Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Antioxidant and Antiproliferative Activities of the Halophyte Angelica japonica Growing in Korean Coastal Area

한국 연안지역에 서식하는 갯강활의 항산화 및 암세포증식 억제 활성

  • Jayapala, Priyanga S. (Division of Convergence on Marine Science, Korea Maritime & Ocean University) ;
  • Oh, Jung Hwan (Nutritional Education, Graduate School of Education, Silla University) ;
  • Kong, Chang-Suk (Marine Biotechnology Center for Pharmaceuticals and Foods, College of Medical and Life Sciences, Silla University) ;
  • Sim, Hyun-Bo (Incheon Academy of Science and Arts) ;
  • Seo, Youngwan (Division of Convergence on Marine Science, Korea Maritime & Ocean University)
  • 사지와니 자야팔라 (한국해양대학교 해양과학융합학부) ;
  • 오정환 (신라대학교 교육대학원 영양교육전공) ;
  • 공창숙 (신라대학교 해양식의약소재융합기술연구소) ;
  • 심현보 (인천과학예술영재학교) ;
  • 서영완 (한국해양대학교 해양과학융합학부)
  • Received : 2022.07.18
  • Accepted : 2022.08.15
  • Published : 2022.10.30
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Abstract

This study evaluated the antioxidizing and antiproliferative effects of Angelica japonica extract and its solvent-partitioned fractions. A dried sample of the halophyte A. japonica was extracted twice using methylene chloride (CH2Cl2) and extracted twice again using methanol (MeOH). The combined crude extracts were then fractionated by solvent polarity into distilled water (water), n-butanol (n-BuOH), 85% aqueous methanol (85% aq.MeOH), and n-hexane fractions. The antioxidant activities of the crude extracts and their solvent-partitioned fractions were assessed according to their DPPH radical and peroxynitrite scavenging abilities, formation of intracellular reactive oxygen species (ROS), DNA oxidation, NO production, and ferric reducing antioxidant power (FRAP). The crude extract showed significant antioxidant activity in the overall antioxidizing bioassay systems. Among solvent-partitioned fractions, good antioxidant activities were observed in n-BuOH and 85% aq.MeOH fractions and significantly correlated with the polyphenol and flavonoid contents of the samples. Furthermore, all samples tested, including the crude extract, not only showed cytotoxic effects against human cancer cells (AGS, HT-29, MCF-7, and HT-1080) but also prevented cell migration in a dose-dependent manner in the wound healing assay using HT 1080. Among the solvent-partitioned fractions, the 85% aq.MeOH fraction most effectively inhibited the invasion of HT-1080 cells. Therefore, these results suggest that A. japonica may be a potential antioxidizing and antiproliferative agent.

본 연구는 갯강활 추출물과 그 용매분획물의 항산화 및 암세포증식 억제효과를 평가하고자 하였다. 갯강활의 건조 시료를 차례대로 메틸렌클로라이드(CH2Cl2)로 2회, 그리고 메탄올(MeOH)로 2회 추출한 다음, 그 조추출물을 합한 후에 다시 용매극성에 따라 n-hexane, 85% 메탄올 수용액(85% aq.MeOH), n-buta- nol(n-BuOH) 및 물 분획층으로 분획하였다. 합해진 조추출물과 용매분획물의 항산화 활성은 DPPH 라디칼과 peroxynitrite 소거능, 세포내 활성산소종(ROS) 생성, DNA 산화, NO 생성, 철이온 환원력(FRAP)에 의해 평가되었다. 조추출물은 모든 항산화활성검색 시스템에서 유의적인 항산화 활성을 보였다. 용매분획들 중에서는 n-BuOH 및 85% aq.MeOH 분획에서 우수한 항산화 활성이 관찰되었으며 이 활성은 시료의 폴리페놀 및 플라보노이드 함량과 유의적인 상관관계가 있었다. 이 뿐만 아니라 조추출물을 포함한 모든 시료들이 인간 암세포(AGS, HT-29, MCF-7, HT-1080)에 대한 세포 독성 효과를 보였으며, HT 1080을 이용한 wound healing assay에서도 농도 의존적으로 세포이동을 억제하였다. 시료 중에는 85% aq.MeOH 용매분획이 HT-1080 세포의 침입을 가장 효과적으로 억제하였다. 그러므로 본 연구결과는 갯강활을 이용하여 항산화제 및 항암제 개발을 위한 기초자료로 활용될 수 있을 것이다.

Keywords

Introduction

The halophytes are native plants growing in habitats with high salinity (salt marsh, coastal sand dunes, and mud flats) [15]. When compared with lycophytes, they are stress-tolerant plants well adapted to a variety of hostile environmental stresses such as constant salinity changes, high temperatures, drought, direct exposure to UV radiation, and waterlogging [7,18,19,21].

Halophytes inhabiting these extreme environments have to deal with frequent change of salinity level which causes the plant to produce and accumulate reactive oxygen species (ROS), eventually leading to cellular damage and biometabolic dysfunction.

In response to such as oxidative damage, the halophytes have developed several defense mechanisms, including synthesis of various bioactive molecules such as antioxidants [9,16,24].

In addition, halophytes readily absorb toxins and heavy metals [5]. As a result, it is highly likely that halophytes will contain secondary metabolites to adapt to these environments [9,16,18]. For many years, halophytes have been widely used in folk medicines in many countries [15].

More than 60 species of the genus Angelica (family Apiaceae) have been used medically to lessen inflammation and to treat arthritis, as well as to heal influenza, hepatitis, bronchitis, and other chronic ailments [27].

The halophyte Angelica japonica is a medicinal perennial herb, which prefers sunny and moist soil and can survive seawater exposure. It has been reported to possess many bioactive compounds such as chromones, coumarins, and polyacetylenes. However, for its biological activities, there have been no reports except for antiproliferative activity [1,5,6,12]. Therefore, in the present study, antioxidant and antiproliferative capacity of A. japonica extract and its solvent-partitioned fractions was evaluated using several kinds of assay systems with different mechanisms or different cancer cell lines, respectively.

Materials and Methods

Chemicals and reagents

The human cancer cell lines were purchased from Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified eagle’s medium (DMEM), Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI-1640) medium, and antibiotic penicillin-streptomycin were purchased from Gibco/BRL (Burlington, ON, Canada). 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), 2’-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5’-bi-1H-benzimidazole trihydrochloridetrihydrate, and dimethyl sulfoxide (DMSO) were produced from Sigma-Aldrich (St. Louis, MO, USA). All chemicals and reagents used in these experiments were analytical grade.

Collection, extraction and fractionation of samples

The samples of the halophyte A. japonicum were collected in coastal area of Jeju Island of South Korea. They were identified by Dr. Hyun-Bo Sim (Incheon Academy of Science and Arts, Republic of Korea). The plant samples were dried in the shade at a room temperature and then successively extracted twice with CH2Cl2 and twice with MeOH, in turn. The combined crude extracts were concentrated under vacuum to obtain a residue, which was partitioned between CH2Cl2 and water. The aqueous layer was further fractionated into n-BuOH and Water. The organic layer was fractionated into 85% aq.MeOH and n-hexane, resulting in solvent-partitioned fractions of n-hexane, 85% aq.MeOH, n-BuOH and water. Samples of the crude extract and solvent fractions were dissolved in 10% DMSO for use in experiment and kept at -20℃.

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay

The scavenging activity of samples on DPPH radicals was measured following the procedure reported by Blois with some modification [2]. A solution of each sample (100 μl) was added to 100 μl of 0.4 mM DPPH in a 96-well plate. The absorbance was read at 517 nm by spectrophotometer (BioTek Instruments, USA) after leaving it in the dark for 5 minute with gently shaking it at room temperature.

Peroxynitrite (PON) scavenging assay

The PON-induced oxidation of DHR 123 was examined as described by Kooy et al [14]. A buffer solution containing 90 mM sodium chloride, 50 mM sodium phosphate (pH 7.4) and 5 mM potassium chloride was prepared and placed on ice. A stock solution of 5 mM DHR 123 in DMF stored at -80℃ was diluted to 5 μM with the prepared buffer solution on ice in the dark immediately before use. Authentic ONOO- (200 µM) or SIN-1 (200 µM) in 0.3 M sodium hydroxide is added to 5 mM DHR 123 (1 µM) in DMF until a final concentration of 10 µM. The degree of oxidation of DHR 123 was expressed as an absorbance measured at excitation 485 nm and emission 530 nm with the fluorescence microplate reader (BioTek Instruments, USA) at room temperature. The final fluorescence and background intensities were measured after 5 min after treatment with authentic ONOO-1 and 1 hr after treatment with SIN-1. Penicillamine and L-ascorbic acid were compared as positive standards.

Ferric reducing antioxidant potential (FRAP) assay

The ferric reduction antioxidant potential (FRAP) assay is an electron transfer-based method that reduces ferric ion (Fe3+)-ligand complexe to dark blue ferrous (Fe2+) complex by antioxidants under an acidic condition. A solution of 0.2 ml of ferricyanide and 0.2 ml of 1% sodium phosphate (pH 6.6) was added to 0.2 ml of sample and then the mixture was reacted at 50℃ for 20 minutes. After addition of 10% trichloroacetic acid (0.2 ml), the mixture was centrifuged for 10 min at a speed of 10,000 rpm. A volume of the supernatant (0.5 ml) was reacted with 0.1% ferric chloride (0.5 ml), and the reducing ability was measured using a spectrophotometer at wavelength of 700 nm. The reducing power was compared with the vitamin C [23].

Cell culture

HT-1080, HT-29 and AGS cells were cultured in RPMI-1640 medium using T-75 tissue culture flask (Nunc, Roskilde, Denmark). RAW 264.7 and MCF-7 cells were cultured in DMEM medium using the same flask. Both culture media were supplemented with 10% FBS and 1% antibiotic (100 μg/ml of penicillin-streptomycin). The cells were cultured under atmosphere of 5% CO2 at 37℃ and washed with 1× PBS buffer 3-4 times before the medium replaced. After dissociating the adherent cells using 0.05% Trypsin-0.02% EDTA solution, they were subcultured. The attached raw cells were detached by using scratches.

Determination of cell viability using MTT assay

RAW 264.7 macrophages were grown into monolayer in T-75 cell culture flask having Dulbecco's Modified Eagle's Medium under a humidified atmosphere with 5% CO2 at 3 7℃. The medium was replaced 2 or 3 times per week, and after treatment of samples for the cultured cells, cell viability was calculated using MTT reduced to MTT-formazan by mitochondrial dehydrogenase [8].

Cells were raised to a density of 5×103 cells/well in 96-well plates and washed with fresh medium after 24 hr and 90 µl medium was added thereto. It was then treated with 10 µl of samples of different concentrations. After incubation for 24 hr, cells were washed again followed by addition of 100 μl of MTT solution (1 mg/ml) and incubated for another 4 hr. Finally, after removal of the medium, DMSO (100 μl) was added thereto to dissolve the formed formazan crystals. The amount of formazan formed was determined by absorbance measured at 540 nm with Synergy HT microplate reader (Bio-Tek Instruments Inc., Wicooski, VT. USA). The 1 X PBS buffer was added to the control medium instead of the sample. Cell viability was recorded by the amount of MTT reduced to formazan and depicted as a percentage relative to absorbance of the control.

Determination of intracellular formation of ROS using DCFH-DA labeling

The intracellular formation of reactive oxygen species (ROS) in RAW 264.7 cells was assessed using the method described by Okimotoa et al with some amendment [22]. RAW 264.7 cells cultured in 96-well plates were reacted with 20 μM DCFH-DA (2',7'-dichlorofluorescein diacetate) in HBSS (Hank's Balanced Salt Solution) in the dark for 30 minutes. After treatment with the sample and further incubation for 1 hr, cells were washed twice with PBS and 500 μM H2O2 in HBSS was added to cells. The effects of the samples on the cells were assessed by comparing them with fluorescence intensity of control and blank. Fluorescence intensity of DCF formed due to oxidation of DCFH-DA was measured in cells after 5, 30, 60, 90 and 120 minutes at an excitation 485 nm and emission 528 nm using a multi-label plate reader (BioTek Instruments, USA).

Genomic DNA isolation and determination of DNAdamage by radical

Genomic DNA was obtained from HT-1080 cells by using Genomic DNA Extraction Kit (Bioneer Inc., Alameda, CA, USA). To 4 µl of genomic DNA (0.5-1.0 µg) were added 4 µl of the sample (prepared for each concentration), 14 µl of each of 600 µM FeSO4 and 0.5 mM H2O2. The mixture was oxidized at room temperature for 30 minutes, and then 130 mM EDTA was added to stop the reaction. It was electrophoresed for 30 minutes at 100 V using agarose gel. Gels were colored with ethidium bromide (1 mg/ml) and then observed with UV light based on Quantity-One image analysis software (Bio-Rad Co., Hercules, California, USA) [20].

Determination of NO production in RAW 264.7 macrophage cells

To determine effects of samples on nitric oxide (NO) production in the cultured media, RAW 264.7 cells were seeded at a density of 2×10 cells/well in 96-well plates using DMEM containing no phenol red, treated with samples for 1 hr and left to attach overnight for 1 hr. Cells were stimulated by addition of LPS 1 μg/ml (final concentration) followed by further incubation for 48 hr. After that, the amount of NO production was confirmed by the Griess reaction. Each of the 50 µl of cultured medium was added to a 96-well microtiter plate. To each well, Griess reagent (50 μl) was added and the mixture stands for 15 minutes at room temperature. The absorbance of the mixture was recorded at 550 nm by a Synergy HT microplate reader (Bio-Tek instrument Inc., Wicooski, VT, USA). The amounts of nitrite were obtained from regression analysis with serial dilutions of sodium nitrite as a standard. All of the samples were prepared at concentrations of 10, 50, 100 and 200 μg/ml respectively before being used in this bioassay test. The blank, which was not treated with the sample and LPS, and the control group treated only with LPS were used for comparison with those treated with the samples [4].

Determination of total polyphenol content

The total polyphenol content of the halophyte extract and its solvent fractions was determined using Folin-Ciocalteu method [10]. Briefly, to 500 µl of Folin-Ciocalteu reagent (Sigma chemical Co., St. Louis, MO, USA) was added 50 µl of each sample and the mixture was then allowed to react in the dark for 3 minutes at room temperature. To this reaction mixture was added 500 µl of 10% Na2CO3 and then it was left in a dark place for 60 minutes at room temperature. All samples were measured in triplicate and their absorbances were recorded with a microplate reader (BioTek Instruments, USA) at 765 nm. The polyphenol content was described as equivalent of tannic acid per gram of dry weight calculated from the calibration curve of tannic acid, a standard substance.

Determination of total flavonoid content

Content of total flavonoid was measured following the procedure of Lee et al [17]. A volume (0.4 ml) of the crude extract in 10% aqueous DMSO (1 mg/ml) was diluted with 4 ml of 90% aqueous diethylene glycol followed by addition of 1 N NaOH (40 ml). After shaking the mixture sufficiently, it was left at room temperature for 1 hr. The absorbance of the solution was recorded at 420 nm with a microplate reader. The total flavonoid content was determined in the same way as the total polyphenol content, and rutin was used as a standard.

Inhibition of cancer cell proliferation

The cells were grown as a monolayer in a culture flask with 5% CO2, under a humidified atmosphere of 37℃. The antiproliferative effects of crude extract and solvent fractions on cultured human cancer cells by their cytotoxicities were determined using MTT. The number of cancer cells was adjusted to 5×1010 cells/well in 96-well plates by incubation for 24 hr. After cells were treated with different concentrations of the sample, they were incubated for 24 hr, then 100 μl of MTT solution (1 mg/ml) was added and it was incubated for another 4 hours. Finally, formazan crystals formed were dissolved by addition of DMSO (100 μl) after removal of the medium, and the absorbance of the formazan was recorded at 540 nm by a synergistic HT microplate reader (Bio-Tek Instruments Inc., Wicooski, VT, USA).To the control group, 1X PBS was added instead of the sample. Cell viability was presented as a percentage relative to the control.

Observation of cell migration by the wound-healing assay

HT-1080 cells were placed in a 12 well culture plate for 24 hr with 5% CO2 and 37℃. The density of cell per well was 80-90%. An injury line was made with a width of 2 mm by scratching vertically center of each well with a sterile plastic tip. After removing the floating cells by washing with 1 X PBS buffer, cell medium was exchanged with fresh serum-free medium. Each well was treated with samples of 100, 50, and 10 µg/ml, respectively. Migration of cell was observed through an inverted microscope. Photographs were taken just after sample treatment and 24 hr later, respectively.

Statistical analysis

The data were presented as average of three different experiments ± SD (n=3). Analysis of the statistically significant difference between two groups was done by the analysis of variance (ANOVA) of SAS (9.1) software (SAS Institute, Cary, NC, USA) followed by Duncan's multiple range tests. Significant differences were defined at the p<0.05 level.

Results

Scavenging activity on DPPH radical

The plant extract and its solvent-partitioned fractions dose-dependently scavenged DPPH radical as depicted in Fig. 1 (p crude extract > 85% aq. MeOH > n-hexane ≒ water at 200 µg/ml. The scavenging abilities of the crude extract and n-BuOH fraction for DPPH radical were higher than those of other solvent fractions. However, they were not high and did not exhibit any significant effect when compared to the controls, vitamin C, BHA, and BHT.

SMGHBM_2022_v32n10_749_f0001.png 이미지

Fig. 1. DPPH radical scavenging effect of A. japonica crude extract and its solvent fractions. 85% aq.MeOH: 85% aqueous methanol, n-BuOH: n-butanol, BHA: butylated hydroxyanisole. a-eMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

Peroxynitrite scavenging activity

The scavenging effect of the plant crude extract and its solvent-partitioned fractions on authentic peroxynitrite is exhibited in Fig. 2. In this experiment, penicillamine and vitamin C were used as positive controls. Crude extract and n-BuOH fraction revealed higher scavenging effect compared with other solvent fractions, even at 50 µg/ml concentration. Among all solvent fractions, the n-BuOH fraction showed the highest scavenging effect. However, the effect was almost close to that of the crude extract (86.9% for n-BuOH and 87.2% for crude extract at 200 µg/ml).

SMGHBM_2022_v32n10_749_f0002.png 이미지

Fig. 2. Scavenging effects of crude extract and its solvent fractions of A. japonica on authentic ONOO- (% of control). a-gMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

The scavenging activity on peroxynitrite derived from degradation of SIN-1 was also estimated. As a result, significant scavenging rates were shown in the crude extract, n-BuOH, 85% aq.MeOH, and Water fractions (Fig. 3).

SMGHBM_2022_v32n10_749_f0003.png 이미지

Fig. 3. Scavenging effects of crude extract and its solvent fractions of A. japonica on SIN-1 (% of control). a-gMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

The strongest scavenging rate was observed in the 85% aq.MeOH fraction of all solvent fractions, but the scavenging rate of 85% aq.MeOH fraction was a little lower than that of the crude extract (86.1% for 85% aq.MeOH and 89.8% for crude extract at 200 µg/ml).

The measurement of ferric reducing antioxidant potential (FRAP)

The principle for FRAP measurement is that iron tripyridyltriazine (Fe3+-TPTZ) complex is reduced to blue tripyridyltriazine iron (Fe2+-TPTZ) by a reducing agent at low pH. Iron(II) sulfate (FeSO4) was used as a standard substance for FRAP measurement. All samples tested, containing crude extract showed the significant ferric ion-reducing antioxidant power, compared with vitamin C at 200 µg/ml as shown in Fig. 4 (96.3%, 91.4%, 90.5%, 85.9%, and 70.5% for crude extract, 85% aq.MeOH, n-BuOH, n-hexane, and Water, respectively).

SMGHBM_2022_v32n10_749_f0004.png 이미지

Fig. 4. Ferric reducing antioxidant power of crude extract and its solvent fractions of A. japonica (% of control). a-eMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

Measurement of intracellular ROS using DCFH-DA

The amount of ROS in the cells was calculated by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). When DCFH-DA passes through the cell membrane, it is hydrolyzed by the intracellular enzyme esterase to non-fluorescent DCFH which is captured inside cells. The DCFH is oxidized to highly fluorescent DCF by intracellular ROS such as hydroxyl radical generated in the cells. Samples of different concentrations were loaded into cells incubated for 1 hr. Fluorescence levels were measured every 30 min over 120 min. The absorbance of the cells to which the sample was added was compared with the control group treated with only hydrogen peroxide and the blank untreated with both samples and hydrogen peroxide, respectively. In the control group, fluorescence value of the DCF increased continuously but in the blank, it hardly changed over time. The influences of crude extract and solvent fractions on RAW 264.7 cell viability were investigated by MTT assay to determine non-toxic doses for intracellular experiments prior to measuring intracellular ROS levels.

No significant cytotoxicities (cell viability of about 80% or more) were observed in RAW 264.7 cell up to 200 μg/ml concentration for all samples (Fig. 5). Based on these results, the scavenging effects of the samples on intracellular ROS generated by hydrogen peroxide at concentrations of 200 μg/ ml or less were investigated. As shown in Fig. 6, clearance rates of crude extract and solvent fractions on ROS were elevated in a dose-dependent mode when compared to both control and blank. The intensity of intracellular ROS scavenging activity was as follows at 200 and 100 μg/ml concentrations: Water< n-hexane< crude extract<85% aq.MeOH

SMGHBM_2022_v32n10_749_f0005.png 이미지

Fig. 5. Effects of crude extract and its solvent fractions of A. japonica on cell viability of RAW 264.7 cells (% of control). a,bMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

SMGHBM_2022_v32n10_749_f0006.png 이미지

Fig. 6. Scavenging effects of crude extract and solvent fractions of A. japonica on intracellular ROS induced by hydrogen peroxide in RAW 264.7 Cell.

Genomic DNA oxidative damage

The inhibitory ability of crude extract and solvent fractions of A. japonica on genomic DNA oxidation was measured using DNA electrophoresis at the concentration of 100 μg/ml. The results showed crude extract, n-BuOH fraction, and 85% aq.MeOH fraction protected significantly the radical-mediated DNA damage, when compared to the control group. Among them, n-BuOH fraction showed the strongest protective effect almost similar to the blank (Fig. 7).

SMGHBM_2022_v32n10_749_f0007.png 이미지

Fig. 7. Protective effect of crude extract and its solvent fractions from A. japonica on oxidative damage of genomic DNA in HT 1080 cells. a-dMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

Inhibitory effect against nitric oxide (NO) production

Nitric oxide is known to produce peroxynitrite (ONOO-), a latent oxidizing agent, by the chemical reaction of NO with superoxide. Various negative effects of ONOO- such as oxidation and nitration of proteins have been described. Therefore, suppressing NO production is a crucial step in control of NO-mediated diseases [3,23-25,28]. The suppressive effect of NO production by crude extract and solvent fractions was observed in LPS-stimulated RAW 264.7 cells.

However, the crude extract of A. japonica significantly reduced the production of NO, but none of the solvent fractions did not reduce the production of NO more strongly than the crude extract. Although each solvent fraction does not significantly inhibit NO production, it is assumed that this result can be obtained due to their synergistic effect when each chemical component contained therein is combined together as a crude extract. The crude extract inhibited NO production by 10.1, 16.8, 35.7 and 43.8%, respectively, when compared to the control at 10, 50, 100, and 200 µg/ml concentrations (Fig. 8).

SMGHBM_2022_v32n10_749_f0008.png 이미지

Fig. 8. Effect of crude extract and its solvent fractions of A. japonica on NO production in LPS induced RAW 264.7 cells (% of control). a-eMeans with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

Determination of total polyphenol and flavonoid contents

Plant extracts containing flavonoids and polyphenols are well-known for their potent antioxidizing capacity [13,26,27]. Therefore, polyphenol and flavonoid contents of the samples were analyzed to confirm the antioxidant component. Contents of phenols and flavonoids were increased in order of n-BuOH > 85% aq.MeOH > crude extract > n-hexane > Water. The total polyphenol content was 148.75 and 293.50 mg/g in the 85% aq.MeOH and the n-BuOH fractions, respectively, and the total flavonoid content was 98.00 and 172.50 mg in the 85% aq.MeOH and the n-BuOH fractions, respectively (Table 1). It was observed that the solvent fraction with the high polyphenol contents tends to have the high flavonoid contents.

Table 1. Total polyphenol and flavonoid contents of crude extract and its solvent fractions of A. japonica

SMGHBM_2022_v32n10_749_t0001.png 이미지

a–eMeans with different letters at the same concentration are significantly different (p<0.05) by Duncan’s multiple range test.

Cytotoxic effect against human cancer cells

The antiproliferative activity of crude extracts and solvent fractions was investigated against human cancer cell lines (HT-1080, HT-29, AGS and MCF-7). Concentrations of the crude extract and solvent fraction were used in the range of 10 to 200 g/ml and antiproliferative effect was measured using MTT assay. The crude extract, n-BuOH fraction, and 85% aq.MeOH fraction significantly showed dose-dependent antiproliferative effects against all human cancer cells (p<0.05).

As a result of comparative analysis on the antiproliferative effect, 85% aq.MeOH fraction showed significant inhibition rates against HT-1080, HT-29 and MCF-7 while n-BuOH fraction against AGS and HT-29 (Fig. 9).

SMGHBM_2022_v32n10_749_f0009.png 이미지

Fig. 9. Effects of crude extract and its solvent fractions of A. japonica on viability of HT-1080 (A), AGS (B), MCF-7 (C), and HT-29(D) cells (% of control). Means with the different letters are significantly different (p<0.05) by Duncan’s multiple range test.

Measurement of cell migration ability by the wound healing assay

Metastasis is a characteristic of cancer cells that induces cancer expansion, and the most important factor in metastasis is cancer cell motility. Motility can be observed with a certain directionality as the cell shape changes. Wound-healing assay is an easy and simple method to determine the rate of cell migration as well as interactions. The effects of crude extract and solvent fractions on invasive ability of HT-1080 cells were assessed by cell migration assay. As the microscopic image showed, cell migration was inhibited in a concentration-dependent fashion after each of all samples was added to the cells and then they were incubated for 24 hr, Of all samples tested, 85% aq.MeOH fraction inhibited most effectively invasion of HT-1080 cells (Fig.10)..

SMGHBM_2022_v32n10_749_f0010.png 이미지

Fig. 10. Effect of crude extract and its solvent fractions of A. japonica on cell migration induced HT-1080 cells.

Discussion

As part of our effort on research to find bioactive compounds from marine resources, screening for antioxidant activity of A. japonica crude extract and its solvent fractions was performed. At 200, 100, 50, and 10 μg/ml, antioxidzing power of each sample was measured in six bioassay methods: scavenging power on peroxynitrite, DPPH radical, and intracellular ROS; inhibition of nitric oxide production; DNA oxidation damage; ferric reduction antioxidant power. The scavenging effects of all samples on DPPH radical were not significant compared to those of the controls. However, for authentic peroxynitrite and 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite, all samples except the n-hexane fraction showed the significant scavenging rates.

The scavenging rates of crude extract, 85% aq.MeOH fraction, and n-BuOH fraction were very effective at a concentration of 200 μg/ml. In the case of FRAP, all samples except for Water fraction revealed the reducing power similar to the control vitamin C at 100 μg/ml or more. The crude extract exhibited the highest ferric-reducing power.

Since RAW 264.7 cells survived more than 80% at concentrations below 200 μg/ml, the scavenging effect on intracellular ROS was investigated below this concentration, which is considered not to be toxic. When the interval between control and blank in Fig. 6 was set to 100%, n-BuOH and 85% aq.MeOH fractions revealed scavenging ratios of 80.6% and 70.7%, respectively, after 2 hr at 200 μg/ml. Even at 100 μg/ml concentration, considerablely high removal rate of 69.8% was observed for n-BuOH fraction after 2 hr.

To determine the inhibitory effect of A. japonica samples on genomic DNA oxidation, degree of its oxidation was evaluated using electrophoresis. All samples of A. japonica showed a significant protection effect against oxidation of genomic DNA, and when absorption rate of the blank was set to 100%, n-BuOH fraction exhibited an absorption rate of 97.5%, which means the strongest protection effect among the samples at 100 μg/ml concentration.

In general, at least one of the solvent fractions obtained after solvent fractionation of the crude extract according to solvent polarity tends to show greater antioxidant activity than the crude extract. But in the current study, the inhibition rate of the crude extract was stronger than those of the solvent fractions, when the inhibitory effect on nitric oxide and peroxynitrite, and the reducing effect on ferric ion were measured. Since previous studies have already published similar results, it is believed that several types of chemical constituents contained therein exhibit synergistic antioxidant effect due to their interaction when they are present together in the crude extract [19,30-32].

When the antiproliferative activities against human cancer cells were investigated using the above-mentioned MTT assay, all activities were concentration-dependent, and 85% aq.MeOH and n-BuOH fractions showed good inhibitory activity among the tested samples. In addition, all samples inhibited cell migration in a dose-dependent fashion after 24 hr incubation. Of all the samples tested, it was the 85% aq.MeOH fraction that inhibited the invasion of HT-1080 cells most effectively. According to a literature review, the substance showing antitumor activity have already been reported from A. japonica [11].

To summarize, the 85% aq.MeOH fraction revealed the strongest effect in both most antioxidant and antiproliferative tests and the n-BuOH fraction also exhibited relatively good antioxidant and antiproliferative activities. As already mentioned earlier, antioxidant activity is closely related to polyphenols, and since polyphenol derivatives (coumarin and chromone) have been previously isolated from A. japonica [5], it is presumed that this is due to polyphenols contained in the solvent fraction. However, the n-BuOH fraction with a higher polyphenol content than the 85% aq.MeOH fraction showed weaker antioxidizing effect than the 85% aq.MeOH fraction in most of antioxidant tests except for genomic DNA oxidation.

Therefore, it is estimated that the 85% aq.MeOH fraction contains other components exhibiting antioxidant activity in addition to polyphenols. The present study suggests that A. japonica could be a beneficial material for development of natural antioxidants and antiproliferative agents.

Acknowledgment

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. 2019R1F1A1059325).

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.

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