DNA Damage Protection and Anti-inflammatory Activity of Different Solvent Fractions from Aruncus dioicus var. kamtschaticus
DNA Damage Protection and Anti-inflammatory Activity of Different Solvent Fractions from Aruncus dioicus var. kamtschaticus
Korean Journal of Plant Resources. 2014. Dec, 27(6): 714-719
Copyright © 2014, The Plant Resources Society of Korea
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : November 11, 2014
  • Accepted : December 12, 2014
  • Published : December 31, 2014
Export by style
Cited by
About the Authors
Qin Zhang
Department of Medical Biotechnology, Kangwon National University, Chuncheon 200-701, Korea
Hye-Young Kim
Department of Dental Hygiene, Kangwon National University, Samcheok 245-711, Korea
This study investigated DNA damage protection and anti-inflammatory activity of different solvent fractions from Aruncus dioicus var. kamtschaticus ( A. dioicus ) aerial parts water extract. As for DNA damage protection, distilled water (H 2 O) fraction displayed the most powerful protection for DNA damage at a concentration of 1 ㎎/㎖. As for anti-inflammatory activity, dichloromethane (CH 2 Cl 2 ) fraction exhibited the highest NO inhibition activity, ranging from 61% to 19% (10-40 ㎍/㎖). Furthermore, the levels of pro-inflammatory cytokines mRNA expressions and intracellular reactive oxygen species (ROS) were employed to verify the anti-inflammatory activity of the CH 2 Cl 2 fraction on further researches. It could be concluded that A. dioicus had a significantly effect of DNA damage protection and anti-inflammatory activity which also as an essential edible vegetable and medicinal species.
Free radicals or, more generally, ROS and reactive nitrogen species (RNS) are regarded as two products of normal cellular metabolism, which play dual roles in human health development ( Valko , 2006 ). When ROS and RNS exist in a moderate concentration, they have beneficial physiological effects on cellular responses to noxia, such as resisting infectious agents, regulating cellular signaling systems and inducting mitogenic responses ( Jayakumar ., 2009 ; Valko ., 2007 ). In contrast, overproduction of ROS and RNS involve deleterious roles in cell structure including proteins, DNA and lipids, resulting in a variety of diseases such as inflammation, cancer, gastric ulcers, cardiovascular disease and diabetes mellitus ( Li , 2010 ). Lipopolysaccharide (LPS) is a primary constituent of the outer cell walls of Gram-negative bacteria which could activate an exaggerated inflammatory response ( Park ., 2014 ). Macrophages play a key pivotal in inflammatory response linking with excessive production of pro-inflammatory cytokines as for an example of interleukin-1β (IL-1β), and inflammatory mediators as for instance of ROS and NO, resulted from activated inducible nitrite oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) ( Heo ., 2010 ). Thus, anti-inflammatory activity can be examined by measuring the suppression activities of pro-inflammatory cytokines iNOS, COX-2 and IL-1β mRNA expressions and the inhibition levels of inflammatory mediators ROS and NO.
Aruncus dioicus var. kamtschaticu s is widely grown in mountainous areas in Korea and is known as an important kind of edible vegetable and medicinal species, which belongs to the Rosaceae family. A. dioicus has been employed in traditional medicine system to treating cancer, obesity, diabetes mellitus, tonsillitis, brain injury and skin aging ( Ahn., 2014 ; Han and Lee, 2011 ; Jeong , 2011 ; Kim ., 2012 ). Although the published works have reported foresaid pharmacological activities, in the scope of my knowledge, there is little information involved in its anti-inflammatory activity. Furthermore, A. dioicus is edible, inexpensive and easily available. The focus of study shifted to its inflammation become more valuable. In this regard, the goal of this study was to investigate DNA damage protection and anti-inflammatory activity of different fractions from the A. dioicus water extract.
Materials and Methods
- Samples preparation
A. dioicus plant was obtained in spring from JeongSun, Korea. The voucher specimen (ADK 2013) was deposited in the lab of molecular of plant biotechnology. The aerial parts were dried in the shade and macerated with distilled water at room temperature for 72 h. The water extract was got by concentrating the filtrates with a rotary evaporator (CCA-110, Eyela, Tokyo, Japan). The water extract (30 g) was dissolved into distilled water and fractionated with dichloromethane, ethyl acetate, n -butanol and distilled water to get dichloromethane (CH 2 Cl 2 ) (0.15 g), ethyl acetate (EtOAc) (1.28 g), n -butanol ( n -BuOH) (5.03 g), and distilled water (H 2 O) (21.45 g) fractions, respectively.
- Chemicals and reagents
1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), 7-dichlorofluorescein-diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), ethidium bromide (EB), lipopolysaccharide (LPS) (E. coli 0111:B4) and phosphate-buffered saline (PBS) were purchased from Sigma (St, Louis, MO, USA). RPMI medium 1640 and fetal bovine serum (FBS) were acquired from Gibco BRL (Grand Island, NY, USA). All culture supplies were obtained from BD-Falcon (BD, Franklin Lakes, NJ). All other chemicals were of analytical grade.
- Cell line and cell culture
Murine macrophage cell RAW 264.7 lines were obtained from the Korean Cell Line Bank (Seoul, Korea). The RAW 264.7 cells were cultured in RPMI medium 1640, supplemented with 10% FBS and 100 U/㎖ of penicillin. The cells were incubated at 37℃ in a humidified atmosphere, 5% CO 2 .
- Genomic DNA isolation
The genomic DNA was isolated from RAW 264.7 cells. The cells were harvested by incubating for three days and washed with PBS. The collected cells were lysed in extraction buffer (1 M Tris-HCl, 0.5 M EDTA, 5 M Sodium chloride and 20% SDS), and then incubated at 64℃ for 10 min. The DNA was centrifuged at 13,000 rpm for 15 min after isolated three times with phenol:chloroform:isoamylalcohol (25:24:1) solution. Finally, the DNA was washed by 70% ethanol and dissolved in distilled water.
- Assay of DNA damage protection
Four μl of genomic DNA was added into 3 μl of 50 mM phosphate buffer (pH 7.4). The mixture solution was incubated at 37℃ for 30 min after addition of 3 μl of 1 mM ferrous sulphate, 10 μl of 1 ㎎/㎖ sample and 4 μl of 0.1 mM hydrogen peroxide. The DNA was analyzed using 1% agarose gels containing 1 μl of EB. The band was observed by a Mini BIS image analysis system (DNR Bio-Imaging Systems Ltd, Kiryat Anavim, Israel). The protective effect of DNA damage was measured by comparing with the genomic DNA control.
- Assay of cell viability
The cytotoxicity of sample on RAW 264.7 cells was evaluated with a conventional MTT assay. The cells were plated at a 96-well plate (1 × 10 5 cells/well) for 16 h. Sample (0, 10, 20, 40 ㎍/㎖) was added into medium for 24 h. After discarding all medium, 10 μl of MTT solution (5 ㎎/㎖ in PBS) and 90 μl of non-FBS medium were mixed sufficiently and added into each well. The mixture was incubated for 4 h. After removing all the supernatant, 200 μl of DMSO was added into each well for solubilization. The absorbance was measured at 550 ㎚ by using an enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Tek, Winooski, VT, USA).
- Determination of NO production
The level of NO production about cell culture supernatant was based on Griess reaction. The RAW 264.7 cells were seeded in 96-well plates (1 × 10 5 cells/well) for 16 h. The cells were pre-treated with sample (0, 10, 20, 40 ㎍/㎖) and stimulated with LPS (1 ㎍/㎖) for 24 h. Subsequently, 100 ㎕ of each supernatant was removed into a new 96-well plated. 100 μl of Griess reagent (1%; sulfanilamide diluted in 5% phosphoric acid, 50 μl; 0.1% naphthyl-ethylenediamine dihydrochloride diluted in distilled water, 50 μl) was added. Rest for 10 min, the absorbance was measured at 550 ㎚ with an ELISA.
- Total RNA extraction and RT-PCR
The total RNA was isolated from the LPS-stimulated RAW 264.7 cells in the pre-treatment of sample (0, 10, 20, 40 ㎍/㎖) by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was obtained from the reverse-transcribed RNA with oligo (dT) and Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Invitrogen). The primer sequences for iNOS, COX-2, IL-1β and GAPDH were listed in Table 1 . The isopyknic of the individual PCR products were analyzed by electrophoresis using 1% agarose gels containing 1 μl of EB. The band was observed by a Mini BIS image analysis system (DNR Bio-Imaging Systems Ltd, Kiryat Anavim, Israel).
PCR primers used in the experiment (F: forward, R: reverse)
PPT Slide
Lager Image
ziNOS: inducible nitrite oxide synthase.yCOX-2: cyclooxygenase-2. xIL-1β: interleukin-1β.
- Determination of intracellular ROS
Intracellular ROS induced by LPS was evaluated using fluorescent DCFH-DA probe. RAW 264.7 cells were plated at a 6-well plate (1 × 10 6 cells/well) for 16 h. After removing medium, the cells were cultivated by sample (0, 10, 20, 40 ㎍/㎖) and stimulated with LPS (1 ㎍/㎖) for 24 h. After discarding all of the culture, 1 ㎖ of DCFH-DA solution (20 μΜ) was added into each well, and then incubated at 37℃ for 30 min. The DCFH-DA solution of each well was removed and 0.5 ㎖ of PBS was added. The level of intracellular ROS was evaluated by flow cytometry (Becton-Dichinson, Franklin Lakes, NJ, USA) at excitation and emission wavelengths of 485 ㎚ and 530 ㎚, respectively.
- Statistical analyses
Each test was carried out in triplicate (n = 3). The results were expressed as mean ± standard deviation (SD). One-way analysis of variance with Duncan’s multiple-range test was used to analyze the significant differences between the groups (p < 0.05 was considered significant). All analyses were analyzed using SPSS 21 (SPSS Institute, Cary, NC, USA).
Results and Discussion
- DNA damage protection
The normal life cycle of the cell and the stability of the genome are closely related to DNA, which has influenced on repair pathways, cell cycle regulation and cell death through a variety of mechanisms ( Packer ., 1999 ). Oxidative DNA damage by ROS influences replication and transcription, and finally causes cell death, which has initiated various degenerative diseases, for instance, cancer, cardiovascular disease and diabetes mellitus ( Rowe ., 2008 ). It has been generally recognized that hydroxyl radicals are capable of causing strand breaks in isolated DNA ( Rhim, 2013 ). The DNA damage stimulated by hydroxyl radicals in the treatment of the four fractions from A. dioicus was shown in Fig. 1 . Line 1 and Line 2 was the genomic DNA incubated without or with Fenton’s reagent as the control. On the contrary, line 3, line 4 and line 5 were absolutely damaged by hydroxyl radicals. Line 6 exhibited protection for DNA damage. According to the result, except the H 2 O fraction, none of the other fractions prevented the DNA from damage, which manifested that the components of protecting DNA from damage may be more likely to dissolve in water.
PPT Slide
Lager Image
Agarose gel electrophoretic patterns of the DNA damage induced by hydroxyl radicals in the treatment of different fractions from A. dioicus water extract. Line 1, DNA incubated without Fenton’s reagent; Line 2, DNA incubated with Fenton’s reagent; Line 3, 4, 5 and 6, DNA incubated with Fenton’s reagent in the presence of 1 ㎎/㎖ CH2Cl2 fraction, EtOAc fraction, n-BuOH fraction, H2O fraction.
- Cell viability and NO production
The cytotoxicity of the four fractions from A. dioicus on RAW 264.7 cells was shown in Fig. 2A . The four fractions at the test concentrations exhibited little toxicity to cell viability. Inflammation is recognized to implicate for many pathological conditions, such as diabetes, cancer and cardiovascular disease ( Jiang , 2013 ). In order to investigate the anti-inflammatory effect, NO is regarded as a primary regulator and mediator of inflammatory responses ( MacMicking , 1997 ). The NO inhibitory ability of the four fractions from A. dioicus was evaluated using the LPS-induced RAW 264.7 cells. As shown in Fig. 2B , the cells stimulated by LPS generated excessively high-level of NO. However, the high-level of NO production was suppressed in a dose-dependent trend as the cells were pretreated with different concentrations of the four fractions. Especially, the CH 2 Cl 2 fraction exhibited the highest NO inhibition activity, ranging from 61% to 19% (10-40 ㎍/㎖), which reflected unusually outstanding NO inhibition ability.
PPT Slide
Lager Image
Cytotoxicity (A) and NO inhibitory ability (B) on LPSstimulated RAW 264.7 cells of different fractions from A. dioicus water extract. Each value is expressed as the mean ± SD (n = 3). Means not sharing the same letter are significantly different at P < 0.05 probability level at same concentration by Duncan’s multiple-range test.
- RT-PCR of inflammatory genes
RT-PCR was employed to determine whether the above NO inhibition ability of CH 2 Cl 2 fraction was correlated with the modulation of specific mRNA expression. During the process of anti-inflammation, massive amounts of the proinflammatory mediators such as NO are produced by the inducible isoforms of pro-inflammatory cytokines ( Kim., 2007 ; Nam ., 2013 ). Housekeeping gene (GAPDH) was essential for basis cell survival and was used to normalize gene expression. Furthermore, we investigated the mRNA expression level of pro-inflammatory cytokines including iNOS, COX-2 and IL-1β. As shown in Fig. 3 , in the LPS-unstimulated RAW 264.7 cells, the expression of iNOS, COX-2 and IL-1β mRNA were not detected; however, the expression was markedly up regulated after the pre-treatment with LPS. Meanwhile, the LPS-stimulated expression was considerably suppressed in a concentration-dependent manner of the CH 2 Cl 2 fraction. According to the result, it could be concluded that the CH 2 Cl 2 fraction had the suppressive activity on iNOS, COX-2 and IL-1β at the transcriptional levels.
PPT Slide
Lager Image
Effect of CH2Cl2 fraction from A. dioicus water extract on iNOS, COX-2, and IL-1β expression in the LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with 1 ㎍/㎖ of LPS and different concentrations of CH2Cl2 fraction from A. dioicus water extract for 24 h. The levels of iNOS, COX-2, IL-1β and GAPDH mRNA expression were determined by RT-PCR.
- Intracellular ROS activity
Inflammatory cells are specifically adept in producing and releasing ROS, which can cause the situation of oxidative stress and is potentially related to a range of diseases such as inflammation, cancer, gastric ulcers ( Ramos ., 1992 ). Non-fluorescent compound DCFH-DA can freely permeate through the cell membrane and supply DCFH as the intracellular esterases in cells. Since the exposure of intracellular ROS, DCFH is easily oxidized to the fluorescent product DCF ( LeBel , 1992 ). Therefore, the fluorescence of DHF can be monitored to quantify the overall reactive oxygen in living cells. LPS was used to generate oxidant stress in RAW 264.7 cells. As shown in Fig. 4 , the intracellular ROS accumulation in presence of LPS was increased remarkably contrasted with untreated cells. Pretreatment with the CH 2 Cl 2 fraction blocked LPS-stimulated intracellular ROS generation in a concentrationdependent trend. This result suggested that CH 2 Cl 2 fraction prevented the oxidative stress in cells and therefore protected cells against oxidative damage.
PPT Slide
Lager Image
Flow cytometric analysis of LPS-stimulated ROS production in RAW 264.7 cells. (A) cells treated without LPS and DCFH-DA; (B) cells treated without LPS; (C) cells treated with LPS; (D) (E) (F) cells treated with LPS and different concentrations of CH2Cl2 fraction, 10 ㎍/㎖, 20 ㎍/㎖, 40 ㎍/㎖.
In conclusion, this study demonstrated that DNA damage protection and anti-inflammatory activity of different fractions from A. dioicus aerial parts water extract. In the DNA damage protection assay, the H 2 O fraction displayed the most powerful protection for DNA damage. In a series of anti-inflammatory assays, the CH 2 Cl 2 fraction exhibited significantly antiinflammatory activity. Moreover, the active ingredients of DNA damage protection and anti-inflammatory activity are needed to do further researches. Additional research can currently underway to identify the active ingredients for DNA damage protection and anti-inflammatory activity in this plant.
Ahn H.C. , Kim J.H. , Kim J.G. , Auh J.H. , Choe E.N. 2014 In vitro α-glucosidase and pancreatic lipase inhibitory activities and antioxidants of Samnamul (Aruncus dioicus) during rehydration and cooking Food Sci. Biotechnol. 23 1287 - 1293
Han H.S. , Lee J.W. 2011 Attenuation of brain injury by water extract of Goat’s-beard (Aruncus dioicus) and its ethyl acetate fraction in a rat model of ischemia-reperfusion J. Food Sci. Nutr. 16 217 - 223
Heo S.J. , Yoon W.J. , Kim K.N. , Ahn G.N. , Kang S.M. , Affan A. , Kang D.H. , Oh C. , Jung W.K. , Jeon Y.J. 2010 Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages Food Chem. Toxicol. 48 2045 - 2051
Jayakumar T. , Thomas P.A. , Geraldine P. 2009 In-vitro antioxidant activities of an ethanolic extract of the oyster mushroom, Pleurotus ostreatus Innov. Food Sci. Emerg. 10 228 - 234
Jeong S.Y. , Jun D.Y. , Kim Y.H. , Min B.S. , Min B.K. , Woo M.H. 2011 Monoterpenoids from the aerial parts of Aruncus dioicus var. kamtschaticus and their antioxidant and cytotoxic activities Bioorg. Med. Chem. Lett. 21 3252 - 3256
Jiang Y.Y. , Noh H. , Wang M.H. 2013 Comparison of biological activities of Synurus deltoids (Aiton) Nakai under different shading conditions Korean J. Plant Res. 26 686 - 694
Kim D.H. , Moon Y.S. , An B.J. , Son J.H. 2012 Potent antiaging activity of Aruncus dioicus, a native plant of Ulleung-do, South Korea, in CCD-986sk fibroblasts via suppression of matrix metalloproteinases J. Nat. Med. 66 631 - 636
Kim J.B. , Han A.R. , Park E.Y. , Kim J.Y. , Cho W. , Lee J. , Seo E.K. , Lee K.T. 2007 Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-κ inactivation in RAW 264.7 macrophage cells Biol. Pharm. Bull. 30 2345 - 2351
LeBel C.P. , Ischiropoulos H. , Bondy S.C. 1992 Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress Chem. Res. Toxicol. 5 227 - 231
Li C.M. , Son H.J. , Huang C.Q. , Lee S.K. , Lohakare J. , Wang M.H. 2010 Comparison of Crataegus pinnatifida Bunge var. typica Schneider and C. pinnatifida Bunge fruits for antioxidant, anti-α-glucosidase, and anti-inflammatory activities Food Sci. Biotechnol. 19 769 - 775
MacMicking J. , Xie Q. , Nathan C. 1997 Nitric oxide and macrophage function Annu. Rev. Immunol. 15 323 - 350
Nam J.H. , Jeong J.C. , Kwon O.K. , Hong S.Y. , Kim S.J. , Soh H.B. , Lee J.N. , Park H.J. 2013 Anti-inflammatory activity of peel extracts in color-fleshed potatoes Korean J. Plant Res. (in Korean) 26 533 - 538
Packer L. , Rimbach G. , Virgili F. 1999 Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (pinus maritima) bark, pycnogenol Free Radical Bio. Med. 27 704 - 724
Park G.H. , Park J.H. , Eo H.J. , Song H.M. , Lee M.H. , Lee J.R. , Jeong J.B. 2014 Anti-inflammatory effect of the extracts from Abeliophyllum distichum Nakai in LPS-stimulated RAW264.7 cells Korean J. Plant Res. 27 209 - 214
Ramos C.L. , Pou S , Britigan B.E. , Cohen M.S. , Rosen G.M. 1992 Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes J. Biol. Chem. 267 8307 - 8312
Rhim T.J. 2013 In vitro antioxidant activity of Sanguisorbae radix ethanol extracts Korean J. Plant Res. (in Korean) 26 149 - 158
Rowe L.A. , Degtyareva N. , Doetsch P.W. 2008 DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae Free Radical Bio. Med. 45 1167 - 1177
Valko M. , Rhodes C.J. , Moncol J. , Izakovic M. , Mazur M. 2006 Free radicals, metals and antioxidants in oxidative stress-induced cancer Chem.-Biol. Interact. 160 1 - 40
Valko M. , Leibfritz D. , Moncol J. , Cronin M.T. , Mazur M. , Telser J. 2007 Free radicals and antioxidants in normal physiological functions and human disease Int. J. Biochem. Cell B. 39 44 - 84