Antioxidative Activity and Anti-inflammatory Effects on the Murine Macrophages of Methanol Extracts Sang-Bum Kim, Min-Ho Chang1, Sang-Hyun Han2 and Hong-Shik Oh2,* Ohyun Middle School, Jeju 690-061, Korea 1National Park Research Institute, Korea National Park Service, Namwon 590-811, Korea 2Department of Science Education, Jeju National University, Jeju 690-756, Korea Abstract - Oxidative stress has been reported to be one of causes of neuritis. This study examined
antioxidative activities of methanol extracts of six amphibian species known to be medicinal animals
(Rana catesbeiana, R. coreana, R. rugosa, R. dybowskii, R. nigromaculata
, and Hyla japonica) and
investigated their effects of inhibiting nitric oxide (NO) production and cytotoxicity on the murine
macrophage RAW264.7 cells. As inflammation is closely associated with reactive oxygen species,
assays on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, xanthine oxidase inhi-
bitory activity, superoxide anion radical scavenging activity and NO scavenging activity of the
extracts of the six species were performed to investigate their antioxidative activity. The results
obtained were as follows; All extracts showed antioxidative activity, and the activity of R. dybowskii
was the highest in comparison among those. Anti-inflammatory effects of the extracts were also
examined, the five extracts except that of R. rugosa
did not show cytotoxicity for RAW264.7 cells at
the maximal concentration (1,000
μg mL-1). Selectivity index, meaning NO scavenging activity com-
pared to cytotoxicity, showed the highest level in the extract of R. dybowskii
. These results will be
very useful basic data for future studies on prevention and treatment of human diseases to under-
stand the biological roles of amphibian extracts throughout the antioxidative or anti-inflammatory

Key words : antioxidative activity, cytotoxicity, anti-inflammatory effect, Korean amphibians
of using amphibians as a biological resource (Erspamer et INTRODUCTION
al. 1986; Rinaldi 2002; Lu et al. 2008; Gomes et al. 2011; Wang et al. 2012). The use as a biological resource empha- For useful biological resources, the interest in their use sizes the importance of gene pool and the use for productive, and the researches for them have focused on plants such as medical and dietary purposes. In particular, the use of amphi- natural medicine and the interest in use and development of bians for a medical purpose has been well known in both of useful animals have been very low around the world. How- Oriental and Western countries and in Korea it also has been ever, diversity and usefulness of animals is currently retho- utilized as a medicinal animal in traditional oriental medi- ught and development of technologies in the agricultural and cine and fork remedies (Mor et al. 1994; Batista et al. 1999; biological industries leads to confirmation of high possibility Je et al. 2007; Qian et al. 2008; Cho et al. 2009; Jin et al. 2009). However, its effects and usage as a material for tradi- tional oriental medicine shown in references and antient * Corresponding author: Hong-Shik Oh, Tel. 064-754-3283, Fax. 064-725-4902, E-mail. sciedu@jejunu.ac.kr books were induced from experiences, fork remedies and ─ 157
Sang-Bum Kim, Min-Ho Chang, Sang-Hyun Han and Hong-Shik Oh
traditional oriental medicine without a scientific analysis 3. DPPH radical scavenging activity assay
(Park and Lee 1998; Park et al. 2005).
To examine antioxidant activity of each sample, the Blois Amphibians are usually called as ‘wa’ in traditional orien- (1958) method measuring radical scavenging effect with tal medicine because it croaks well. In [Donguibogam] its DPPH (Sigma, USA) was used. DPPH solution was made cold characteristic was reported to control fissure and foods by dissolving around 2 mg DPPH in 15 mL EtOH. After of children and in [Pen-Tsao-Kang-Mu] it was written to adding 6.25 mL dimethylsulfoxide (DMSO) to 12 mL of the relieve diarrhea and pathology of fever. In traditional orien- solution, it was diluted with EtOH for absorbance of the con- tal medicine, it is used for nephropathy, diuresis, nutrition trol to be 0.94~0.97 at 517 nm wavelength and was shaken and flatulence, dried powder in warm honey water or boiled for 10 sec. In addition, 100 μL samples of each concentra- amphibian bodies by itself is eaten as a special efficient medi- tion dissolved in MeOH were put on 96 well plate and a cine of pulmonary tuberculosis, roborant and asthma. This same amount of 0.4 mM DPPH was added. After 10 min study was conducted to provide necessary data for researches incubation at room temperature, absorbance was measured on antioxidants and anti-inflammatory materials and on their isolation and mechanism to prevent and cure diseases by in- vestigating biological activity such as antioxidant activity 4. Xanthine oxidase inhibitory activity assay
and anti-inflammatory effect of methanol (MeOH) extracts of six amphibian species collected in Korea, which has been The production of uric acid caused by xanthine/xanthine rarely studied and has been reported to be used as a fork oxidase was measured with the increased absorbance at 290 nm (Cheng et al. 1998) and allopurinol (Sigma, USA) was used as the control. For the mixture, each samples of various concentrations, 0.5 mM xanthine and 1 mM EDTA were pre- MATERIALS AND METHODS
pared in 200 mM phosphate buffer (pH 7.5) and 50 units μL-1xanthine oxidase was added to induce production of uric 1. Animals and preparation of extracts
acid. Xanthine oxidase inhibitory activity was presented with the decreased rate of absorbance of the produced uric Six amphibian species (R. catesbeiana, R. coreana, R. rugosa, R. dybowskii, R. nigromaculata, and H. japonica) were used for this study obtained from American Bullfrog 5. Superoxide anion scavenging activity assay
Capture Operation Division (Jeongeup, Jeollabuk-do, Repub- lic of Korea) and Frog Village (Muju, Jeollabuk-do, Repub- The amount of superoxide anion formed by using phena- lic of Korea). After lyophilization of animal specimens, zine methosulfate (PMS)/NADH system was measured at samples were subsequently grinded, deposited on 500 mL 517 nm with nitroblue tetrazolium reduction method (Frido- 80% MeOH, and extracted three times by using a sonicator.
vich 1970; Nishikimi et al. 1972; Liu et al. 1997). The mix- And then, the supernatant was isolated and evaporated and ture was prepared with each sample, 125 μM NADH and after frozen drying it was used with diluted with 100 mg 63 μM NBT in 200 μL PBS (pH 8.4) and 8 μM PMS was mL-1 1 : 1 ethanol (EtOH) : phosphate-buffered saline (PBS) added to provoke production of superoxide. Superoxide anion scavenging activity was shown with the decreased rate of absorbance of the produced superoxide.
2. Cell culture
6. NO scavenging activity assay
Murine macrophage cell line RAW264.7 was obtained from Korean Cell Line Bank (Seoul, Republic of Korea) and was NO scavenging activity was analyzed by using sodium C with 5% CO2 conditions using Dulbecco’s nitroprusside (SNP) forming naturally NO (Green et al. 1982; modified Eagle’s medium (DMEM) including 100 units mL-1 Marcocci et al. 1994). Each samples of various concentra- penicillin-streptomycin and 10% fetal bovine serum (FBS).
tions were added to 10 mM SNP and was incubated at 25� Subcultures were conducted every 3~4 days.
for 3 hr. After the reaction, Griess solution [1% (w/v) sul- Antioxidative Activity of the Korean Amphibians
fanilamide, 0.1% N-1-naphylethylen diamine in 2.5% (v/v) 9. Statistical analysis
phosphoric acid] of a same amount with the mixture was The results were presented with a mean and standard devia- added. It was at room temperature for 10 min and its absor- tion and a statistical significance was analyzed with Student’s bance was measured at 540 nm. NO scavenging activity was calculated with the amount of residual nitrite. The activity was presented with % of the scavenging activity at 500 μg mL-1.
7. Cytotoxicity assay
RAW264.7 cells were put into 96 well microplates with 2 1. Antioxidative activity of amphibian extracts
×105 cells well-1 by using the DMEM and were incubated The antioxidant activities of MeOH extracts of R. cates- for 18 hr. After each samples of different concentrations and beiana, R. coreana, R. rugosa, R. dybowskii, R. nigromacu- 100 ng mL-1 lipopolysaccharide (LPS) (Sigma, USA) were lata, and H. japonica were presented in Table 1 and Fig. 1.
added and incubated for 24 hr. 2 mg mL-1 3-(4,5-dimehtyl- DPPH free radical scavenging activity was increased with thiazol)-2,5-diphenyl-tetrazolium bromide (MTT) was added depending on the treated concentrations (Fig. 1A) and the and was incubated for 1 hr and the media was removed. After IC50 values showing a concentration with 50% scavenging the formazan sediment produced by reduction of MTT by activity of each sample was the lowest in R. dybowskii by adding 200 μL DMSO was dissolved and absorbance was recording 1,570 μg mL-1 and the highest in R. catesbeiana measured at 540 nm with a microplate reader (Biotek, USA).
(Table 1). Xanthine oxidase inhibitory activity of the amphi- By comparing the absorbance of each sample at each con- bian MeOH extracts was measured with xanthine/xanthine centration with that of the untreated sample, cytotoxicity of oxidase system. Xanthine oxidase inhibitory effect at 625 the sample to RAW264.7 cells was evaluated.
μg mL-1 showed the highest level, 95.3% in R. dybowskiiand the lowest level, 53.3% in R. rugosa (Table 1, Fig. 1).
8. NO production inhibition assay
Superoxide anion radical scavenging activity of the extracts RAW264.7 cells (2×105 cells well-1) were put into 96 well showed low levels in all of the samples by recording less plates and each samples of various concentrations were treat- than 5% (Table 1). NO is an active species with a strong ed. After adding LPS (100 ng mL-1), it was incubated for cytotoxicity and the production of much NO provokes indirect 24 hr. After mixing 100 μL supernatant of the media with effects including nitrosation and nitration and oxidation to 100 μL Griess solution they were incubated on 96 well plates induce harmful effects. NO scavenging activity of the MeOH for 10 min and absorbance was measured at 530 nm. The extracts of the six species of Amphibia was measured with amount of NO was compared with that of sodium nitrite as the amount of nitrite by using SNP forming NO. At 2.5 mg mL-1 each sample showed 48~15% scavenging activity Table 1. Comparison of antioxidative potential of MeOH extracts of amphibians
*IC50 values were calculated from regression lines using seven different concentration in triplicate experiments.
**1.25 mg mL-1 scavenging activity (% control).
***2.5 mg mL-1 scavenging activity (% control).
- indicates ⁄5% radical scavenging activity in maximum concentration.
Sang-Bum Kim, Min-Ho Chang, Sang-Hyun Han and Hong-Shik Oh
Fig. 1. Dose-dependent scavenging effects on DPPH radical, NO and xanthine oxidase inhibitory activity by amphibian extracts. The data
indicate the mean±S.D. of triplicate experiments. A: R. catesbeiana; B: R. coreana; C: R. rugosa; D: R. dybowskii; E: H. japonica;F: R. nigromaculata.
and the scavenging effect of R. dybowskii, R. nigromaculata tion of lymphocytes inside of connective tissues. Interlukin and H. japonica was found to be good by recording 48.1, secreted from the lymphocytes by these changes, actives 42.5 and 35.1%, respectively while that of R. coreana, R. effector lymphocytes and promotes various enzyme such as catesbeiana and R. rugosa was relatively low by recording hyaluronidase, elastase and collagenase and inflammatory 26.7, 20.2 and 15.6%, respectively. Like previous studies mediators like prostaglandin to trigger inflammation destroy- on antioxidant activity of medicinal insect extracts revealing ing connective tissues (Deby 1988; Shimizu and Wolfe 1990).
that the extracts of Anomala albopilosa, Sympetrum eroticum NO is a free radial produced from L-arginine with nitric and Anax parthenope had high antioxidant activity (Kim et oxide synthases (NOSs) (Palmer et al. 1988) and it shows al. 2004; Yoon et al. 2007), this study also found that amphib- many biological functions including body defence, signal ians produced a superior antioxidant activity.
transduction and a function as a secondary messenger of vasodilation (Monacada et al. 1991; Knowles and Mocada 2. Anti-inflammatory activity of amphibian
1992; Nathan 1992). Constitutive NOS (cNOS) contains extracts
neuronal- and endothelial-NOS expressed in neurons and endothelial cells, respectively. The production of NO caused For vertebrates, infection and damage of capillary vessels by cNOSs play a key role in managing homeostasis of a body of skin tissues provoke proliferation of synovial cells and (Kawamata et al. 2000). However, contrary to cNOSs, indu- increase of fibroblasts and macrophages along with satura- cible NOS (iNOS) is presented on macrophages, vascular Antioxidative Activity of the Korean Amphibians
smooth muscle cells, endothelial cells, hepatocytes and myo- cardial cells by following the stimuli of LPS, interferon-g, Macrophage is known to be related with homeostasis by interleukin (IL)-1b and tumor necrosis factor (TNF)-α (Lee affecting many host responses such as acquired immunity as et al. 2000). The iNOS expressed on these tissues leads to well as innate immunity and it is very critical for body def- inflammation, tumor (Nathan 1992), damage of tissues, gene ense in the early infection by making NO and cytokine dur- variation and damage of nerves by producing much NO for ing inflammatory reaction (Higuchi et al. 1990). LPS exist- a long time (Stuehr et al. 1991; Weisz et al. 1996). Like this, ing on the outer membrane of gram positive bacteremia is inflammation-related NO and prostaglandin E2 are formed reported to be an endotoxin and trigger sepsis and shock with Fig. 2. Effects of MeOH extracts on the NO production in LPS-stimulated RAW264.7 cells. Cells were treated with LPS (100 ng mL-1) alone
or LPS plus the indicated concentrations of MeOH extracts for 24 hr. A: R. catesbeiana; B: R. coreana; C: R. rugosa; D: R. dybowskii;E: H. japonica; F: R. nigromaculata.
Sang-Bum Kim, Min-Ho Chang, Sang-Hyun Han and Hong-Shik Oh
Table 2. Cell toxicity and the effects on LPS-induced NO produc-
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