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Logo of itxAboutfor AuthorsVersitaInterdisciplinary Toxicology
Interdiscip Toxicol. 2009 December; 2(4): 245–249.
Published online 2009 December 28. doi:  10.2478/v10102-009-0022-2
PMCID: PMC2984114

Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages by Jeju plant extracts


Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be responsible for the vasodilation and hypotension observed during septic shock and inflammation. Thus, inhibitors of iNOS may be useful candidates for the treatment of inflammatory diseases accompanied by the overproduction of NO. In this study, we prepared alcoholic extracts of Jeju plants and screened them for their inhibitory activity against NO production in lipopolysaccharide (LPS)-activated macrophages. Among the 260 kinds of plant extract tested, 122 extracts showed potent inhibitory activity towards NO production by more than 25% at a concentration of 100 µg/mL. Plants such as Malus sieboldii, Vaccinium oldhamii, Corylus hallaisanensis, Carpinus laxiflora, Styrax obassia, and Securinega suffruticosa showed the most potent inhibition (above 70%) at a concentration of 100 µg/mL. The cytotoxic effects of the plant extracts were determined by colorimetric MTT assays and most plant extracts exhibited only moderate cytotoxicity at 100 µg/mL. Therefore, these plants should be considered promising candidates for the further purification of bioactive compounds and would be useful for the treatment of inflammatory diseases accompanying overproduction of NO.

Keywords: cytotoxicity, inflammation, nitric oxide, plant extract


Nitric oxide (NO), which is synthesised by nitric oxide synthase (NOS) from L-arginine using NADPH and molecular oxygen, is a short-lived free radical and an intercellular messenger produced by a variety of mammalian cells, which include macrophages, neutrophils, platelets, fibroblasts, endothelium, neuronal, and smooth muscle cells. NO mediates a variety of biological actions ranging from vasodilatation, neurotransmission, inhibition of platelet adherence and aggregation, as well as the macrophage- and neutrophil-mediated killing of pathogens (Moncada et al., 1991, MacMicking et al., 1997; Oh et al., 2008). Chronic inflammation and infections lead to the up-regulation of a series of enzymes and signaling proteins in affected tissues and cells. The inducible forms of NOS are the most important pro-inflammatory enzymes responsible for increasing the levels of NO. Three isoforms of NOS have been identified and are classified into the following two major categories: constitutive and inducible. Expression of iNOS catalyses the formation of large amounts of NO, which plays a key role in the pathogenesis of a variety of inflammatory diseases. Therefore, the level of NO induced by iNOS may reflect the degree of inflammation and provides an indicator to assess inflammatory processes. Recently, several iNOS inhibitors have been reported as being isolated from plants such as 4-O-methylhonokiol (Oh et al., 2009), fraxinellone (Kim et al., 2009), 6-gingerol (Lee et al., 2009), tanshinone IIA (Fan et al., 2009), and arctigenin (Zhao et al., 2009). In addition, most of the inhibitory activity of these compounds towards NO production has been demonstrated to be through the inhibition of iNOS expression.

Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years across the globe. These plant based medicinal systems continue to play an essential role in health care today, and it has been estimated by the World Health Organization that approximately 80% of the world's inhabitants rely mainly on traditional medicines for primary health care (Cragg and Newman, 2008; Hsieh et al., 2008). Owing to its unique ecosystem, Jeju Island is famous for the richness and diversity of its flora with over 7800 species classified to date. Over the past few years, we have systematically evaluated and characterised selected plant species for their putative bioactivities or potential medicinal applications. In order to find new iNOS inhibitors from endemic Jeju plants, we have established a screen for the inhibitory activity towards NO production by measuring its production in LPS-stimulated RAW 264.7 cells.

Materials and methods

Plant materials and solvent extraction

The plants were collected from Jeju Island, Korea from 2006 to 2008. Voucher specimens were deposited at the herbarium of Jeju Biodiversity Research Institute. Verification of vouchers or living plants was performed by Dr. Gwanpil Song. Plant materials were air-dried, ground and extracted three times with 80% ethanol at room temperature. After the sample was filtered through two layers of cheesecloth, the filtered cakes were extracted and filtered three more times to increase the extraction yield. The filtrates were concentrated under reduced pressure, freeze-dried, and stored in a closed container until testing.

Cell culture

Murine macrophage RAW 264.7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). They were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine, 10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid (HEPES), penicillin (100 units/mL), streptomycin (100 µg/mL) and 10% foetal bovine serum. Cells were cultured at 37 °C in a humidified incubator with an atmosphere of 5% CO2.

MTT assay for cell viability

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a pale yellow substrate that is reduced by living cells to yield a dark blue formazan product. This process requires active mitochondria, and only freshly dead cells do not reduce significant amounts of MTT. RAW 264.7 cells were cultured in 96-well plates for 18 hr, followed by treatment with LPS (1 µg/mL) in the presence of plant extracts at concentrations of 100 µg/mL. After a 24 hr incubation, MTT was added to the medium for 4 hr. Finally, the supernatant was removed and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 540 nm. The percentage of dead cells was determined relative to the control group.

Nitric oxide assay

The nitric oxide assay was performed as described previously with slight modification (Yoon et al., 2009). After pre-incubation of RAW 264.7 cells (1.5 × 105 cells/mL) with LPS (1 µg/mL) for 24 h, the quantity of nitrite in the culture medium was measured as an indicator of NO production. Amounts of nitrite, a stable metabolite of NO, were measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid). Briefly, 100 µL of cell culture medium was mixed with 100 µL of Griess reagent. Subsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader. Fresh culture medium was used as a blank in every experiment. The quantity of nitrite was determined from a sodium nitrite standard curve.

Results and discussion

In murine macrophage RAW 264.7 cells, LPS stimulation alone has been demonstrated to induce iNOS transcription and its protein synthesis, with a corresponding increase in NO production. Furthermore, LPS stimulation has also been shown to induce IκB proteolysis and NF-κB nuclear translocation (Xie et al., 1994; Henkel et al., 1993). Therefore, this cell system is an excellent model for drug screening and the subsequent evaluation of potential inhibitors against iNOS and NO production. In our search for natural products with anti-inflammatory activity, we prepared 80% ethanol crude extracts of 260 native plants from Jeju Island, Korea. All the plant samples were dissolved in 80% ethanol and diluted with sterile water to normalise the concentration of the test sample. The Griess reaction, a spectrophotometric determination for nitrite, was carried out to quantify the nitrite levels in the conditioned medium of RAW 264.7 cells treated with LPS. The final concentration of ethanol in the culture media was 0.1% and this concentration of ethanol did not show any effect on the assay systems. Table 1 shows the inhibitory activity by plant extracts towards NO production by LPS-activated macrophages. Of the 260 kinds of extracts, 122 extracts showed greater than 25% inhibition of NO production at the concentration of 100 µg/mL in the culture media. Among these 122 extracts, Acer pictum, Viburnum dilatatum, Melia azedarach, Lonicera japonica, Osmunda japonica, Alnus firma, Lindera erythrocarpa, Platycarya strobilacea, Rhododendron werrichii, Weigela subsessilis (, Salix koreensis, Magnolia kobus, Corylus sieboldiana, Cornus walteri, Ulmus parvifolia, Morus bombycis, Aria alnifolia, Neoshirakia japonica, Actinodaphne lancifolia, Triadica sebifera, Elaeagnus umbellata, Oenothera glazioviana, Ficus erecta var. sieboldii, Rubus buergeri, Orixa japonica, and Cnidium japonicum showed the most potent inhibition (greater than 70% inhibition) at the concentration of 100 µg/mL. The numbers of viable activated macrophages were not significantly altered by the plant extracts as determined by MTT assays, thereby indicating that the inhibition of NO synthesis by the plant extracts was not simply due to cytotoxic effects. Although some plant extracts such as Idesia polycarpa, Artemisia scoparia, and Elsholtzia splendens also exhibited potent inhibition (above 70%) towards NO synthesis at 100 µg/mL, there were some cytotoxic effects.

Table 1
Nitric oxide inhibition and cytotoxicity of Jeju plant extracts.

To conclude, these data suggest that extracts from the plant species examined in this study deserve further investigation in order to isolate the bioactive secondary metabolites with anti-inflammatory properties. Currently, experiments are in progress to analyse the active fractions from the extracts in order to determine the chemical structure of those compounds and to perform more extensive biological evaluations. Many compounds from medicinal plants have been demonstrated as inhibitors of the expression of iNOS in LPS-activated macrophages. Their structures can be categorised as sesquiterpene (Reddy et al., 2006; Choi et al., 2009), flavonoid (Chen et al., 2008; Paoletti et al., 2009), polyacetylenes (Kim et al., 2003), and lignans (Kim et al., 2008). Thus, plants demonstrating inhibitory activities against NO production will be promising candidates for the activity-guided isolation of active components exhibiting iNOS inhibitory activity, which may have therapeutic potential for the treatment of inflammation accompanying overproduction of NO. Further investigations are underway to characterise the active constituents present in these plant extracts.


This research was supported by the Regional Technology Innovation Program (RTI04-02-07), which is managed by the Ministry of Knowledge and Economy, Korea.


  • Chen CC, Tsai PC, Wei BL, Chiou WF. 8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages. Eur J Pharmacol. 2008;590:430–436. [PubMed]
  • Choi Y, Lee MK, Lim SY, Sung SH, Kim YC. Inhibition of inducible NO synthase, cyclooxygenase-2 and interleukin-1beta by torilin is mediated by mitogen-activated protein kinases in microglial BV2 cells. Br J Pharmacol. 2009;156:933–940. [PMC free article] [PubMed]
  • Cragg GM, Newman DJ. 2nd ed. Boca Raton, F.L: CRC press; 2008. Detection, isolation and structural determination in Bioactive natural products; p. 324.
  • Fan GW, Gao XM, Wang H, Zhu Y, Zhang J, Hu LM, Su YF, Kang LY, Zhang BL. The anti-inflammatory activities of Tanshinone IIA, an active component of TCM, are mediated by estrogen receptor activation and inhibition of iNOS. J Steroid Biochem Mol Biol. 2009;113:275–280. [PubMed]
  • Henkel T, Machleidt T, Alkalay I, Krönke M, Ben-Neriah Y, Baeuerle PA. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993;365:182–185. [PubMed]
  • Hsieh YH, Kuo PM, Chien SC, Shyur LF, Wang SY. Effects of Chamaecyparis formosensis Matasumura extractives on lipopolysaccharide-induced release of nitric oxide. Phytomedicine. 2007;14:675–680. [PubMed]
  • Kim BH, Hong SS, Kwon SW, Lee HY, Sung H, Lee IJ, Hwang BY, Song S, Lee CK, Chung D, Ahn B, Nam SY, Han SB, Kim Y. Diarctigenin, a lignan constituent from Arctium lappa, down-regulated zymosan-induced transcription of inflammatory genes through suppression of DNA binding ability of nuclear factor-kappaB in macrophages. J Pharmacol Exp Ther. 2008;327:393–401. [PubMed]
  • Kim JH, Park YM, Shin JS, Park SJ, Choi JH, Jung HJ, Park HJ, Lee KT. Fraxinellone inhibits lipopolysaccharide-induced inducible nitric oxide synthase and cyclooxygenase-2 expression by negatively regulating nuclear factor-kappa B in RAW 264.7 macrophages cells. Biol Pharm Bull. 2009;32:1062–1068. [PubMed]
  • Kim JM, Lee P, Son D, Kim H, Kim SY. Falcarindiol inhibits nitric oxide-mediated neuronal death in lipopolysaccharide-treated organotypic hippocampal cultures. Neuroreport. 2003;14:1941–1944. [PubMed]
  • Lee TY, Lee KC, Chen SY, Chang HH. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-alpha and NF-kappaB pathways in lipopolysaccharide-stimulated mouse macrophages. Biochem Biophys Res Commun. 2009;382:134–139. [PubMed]
  • MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Ann Rev Immunol. 1997;15:323–350. [PubMed]
  • Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109–142. [PubMed]
  • Paoletti T, Fallarini S, Gugliesi F, Minassi A, Appendino G, Lombardi G. Anti-inflammatory and vascularprotective properties of 8-prenylapigenin. Eur J Pharmacol. 2009;620:120–130. [PubMed]
  • Reddy AM, Lee JY, Seo JH, Kim BH, Chung EY, Ryu SY, Kim YS, Lee CK, Min KR, Kim Y. Artemisolide from Artemisia asiatica: nuclear factor-kappaB (NF-kappaB) inhibitor suppressing prostaglandin E2 and nitric oxide production in macrophages. Arch Pharm Res. 2006;29:591–597. [PubMed]
  • Oh JH, Kang LL, Ban JO, Kim YH, Kim KH, Han SB, Hong JT. Anti-inflammatory effect of 4-O-methylhonokiol, a novel compound isolated from Magnolia officinalis through inhibition of NF-kappaB. Chem Biol Interact. 2009;180:506–514. [PubMed]
  • Oh JH, Lee TJ, Park JW, Kwon TK. Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells. Eur J Pharmacol. 2008;599:11–17. [PubMed]
  • Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994;269:4705–4708. [PubMed]
  • Yoon WJ, Kim SS, Oh TH, Lee NH, Hyun CG. Abies koreana essential oil inhibits drug-resistant skin pathogen growth and LPS-induced inflammatory effects of murine macrophage. Lipids. 2009;44:471–476. [PubMed]
  • Zhao F, Wang L, Liu K. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J Ethnopharmacol. 2009;122:457–462. [PubMed]

Articles from Interdisciplinary Toxicology are provided here courtesy of Slovak Toxicology Society SETOX & Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences