Preventive effect of Ligularia fischerion inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264.7 macrophages depending on cooking method
© Park et al.; licensee BioMed Central. 2014
Received: 21 January 2014
Accepted: 13 November 2014
Published: 15 December 2014
Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated.
Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 μg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE2 production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF.
Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.
KeywordsChlorogenic acid Inflammation Ligularia fischeri Macrophage
Ligularia fischeri (common name Gomchwi) belongs to the family Compositae, which are perennial vegetable plants found mainly in damp shady regions besides brooks and sloping fields in Europe and Asia . In Korea, Gomchwi is generally consumed as salted or fried after a blanching process and is then called Chinamul. The leaves of L. fischeri have been used for their pharmaceutical properties in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases . Antioxidant activity of this plant has been demonstrated by several independent methods, indicating that the plant contains high amounts of antioxidant constituents [1, 3, 4]. In our previous study, L. fischeri exhibited a preventive myoglobin ratio against various reactive oxygen species (ROS) and reactive nitrogen species (RNS) . In another study, L. fischeri leaf tea prepared by blanching fresh leaves in boiling water was recognized as a value-added functional food that contains biological constituents such as caffeoylquinic acid [6, 7]. Leaves of L. fischeri contain caffeoylquinic acid derivatives (CQA) as major phenolic constituents [7, 8]. Shang et al.  reported that a number of caffeoylquinic acid derivatives (CQAs) have been isolated and suggested that these represent the major phenolic constituents in the leaves of L. fischeri.
Inflammation, a central feature of many pathophysiological conditions, occurs in response to tissue injury and results in the development of various human diseases such as cancer and diabetes . During an inflammatory response, macrophages regulate different intracellular signaling pathways and this result in the release of several inflammatory mediators such as cytokines . These in turn induce pro-inflammatory enzymes including the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX), which are responsible for increasing the levels of NO and prostaglandins (PGs) respectively . Santos reported that plant natural polyphenols, namely caffeoylquinic acid derivatives, stimulated inflammatory mediator production (Dos Saontos et al. ). Lemongrass, which contained chlorogenic acid (3-caffeoylquinic acid), has anti-inflammatory activities via inhibition of cytokine expression . Supplementation with anti-inflammatory materials is a possible preventive and therapeutic strategy for inflammation induced-diseases . The objective of this study is to determine the therapeutic effects of Ligularia fischeri that has been subject to cooking processes involved in anti-inflammatory activities.
Effect of cooked LF on cell viability
Effects of cooked LF on LPS-induced NO production and expression of iNOS in RAW264.7 cells
Effects of cooked LF on LPS-induced PGE2production and expression of COX2 in RAW264.7 cells
Effect of chlarogenic acid as a bioactive component from LF on inflammatory responses
3-O-caffeoylquinic acid content of Ligularia fischeri
Content (μg/100 μg/mL of ext.)
Change ratio (fold) (over uncooked LF)
For vegetables, cooking (boiling, microwaving, pressure-cooking, grilling, baking, and frying) can have a profound effect on both the cell walls and nutritional value [14, 15]. Cooking processes bring about a number of changes in the chemical composition of vegetables . In this study, when L. fischeri was submitted to blanch and pan-fry variations appeared in the concentration of 3-O-caffeoylquinic acid (Table 1). It was observed that the lower the initial 3-O-caffeoylquinic acid content, the higher the increase caused by the cooking treatment. The concentration of phenolic acids is highest in the outer layers of some vegetables and these areas are exposed to water . Although total phenolics are usually stored in vegetables in pectin or cellulose networks and can be released during thermal processing, individual phenolic compounds may sometimes increase because heat can break the supramolecular structure . Considering the above, the cooking process could have had a significant influence on the concentration of 3-O-caffeoylquinic acid through cell tissue distribution in L.fischeri.
Many studies have reported the isolation of bioactive components from extracts of L. fischeri and have evaluated their antioxidant activities [2, 7, 19, 20]. In our previous study, the antioxidant activities of extracts of LF were changed by cooking processes . Lee and Choi reported that LF showed anti-inflammatory activities using carrageenan in formalin-induced experimental animal models . Also LF modulated the inflammatory process by suppressing various genes in human synovial cells . In this study, blanched LF showed greater inhibition of NO production in LPS-induced RAW264.7 cells compared with uncooked and pan-fried LF (Figure 2A). As a bioactive component of LF, 3-O-caffeoylquinic acid significantly inhibited NO production and iNOS and COX2 expression in the 0 ~ 20 μM range . In this study, although LF did not cause any decline of COX2 expression, it inhibited NO production and iNOS expression. These results might anti-inflammatory effects of LF were affected through COX2-independent signaling in LPS-induced macrophage.
Regardless of the cooking method, L. fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells. This indicates that the anti-inflammatory effects of LF were not only caused by the 3-O-caffeoylquinic acid content in LF and that, after going through the cooking process, LF may influence the anti-inflammatory response. Based on these results, L. fischeri may be beneficial for the prevention of anti-inflammatory diseases.
Ligularia fischeri (LF) was collected at Inje-gun, Gangwon-do, Korea. RAW264.7 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Low glucose (1000 mg/ml) Dulbeco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin cocktail were purchased from WELLGENE (Daegu, Korea). Lipopolysaccharide (LPS, Escherichia coli O55:B5), 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT), and 3-O-caffeoylquinic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Griess reagent was obtained from Promega (Madison, WI, USA). Nitric oxide (NO) and prostaglandin E2 (PGE2) assay kit were purchased from R&D System (Minnesota, USA). Reverse transcription and polymerase chain reaction premixes were from Applied Biosystems (Carlsbad, CA, USA).
Blanching in a stainless steel vessel: Washed LF (200 g) was added to water (2L) and blanched for 3 min.
Pan-frying: Washed LF (200 g) was placed in a frying pan with oil and stirred for 3 min.
LF extracts were obtained using 70% ethanol with sonication (POWERSONIC 420, 700 W, 50/60 Hz, Hwanshin Tech.) for 40 min twice. Then the extracts of LF were filtered, evaporated (ELISA EVAPORATOR NVC-2000, SB-1000, DPE-1210, CA-1112, ELISA, Japan), and freeze-dried (FD5510, IlShin Lab Co., Ltd., Korea) to make powder samples. All samples were diluted to a 10 mg/mL concentration and used for the anti-inflammatory sample.
Reverse-phase high performance liquid chromatography (HPLC) was conducted using a Dianex u-300 system (Milford, MA, USA) that consists of Ultimate 3000 pumps, autosampler, and UV detector. The Chromeleon chromatographic system was employed to analyze the HPLC data. Chromatographic separation was accomplished using an Atlantis dC18 reverse phase column (Waters, 4.6 × 150 mm, 5 μm) and the elution was monitored at 300 nm. For separation, solvents A (Acetonitriles, ACN) and B (0.02% aqueous phosphoric acid, v/v) were used. The gradient program used was as follows: initial 0-6min, linear change from A-B (13:87, v/v) to A-B (15: 85, v/v); then held for 3 min; 9–17 min, linear change from A-B (15:85, v/v) to A-B (19:81, v/v); 17–28 min, linear change from A-B (17: 83, v/v) to A-B (28: 72, v/v); and then held for 9 min. The flow rate was 0.6 mL/min and an aliquot of 10 μL was injected.
Murine RAW264.7 macrophages were cultured in DMEM medium containing 10% FBS, peniciln, and streptomycin in a 5% CO2 humidified incubator at 37°C. RAW 264.7 cells were grown in 48-well plates at a density of approximately 5 × 104 cells per well.
Cells were treated with different concentrations (10, 50, 100 μg/mL) of LF for 24 hr. After that, the cells were incubated with MTT reagent, which was added to the culture medium at a final concentration of 0.5 mg/mL, for 4 hr in a 5% CO2 humidified incubator at 37°C. The resultant dark blue crystals were dissolved using dimethyl sulfoxide (DMSO) and absorbance values were measured at 540 nm.
Measurement of nitric oxide (NO) production
The production of NO was determined by measuring the accumulated level of nitrite, an indicator of NO in the supernatant. The RAW 264.7 cells were pretreated with or without LF for 1 hr. After LPS (1 μg/mL) was added to the cultured medium for 24 hr, nitrate levels were measured in cell culture supernatants according to the Griess reaction (1% sulfanilamide, 0.1% N-[naphthyl] ethylenediamine dihydrochloride, and 5% phosphoric acid) at room temperature for 10 min. Absorbance of the mixture at 550 nm was measured in a micro-plate reader (SpectraMax M2, Molecular Devices, USA). Nitrate concentration was calculated by comparison with a nitrite standard curve.
Measurement of prostaglandin E2 (PGE2) production
RAW 264.7 cells were seeded in 48-well plates (5 × 104 cells per wells) and incubated for 24 hr. The cells were treated with LF or a vehicle in the presence of LPS (1 μg/μL) for an additional 24 hr. PGE2 production in the cell supernatant was evaluated by PGE2 Parameter Assay kit following the manufacturer’s instructions.
Real-time quantitative polymerase chain reaction (RT-PCR)
RAW 264.7 cells were cultured in 6-well plates (5 × 105 cells per well) for 24 hr. The cells were treated with LF or a vehicle in the presence of LPS (1 μg/μL) for an additional 24 hr. Total RNA was isolated from the cells using RNAeasy kit (Qiagen) and then total RNA reverse-transcribed using a High Capacity RNA-to-cDNA™ Kit (Applied Biosystems) in order to produce cDNAs. Quantitative RT-PCR was performed in a Power SYBR® Green Master Mix (Applied Biosystems) under a STEPONE PLUS (Applied Biosystems), and the results were analyzed with the Stepone Software VER. 2.1 supplied with the machine. The housekeeping gene β-actin was used as an internal standard to quantify the levels of iNOS and COX2 mRNA. Parameters of RT-PCR reaction were 95°C for 5 min for one cycle, then 95°C for 15 sec, 61°C for 30 sec, and 72°C for 30 sec for 40 cycles. The fluorescence signal was detected at the end of each cycle. The primers used in the experiment were iNOS, forward: 5'- CCC TTC CGA AGT TTC TGG CAG CAG C -3', reverse: 5'- GGC TGT CAG AGC CTC GTG GCT TTG G -3'; COX2, forward: 5'- TCT CCA ACC TCT CCT ACT AC -3', reverse: 5'- GCA CGT AGT CTT CGA TCA CT -3'; and β-actin, forward: 5'- CCG TCT TCC CCT CCA TCG T -3', reverse: 5'- ATC GTC CCA GTT GGT TAC AAT GC -3'.
All experiments were repeated three times. All data are expressed as mean values standard deviation (SD). Statistical evaluations were made by ANOVA followed a Tukey’s HSD multiple comparison test. A value of p < 0.05 was considered significant.
This research was supported by the Globalization of Korean Foods R&D program, funded by the Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea (No. 912024–1) in part from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2009-0094017).
- Choi EM: Ligularia fischeri leaf extract prevents the oxidative stress in DBA/1J mice with type II collagen-induced arthritis. J Appl Toxicol. 2007, 27: 176-182. 10.1002/jat.1190.View ArticlePubMedGoogle Scholar
- Kim SM, Jeon JS, Kang SW, Jung YJ, LY LN, Um BH: Content of antioxidative caffeoylquinic acid derivatives in field-grown Ligularia fischeri (Ledeb.) Turcz and responses to sunlight. J Agric Food Chem. 2012, 60: 5597-5603. 10.1021/jf300976y.View ArticlePubMedGoogle Scholar
- Choi EM, Ding Y, Nguyen HT, Park SH, Kim YH: Antioxidant Activity of Gomchi (Ligularia fischeri) leaves. Food Sci Biotech. 2007, 16: 710-714.Google Scholar
- Bae JY, Yu SO, Kim YM, Chon SU, Kim BW, Heo BG: Physiological activity of methanol extracts from Ligularia fischeri and their hyperplasia inhibition activity of cancer cell. J Bio-Enviro Cont. 2009, 18: 67-73.Google Scholar
- An SJ, Park HS, Kim GH: Evaluation of the antioxidant activity of cooked Gomchwi (Ligularia fischeri) using the myoglobin methods. Prev Nutr Food Sci. 2014, 19: 34-39. 10.3746/pnf.2014.19.1.034.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim SM, Kang SW, Um BH: Extraction conditions of radical scavenging caffeolyquinc acids from gomchui (Ligularia fischeri) Tea. J Kor Soc Food Sci Nutr. 2010, 39: 399-405. 10.3746/jkfn.2010.39.3.399.View ArticleGoogle Scholar
- Shang YF, Kim SM, Song DG, Pan CH, Lee WJ, Um BH: Isolation and identification of antioxidant compounds from Ligularia fischeri. J Food Sci. 2010, 75: C530-C535. 10.1111/j.1750-3841.2010.01714.x.View ArticlePubMedGoogle Scholar
- Choi J, Park JK, Lee KT, Park KK, Kim WB, Lee JH, Jung HJ, Park HJ: In vivo antihepatotoxic effects of Ligularia fischeri var. spiciformis and the identification of the active component, 3,4-dicaffeoylquinic acid. J Med Food. 2005, 8: 348-352. 10.1089/jmf.2005.8.348.View ArticlePubMedGoogle Scholar
- Yu T, Ahn HM, Shen T, Yoon K, Jang HJ, Lee YJ, Yang HM, Kim JH, Kim C, Han MH, Cha SH, Kim TS, Kim SY, Lee J, Cho JY: Anti-inflammatory activity of ethanol extract derived from Phaseolus angularis beans. J Ethnopharmacol. 2011, 137: 1197-1206. 10.1016/j.jep.2011.07.048.View ArticlePubMedGoogle Scholar
- Francisco V, Costa G, Figueirinha A, Marques C, Pereira P, Miguel Neves B, Celeste Lopes M, Garcia-Rodriguez C, Teresa Cruz M, Teresa Batista M: Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-kappa B pathway inhibition: contribution of chlorogenic acid. J Ethnopharmacol. 2013, 148: 126-134. 10.1016/j.jep.2013.03.077.View ArticlePubMedGoogle Scholar
- Seo CS, Lee MY, Shin IS, Lee JA, Ha H, Shin HK: Spirodela polyrhiza (L.) Sch. ethanolic extract inhibits LPS-induced inflammation in RAW264.7 cells. Immunopharmacol Immunotoxicol. 2012, 34: 794-802. 10.3109/08923973.2012.656273.View ArticlePubMedGoogle Scholar
- Dos Santos MD, Chen G, Almeida MC, Soares DM, De Souza GE, Lopes NP, Lantz RC: Effects of caffeoylquinic acid derivatives and C-flavonoid from Lychnophora ericoides on in vitro inflammatory mediator production. Nat Prod Commun. 2010, 5: 733-740.PubMedGoogle Scholar
- Yang YZ, Tang YZ, Liu YH: Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells. J Ethnopharmacol. 2013, 148: 271-276. 10.1016/j.jep.2013.04.025.View ArticlePubMedGoogle Scholar
- Zhang D, Hamauzu Y: Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. Food Chem. 2004, 88: 503-509. 10.1016/j.foodchem.2004.01.065.View ArticleGoogle Scholar
- Young G, Jolly P: Microwaves: the potential for use in dairy processing. Aust J Dairy Tech. 1990, 45: 34-37.Google Scholar
- Miglio C, Chiavaro E, Visconti A, Fogliano V, Pellegrini N: Effects of different cooking methods on nutritional and physicochemical characteristics of selected vegetables. J Agric Food Chem. 2008, 56: 139-147. 10.1021/jf072304b.View ArticlePubMedGoogle Scholar
- Andlauer W, Stumpf C, Hubert M, Rings A, Furst P: Influence of cooking process on phenolic marker compounds of vegetables. Int J Vitam Nutr Res. 2003, 73: 152-159. 10.1024/0300-9822.214.171.124.View ArticlePubMedGoogle Scholar
- Bunea A, Andjelkovic M, Socaciu C, Bobis O, Neacsu M, Verhe R, Camp JV: Total and individual carotenoids and phenolic acids content in fresh, refrigerated and processed spinach (Spinacia oleracea L.). Food Chem. 2008, 108: 649-656. 10.1016/j.foodchem.2007.11.056.View ArticlePubMedGoogle Scholar
- Lee BI, Nugroho A, Bachri MS, Choi J, Lee KR, Choi JS, Kim WB, Lee KT, Lee JD, Park HJ: Anti-ulcerogenic effect and HPLC analysis of the caffeoylquinic acid-rich extract from Ligularia stenocephala. Biol Pharm Bull. 2010, 33: 493-497. 10.1248/bpb.33.493.View ArticlePubMedGoogle Scholar
- Piao X, Mi XY, Tian YZ, Wu Q, Piao HS, Zeng Z, Wang D, Piao X: Rapid identification and characterization of antioxidants from Ligularia fischeri. Arch Pharm Res. 2009, 32: 1689-1694. 10.1007/s12272-009-2204-z.View ArticlePubMedGoogle Scholar
- Lee KH, Choi EM: Analgesic and anti-inflammatory effects of Ligularia fischeri leaves in experimental animals. J Ethnopharmacol. 2008, 120: 103-107. 10.1016/j.jep.2008.07.038.View ArticlePubMedGoogle Scholar
- Choi EM, Suh KS: Ligularia fischeri leaf extract suppresses proinflammatory mediators in SW982 human synovial cells. Phytother Res. 2009, 23: 1575-1580. 10.1002/ptr.2823.View ArticlePubMedGoogle Scholar
- Hwang SJ, Kim YW, Park Y, Lee HJ, Kim KW: Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflamm Res. 2013, 63: 81-90.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.