 37 ° C. for 24 hours. After removing the medium, 5 mg of MTT reagent was dissolved in 1 mL PBS, diluted with 9 mL of DMEM (AGS; RPMI) medium containing 10% FBS, treated with 100 μL in each well, and incubated for 3 to 4 hours. Formation was observed. When Formazan was formed, MTT reagent treatment medium was removed, and 100 μL of DMSO was added and reacted for 10 minutes. The absorbance was measured at 540 nm.

Raw 264.7 cell For Crude extract Solvent fractions MTT assay effect

To determine the antioxidant activity of crude extracts and solvent fractions, the effect of samples on viability of Raw 264.7 and HT1080 cells was determined by MTT assay. All sample concentrations were treated at 200, 100, 50, 10, 1 μg / mL.

As shown in FIG. 7, the cell viability of the Raw 264.7 cell showed more than 90% cell viability at 100, 50, 10, and 1 μg / mL of the n -BuOH and H ₂ O fractions compared to the control group. 85 % aq. MeOH did not appear to affect cell survival at concentrations below 50 μg / mL. Crude extracts and n- hexane fractions inhibited cell survival at concentrations above 10 μg / mL.

Based on the measurement results of the cell viability obtained by measuring the MTT assay, the expression of ROS, NO contents and iNOS mRNA was confirmed in the raw cells.

Example 5. Antioxidant Activity Test at the Cell Level

ROS (total free radical measurement) method

Free radical production was measured by DCFH-DA assay (Okimotoa., 2000). DCFH-DA (fluorescence probe 2,7-dichlorodihydrofluorescein diacetate, sigma) is a reaction that reacts with free radicals in the cell to produce a fluorescence, which can be measured by measuring the fluorescence generated by adding this reagent into the cell. have. Raw 264.7 cells were dispensed into 96 wells and incubated for 24 hours. After washing with PBS buffer, 20 μM DCFH-DA was injected into each well and pre-incubated in 37 ° C. 5% CO ₂ incubator for 20 minutes. Samples were treated by concentration in each well for 1 hour incubation at 37 ° C 5% CO ₂ incubator, then DCFH-DA was removed and the cells were washed with PBS buffer again and treated with 500 μM H ₂ O ₂ to exert DCF fluorescence over time. . 485 nm, em. It was measured by a fluorescence analyzer at 530 nm.

ROS (total free radical measurement) scavenging activity of crude extracts and solvent fractions

The active oxygen species present in the cell were measured by DCF fluorescence using DCFH-DA, which reacts with the intracellular active oxygen to produce a fluorescent substance. After treatment with 500 μM of H ₂ O ₂ , measurements were performed for 120 minutes at intervals of 0 to 30 minutes to detect the activity of Ecklonia cava. All sample is to process all of _the 100, 50, 10, 1 μg / mL concentration were measured in the control group as is the control and sample with 500 μM H ₂ O ₂ treated with 500 μM H ₂ O ₂ without placing a sample, respectively Blank was used. The control treated with 500 μM H ₂ O ₂ rapidly increased the DCF flourescence value with time, and the blank without treatment with 500 μM H ₂ O ₂ showed little change of DCF flourescence value with time. All samples except H ₂ O fractions at the concentration of 100 μg / mL showed high activity similar to the blank not treated with 500 μM H ₂ O ₂ for ₂ hours, and control at 1 μg / mL The free radicals produced were significantly erased when compared to. From these results, it was confirmed that Ecklonia cava extract exhibited a very good effect on intracellular ROS scavenging activity at all concentrations measured.

Example 6. Anti-inflammatory activity at the cellular level

How to measure NO production inhibitory effect

Nitric Oxide (NO) reduction assay is used as a method of indirect activity measurement related to phagocytosis, one of the main functions of macrophge. 1-Naphtylenediamine, Sulfanilamide and NO ₂ ^- react to form azocoupling.The two rings form the maximum absorbance at the wavelength of 550 nm, and indirectly quantify the amount of NO produced (Beda et al. , 2005).

Raw 264.7 cells were inoculated in 100 μL at 1 × 10 ⁴ cells / mL in 96 well micro-plate and incubated for 24 hours in a CO ₂ incubator at 37 ℃. After replacing the culture with Modified Eagle MEdion (MEM) containing 10% FBS, the prepared sample was pretreated for 1 hour, treated with 1 μg / mL (1 ppm) of LPS to induce NO production, and then 48 hours. Were incubated in a CO ₂ incubator at 37 ° C. Absorbance was measured at 570 nm by reacting 50 μL of Griess reagent (0.1% N-1-naphtylethylenediamine: 1% sulfanilamide = 1: 1) with 50 μL of NO containing medium produced from LPS-stimulated Raw 264.7 cells. . The absorbance of each concentration of NO was obtained by preparing a standard curve using the absorbance measured using sodium nitrate, and the amount of NO produced was quantified by applying the standard curve to the experimental results.

NO production inhibitory effect

L-arginine, a substrate of NO, is converted to L-cythroline and NO, which rapidly turn into stabilized nitrogen dioxide, nitrites, and nitrates. Since the Griess reagent chemically reacts with nitrite to form a purple azo salt, which matches the concentration of NO, the absorbance of the nitrite was measured at 540 nm from the concentration of the azo salt. All samples were measured at concentrations of 100, 50, 10, and 1 μg / mL, respectively. The control group was treated with 1 μg / mL LPS without adding sample, and the blanks without both sample and LPS were used. As a result of measuring the production of NO, as compared with the control stimulated with LPS, as shown in FIG. 10, it was confirmed that the NO production decreases in a concentration-dependent manner in the n- BuOH fraction having a high polyphenol content. In particular, at the concentration of 100 μg / mL, the results were excellent, showing almost the same value as the blank not stimulated with LPS. And 85% aq. NO concentrations were reduced in a concentration-dependent manner even at concentrations below 50 μg / mL of the MeOH fraction. The inhibitory effects of NO production on crude extracts were probably due to cytotoxicity, and the n -Hexane and H ₂ O fractions did not effectively inhibit NO production.

Reverse transcriptase - 중합체 chain reaction ( RT - PCR )

Expression of iNOS mRNA was confirmed via RT-PCR.

Total RNA from Raw 264.7 cells was isolated using Trizol (Invitrogen Co., CA, USA), quantified RNA isolated, oligo dT primer (GIBCO, USA) and

CDNA was synthesized from 2 μg of RNA using SuperScript ™ II reverse transcriptase (Invitrogen). Using the synthesized cDNA as a template, the iNOS gene was amplified by the polymerase chain reaction (PCR) method. The results are shown in Table 4 below.

Housekeeping gene GAPDH was used as interanl coltrol. Each PCR product was electrophoresed using 1% agarose gel, stained with ethidium bromide (EtBr, Sigma), and confirmed by UV.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

In order to confirm the effect of Ecklonia cava extract on proinflammatory factors in Raw 264.7 cells, crude extracts and four fractions were treated with 100 μg / mL to confirm iNOS gene expression. GAPDH was used as a housekeeping gene, and the expression level thereof was measured in density and expressed as% of iNOS / GAPDH. As shown in FIG. 11, it was confirmed that iNOS mRNA expression was significantly reduced in the n -BuOH fraction, which was shown to be the same as the inhibitory effect seen in the measurement of NO content. Crude extract and 85% aq. In the MeOH fraction, iNOS gene expression was suppressed.

Example 7. HT-1080 Invasion Inhibitory Effect

Sample Preparation

Gelatin zymography was performed to detect the enzymatic activity of MMP-2 and MMP-9 of active substance 2-6 secondary metabolites extracted from E. coli n-butanol in detail (Herron et al., 1986). HT-1080 cells were dispensed in 500 μL at 1 × 10 ⁷ cells / mL in a 24 well plate and incubated in a CO ₂ incubator at 37 ° C. for 24 hours. After exchange with DMEM medium without FBS and penicillin, the sample was treated with 200, 100, 50, 10, 1 μM concentration in each well, and after 1 hour, 10 ng / mL of PMA was treated, followed by CO ₂ at 37 ° C. The reaction was carried out for 72 hours in an incubator. As a control, a control without treatment and a blank without treatment with both samples and PMA were used. After transferring the medium to the e-tube, the supernatant obtained by centrifugation at 4 ° C. and 8000 rpm for 5 minutes was transferred to a new e-tube. The samples were treated by concentration, lyophilized, concentrated and zymography was performed, and pellets were used for RT-PCR.

MTT assay effect of crude extract and solvent fraction on HT-1080 cells

As shown in FIG. 12, the cell viability measurement for HT-1080 cells showed similar absorbance to the control at each concentration of all samples, indicating that it did not affect HT-1080 cell viability, n -hexane, n- The BuOH, H ₂ O fraction showed a high value of 80% or more at the concentration of 200 μg / mL, which hardly affected the cell viability. Gelatin zymography and expression of MMP-2 and MMP-9 were confirmed in HT-1080 cells based on the measurement results of cell viability obtained by measuring MTT assay.

Gelatin zymography method

Samples dissolved in 10 μl SDS-PAGE (sodium dodecyl sulfate; SDS, polyacrylamide gel; PAGE) with 25 μL H ₂ O per lane were mixed with 4X loading buffer and electrophoresed. After electrophoresis, the gel was washed twice with renaturing buffer (2.5% Triton X-100) twice for 30 minutes and then developed with developing buffer (50 mM Tris base, 200 mM NaCl, 5 mM CaCl ₂ , 0.01% NaN ₃ ). After incubation for 30 minutes, substrate degradation was induced by incubation with fresh developing buffer for 48 hours. After the reaction was immersed in staining buffer (2.5% Comassive blue, 45% methanol, 10% acetic acid) and salted for 20 minutes and destained with destaining buffer (methanol 25%, acetic acid 10%). The activity of MMP-2 and MMP-9 was observed through the size of the band where gelatin became transparent.

Gelatin zymography

To detect the effect of Ecklonia cava fraction on tumor cell invasion and metastasis, 85% aq. MeMP, n -BuOH, and H ₂ O fractions were treated at concentrations of 200, 100, 50, 10, and 1 μg / mL, respectively. As shown in FIG. 13, the n- BuOH fraction was found to decrease MMP activity in a concentration dependent manner. 85% aq. Although the MeOH fraction showed activity in a concentration dependent manner, it was not significant when compared to n -BuOH. In the case of H ₂ O fraction, there was no change in MMP activity with concentration.

RT-PCR of MMP-2 and MMP-9

RT-PCR confirmed that the secondary metabolite of Ecklonia cava inhibited the expression of MMP-2 and MMP-9 mRNA.

72 hours after PMA treatment, total RNA from HT-1080 cells was isolated using Trizol (Invitrogen Co., CA, USA), followed by quantification of isolated RNA, followed by oligo dT primer and SuperScript ™ II reverse transcriptase. CDNA was synthesized from 2 μg of RNA. Using the synthesized cDNA as a template, the MMP-2 and MMP-9 genes were amplified by PCR method and the results are shown in Table 5 below.

Housekeeping gene GAPDH was used as interanl coltrol. Each PCR product was electrophoresed with 1% agarose gel, stained with EtBr, and confirmed by UV.

MMP -2 and MMP -9 RT - PCR

To investigate the effects of Ecklonia cava extract on HT-1080 cell invasion factor, crude extracts and four solvent fractions were treated at concentrations of 50, 10, and 1 μg / mL to confirm the expression of MMP-2 and MMP-9 genes. It was. GAPDH was used as a housekeeping gene, and the expression level thereof was measured by density and expressed as% of MMP-2, 9 / GAPDH. As shown in Figure 14 and Figure 15, the crude extract and 85% aq. The expression of MMP-2 and MMP-9 was significantly reduced at 50 μg / mL concentration of MeOH fraction and n -BuOH fraction, especially at 50 μg / mL concentration of MMP-9 at n -BuOH fraction. It was confirmed that the decrease by more than%. As a result of the above experiments, it can be seen that the n-butanol extract is sufficiently effective as a cancer metastasis inhibitor.

Example 8 . Ecstatic n- Butanol Active extract isolated from extract ( compounds 2-6 activity

HT -1080 cell For compounds 2-6 MTT assay effect

The cell viability of five compounds isolated from n -BuOH fraction was measured for HT-1080 cells. As measured in concentrations of 200, 100, 50, 10, and 1 μM, as shown in FIG. 16, compounds 2 and 5 did not affect cell viability at concentrations of 100 μM or less, and compound 3 was 80. Survival rate of more than% did not significantly affect cell viability. Compound 4 showed 69% cell viability at the concentration of 100 μM and over 80% at the concentrations of 50, 10, and 1 μM. Compound 6 showed 72% cell viability at the concentration of 100 μM and more than 90% cell viability at the concentrations of 50, 10, and 1 μM. Overall, the concentration below 100 μM did not affect viability of HT-1080 cells.

Gelatin zymography experiments were performed based on the results obtained through the MTT assay.

Gelatin zymography

To investigate the effects of compound 2-6 on tumor cell invasion and metastasis, HT-1080 cells were treated with compound 2-6 at concentrations of 200, 100, 50, 10, and 1 μM, respectively. Post MMP activity was examined by gelatin zymography. As shown in FIG. 17, all compounds were found to inhibit the activity of MMP-2 and 9 in a concentration-dependent manner. In particular, compound 5 was found to inhibit MMP-2 activity to a high level at a concentration of 100 μM which was found to be non-cytotoxic.

MMP -2 and MMP -9 RT - PCR

To determine the effect of Compound 2-6 on HT-1080 cell invasion factor, five compounds were treated at concentrations of 100, 50, 10, and 1 μM, respectively, to confirm the expression of MMP-2 and MMP-9 genes. GAPDH was used as a housekeeping gene, and the expression level thereof was measured by density and expressed as% of MMP-2, 9 / GAPDH. As shown in FIG. 18, the activity of cell infiltration inhibition was observed in all compounds. In particular, compound 3, was both MMP-2 and MMP-9 eseo 6 confirmed to be suppressed at a high level, while the case of compound 4 and compound 5 are respectively inhibitory effect on MMP- 9 and MMP- 2 is indicated compound 4 In MMP-2 showed no inhibitory effect on MMP-9 in compound 5 .

Summary and conclusion

As described above, Ecklonia cava , a brown algae, has recently been known to contain a large amount of seaweed-derived polyphenols, which are rapidly emerging as antioxidants, but it was further subdivided that n-butanol extract was most effective. Based on the NMR data analysis, the active compounds 2-6 could be separated by preferentially separating compounds for the n -BuOH fraction, and it was confirmed that the inhibitory effect of HT-1080 cell infiltration was shown in all. In particular, although compound 2 has been reported to have an excellent inhibitory effect on HT-1080 cell infiltration, the newly tested compounds 3 and 6 exhibit similar or higher inhibitory effects on MMP-2 and MMP-9. It was confirmed. It is expected to be developed as an excellent anticancer material through more anticancer experiments and clinical trials including MMP-2 and MMP-9 as well as antioxidants and superior anti-inflammatory agents that directly remove active oxygen species.

Claims

Brown algae Ecklonia cava (Ecklonia cava) after drying, crushed to produce a powder, crude extract extracted from methanol, the second n-butanol extract of brown algae Ecklonia characterized in that the extract extracted again from n-butanol.

The method of claim 1,
The n-butanol extract,
The Ecklonia cava powder is extracted by heating a methanol (MOH) solvent to a boiling point, and the obtained extract is dissolved in methanol (MeOH) at room temperature, and then concentrated to remove the desalted crude extract sequentially according to the polarity of the solvent. N -hexane ( n -hexane), 85% aqueous methanol (85% aq. MeOH), n-butanol ( n -BuOH), water (H ₂ O) fraction obtained in the n-butanol extract, characterized in that N-butanol extract of brown algae.

3. The method according to claim 1 or 2,
The n-butanol extract is n-butanol extract of brown algae Eckloniasis, characterized in that it has an antioxidant activity having a radical scavenging activity and hydrogen peroxide scavenging activity.

3. The method according to claim 1 or 2,
The n-butanol extract is n-butanol extract of brown alga Eckloniasis, characterized in that it has anti-inflammatory activity having NO production inhibitory activity.

3. The method according to claim 1 or 2,
The n-butanol extract is n = butanol extract of brown algae sensation, characterized in that it has a cancer metastasis inhibiting ability to inhibit the expression of MMP-2 and MMP-9 at the cellular level.

The n-butanol extract of brown algae was divided into detailed fractions using silica column chromatography, each fraction was dissolved in an appropriate concentration of MeOH in CHCl ₃ , and the active extract was separated through sephadex LH20 column chromatography. N-butanol extract of brown algae cherries, characterized by

The method according to claim 6,
The isolated active extract is n-butanol extract of brown algae Eckloniasis, characterized in that triloresol-A (Triphlorethol-A).

The method according to claim 6,
The isolated active extract is n-butanol extract of brown algae sensation, characterized in that Eckol (Eckol).

The method according to claim 6,
The isolated active extract is n-butanol extract of brown algae sensation, characterized in that it is Extoronol (Eckstolonol).

The method according to claim 6,
The isolated active extract is n-butanol extract of brown algae Eckloniasis, characterized in that the phlorofucoloekol (Phlorofuroeckol).

The method according to claim 6,
The isolated active extract is n-butanol extract of brown algae sensation, characterized in that Dieckol (Dieckol).

The method according to any one of claims 6 to 11,
Each of the active extracts, n-butanol extract of brown algae Ecklonia cava was divided into detailed fractions by silica column chromatography, and 10% MeOH in CHCl ₃ fractions in each fraction were separated by sephadex LH20 column chromatography. Separate the ckstolonol,
The remaining fractions were combined with 20% MeOH in CHCl ₃ and 30% MeOH in CHCl ₃ subfractions, followed by silica column chromatography. The fractions were separated into seven fractions, and 15% MeOH in CHCl ₃ subfraction was sephadex LH20 column chromatography. Separate the Triphlorethol-A,
Sephadex LH20 column chromatography was also performed on 20% MeOH in CHCl ₃ fractions to further separate triphlorethol-A and ectolonol, and dieckol was newly separated.
For 25% MeOH in CHCl ₃ subfraction, sephadex LH20 column chromatography and reverse-phased HPLC were performed sequentially to further separate trichlorlorethol-A, Eckol and Phloofuroeckol. N-butanol extract of brown algae sieving.

13. The method of claim 12,
Each of the active extract is n-butanol extract of brown algae sensation, characterized in that it has a cancer metastasis inhibiting ability to inhibit the expression of MMP-2 and MMP-9 at the cellular level.