RAW 264.7 Macrophage Cell Line: In Vitro Model for the Evaluation of the Immunomodulatory Activity of Zingiberaceae http://.www.doi.org/10.26538/tjnpr/v7i2.3

Main Article Content

Anami Riastri
Dyaningtyas D.P. Putri
Miftahus Sa’adah
Andayana P. Gani
Retno Murwanti


The immune response plays an essential role in the body's defense against infection. Macrophages are promising targets against which to screen agents that modulate immune responses. In vitro analysis, primarily using cell lines, is preferred to screen bioactivity initially. RAW 264.7 cells comprise a model macrophage cell line close to primary murine macrophages. Indonesian people often use the Zingiberaceae family or “empon-empon" in the favorable treatment of several diseases, including immune disorders. This review highlights several studies of Zingiberaceae using RAW 264.7 cells, focusing on the observed immunomodulatory activity and assay methods. The research on RAW 264.7 macrophage cell line in Zingiberaceae was gathered using Scopus, PubMed, ScienceDirect, and Google Scholar from 2012-2021. Based on the reviews, the Zingiberaceae family has immunomodulatory effects by increasing or  decreasing inflammatory mediators such as NO, IL-6, TNF-α, IL-1β, IL-10, and phagocytosis activity in RAW 264.7 cells. Several methods can assess the activation of RAW 264.7 cells, including cell viability assays using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT); measuring nitric oxide production (Griess reaction), cytokine production (enzyme-linked immunosorbent assay [ELISA]), and phagocytosis (neutral red uptake assay); and detecting DNA (real-time quantitative polymerase chain reaction [qPCR]). The information provided in this review presents avenues for future investigations of the immunomodulatory activity of Zingiberaceae.

Article Details

How to Cite
Riastri, A., Putri, D. D., Sa’adah, M., Gani, A. P., & Murwanti, R. (2023). RAW 264.7 Macrophage Cell Line: In Vitro Model for the Evaluation of the Immunomodulatory Activity of Zingiberaceae: http://.www.doi.org/10.26538/tjnpr/v7i2.3. Tropical Journal of Natural Product Research (TJNPR), 7(2), 2316–2324. Retrieved from https://tjnpr.org/index.php/home/article/view/1615
Author Biographies

Andayana P. Gani, Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.

Medicinal Plants and Natural Products Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.

Retno Murwanti, Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.

Medicinal Plants and Natural Products Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia.


Marshall JS, Warrington R, Watson W, Kim HL. An introduction to immunology and immunopathology. Allergy Asthma Clin Immunol. 2018; 14(S2):49.

Watanabe S, Alexander M, Misharin AV, Budinger GRS. The role of macrophages in the resolution of inflammation. J Clin Invest. 2019; 129(7):2619-28.

Sreejit G, Fleetwood AJ, Murphy AJ, Nagareddy PR. Origins and diversity of macrophages in health and disease. Clin Transl Immunol. 2020; 9(12). e1222.

Elisia I, Pae HB, Lam V, Cederberg R, Hofs E, Krystal G. Comparison of RAW 264.7, human whole blood and PBMC assays to screen for immunomodulators. J Immunol Methods. 2018; 452:26-31.

Khokra SL, Parashar B, Dhamija HK, Bala M. Immunomodulators: Immune System Modifiers. Res J Pharm Technol. 2012; 5:169-74.

Subositi D, Wahyono S. Study of the genus Curcuma in Indonesia used as traditional herbal medicines. Biodiversitas J Biol Divers. 2019; 20(5):1356-1361.

Singh N, Tailang M, Mehta SC. A Review on Herbal Plants as Immunomodulators. Int J Pharm Sci Res. 2016; 7(9):3602-3610.

Hartley JW, Evans LH, Green KY, Naghashfar Z, Macias AR, Zerfas PM, Ward JM. Expression of infectious murine leukemia viruses by RAW 264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line. Retrovirology. 2008; 5(1):1-6.

Hwang J, Ma J, Park J, Jung H, Park Y. Anti-inflammatory and antioxidant effects of MOK, a polyherbal extract, on lipopolysaccharide-stimulated RAW 264.7 macrophages. Int J Mol Med. 2018; 43:26-36.

Madhvi A, Mishra H, Leisching G, Mahlobo P, Baker B. Comparison of human monocyte derived macrophages and THP1-like macrophages as in vitro models for M. tuberculosis infection. Comp Immunol Microbiol Infect Dis. 2019; 67:101355.

Deng Y, Govers C, Beest E ter, van Dijk AJ, Hettinga K, Wichers HJ. A THP-1 Cell Line-Based Exploration of Immune Responses Toward Heat-Treated BLG. Front Nutr. 2021; 7:612397.

Taciak B, Bia?asek M, Braniewska A, Sas Z, Sawicka P, Kiraga ?, Rygiel T, Krol M, Roberts DD. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. PLOS ONE. 2018; 13(6):e0198943.

Hendrickx S, Van Bockstal L, Caljon G, Maes L, Kita K. Indepth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates. PLoS Negl Trop Dis. 2019; 13(12):e0007885.

Geraghty RJ, Capes-Davis A, Davis JM, Downward J, Freshney RI, Knezevic I, Lovell-Badge R, Masters JR, Meredith J, Stacey GN, Thraves P, Vias M. Guidelines for the use of cell lines in biomedical research. Br J Cancer. 2014; 111(6):1021-46.

Guo D, Zhang X, Huang Z, Zhou X, Zhu L, Zhao Y, Gu N. Comparison of cellular responses across multiple passage numbers in Ba/F3-BCR-ABL cells induced by silver nanoparticles. Sci China Life Sci. 2012; 55(10):898-905.

Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013; 496(7446):445-55.

Collin-Osdoby P, Osdoby P. RANKL-Mediated Osteoclast Formation from Murine RAW 264.7 cells. Methods Mol Biol. 2012; 816:187-202.

Günther J, Koy M, Berthold A, Schuberth HJ, Seyfert HM. Comparison of the pathogen species-specific immune response in udder derived cell types and their models. Vet Res. 2016; 47(1):22.

Fard M, Arulselvan P, Karthivashan G, Adam S, Fakurazi S. Bioactive extract from Moringa oleifera inhibits the proinflammatory mediators in lipopolysaccharide stimulated macrophages. Pharmacogn Mag. 2015; 11(44):556-63.

Park SH, Kyeong MS, Hwang Y, Ryu SY, Han SB, Kim Y. Inhibition of LPS binding to MD-2 co-receptor for suppressing TLR4-mediated expression of inflammatory cytokine by 1-dehydro-10-gingerdione from dietary ginger. Biochem Biophys Res Commun. 2012; 419(4):735-40.

Tan WS, Arulselvan P, Karthivashan G, Fakurazi S. Moringa oleifera Flower Extract Suppresses the Activation of Inflammatory Mediators in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages via NF-?B Pathway. Mediators Inflamm. 2015; 2015:1-11.

Yudhawan I, Ediati S, Puspitasari I. Immunomodulatory effect of Standardized Polysaccharide Fraction syrup from Noni fruit (Morinda citrifolia) on Cytokines level (IL-2 and IFN-?) and Its Histological Evaluation in Rats Vaccinated with Hepatitis-B. Res J Pharm Technol. 2020; 13(2):882-8.

Aki T, Funakoshi T, Noritake K, Unuma K, Uemura K. Extracellular glucose is crucially involved in the fate decision of LPS-stimulated RAW 264.7 murine macrophage cells. Sci Rep. 2020; 10(1):10581.

Li D and Wu M. Pattern recognition receptors in health and diseases. Signal Transduct Target Ther. 2021; 6(1):291.

George G, Shyni GL, Abraham B, Nisha P, Raghu KG. Downregulation of TLR4/MyD88/p38MAPK and JAK/STAT pathway in RAW 264.7 cells by Alpinia galangareveals its beneficial effects in inflammation. J Ethnopharmacol. 2021; 275:114132.

Karunarathne WAHM, Lee KT, Choi YH, Jin CY, Kim GY. Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-?B signaling pathway. Phytomedicine. 2020; 76:153237.

Cho YC, Vuong HL, Ha J, Lee S, Park J, Wibow AE, Cho S. Inhibition of Inflammatory Responses by Centella asiaticavia Suppression of IRAK1-TAK1 in Mouse Macrophages. Am J Chin Med. 2020; 48(05):1103-20.

Bai Y, Jiang Y, Liu T, Li F, Zhang J, Luo Y, Zhang L, Yan G, Feng Z, Li X, Wang X, Hu W. Xinjiang herbal tea exerts immunomodulatory activity via TLR2/4-mediated MAPK signaling pathways in RAW 264.7 cells and prevents cyclophosphamide-induced immunosuppression in mice. J

Ethnopharmacol. 2019; 228:179-87.

Ahmed SMU, Luo L, Namani A, Wang XJ, Tang X. Nrf2 signaling pathway: Pivotal roles in inflammation. Biochim Biophys Acta BBA-Mol Basis Dis. 2017; 1863(2):585-97.

Karunatilleke NC, Fast CS, Ngo V, Brickenden A, Duennwald ML, Konermann L, Choi WY. Nrf2, the Major Regulator of the Cellular Oxidative Stress Response, is Partially Disordered. Int J Mol Sci. 2021; 22(14):7434.

Matole V, Thorat Y, Ghurghure S, Ingle S, Birajdar A, Nangare G, Safwan M, Saili M, Patil S, Zainab B, Aishwarya S. A Brief Review on Herbal Medicines. Res J Pharmacogn Phytochem. 2021; 13(2):101-2.

Wanjari AS, Wanjari DS. An Overview on Herbal Medicine. Res J Pharmacogn Phytochem. 2019; 11(1):14.

Andrina S, Churiyah C, Nuralih N. Anti-Inflammatory Effect of Ethanolic Extract of Curcuma aeruginosa Roxb Rhizome, Morinda Citrifolia Fruit and Apium graveolens Leaf on Lipopplysaccharide-induce RAW 264.7 Cell Lines. Indones J Cancer Chemoprevention. 2015; 6(3):84-8.

Samuel SM, Pramod K, Bijin EN, Ajithkumar KC, Jijith US. Herbal Remedies for Rheumatoid Arthritis. Res J Pharmacogn Phytochem. 2016; 8(1):32-26.

Lee JA, Lee MY, Shin IS, Seo CS, Ha H, Shin HK. Antiinflammatory Effects of Amomum compactum on RAW 264.7 cells via induction of heme oxygenase-1. Arch Pharm Res. 2012; 35(4):739-46.

Park JH, Jung YJ, Shrestha S, Lee SM, Lee TH, Lee CH, Han D, Kim J, Baek NI. Inhibition of NO Production LPSStimulated RAW 264.7 Macrophage Cells with Curcuminoids and Xanthorrhizol from the Rhizome of Curcuma xanthorrhiza Roxb and Quantitative Analysis Using HPLC. J Korean Soc Appl Biol Chem. 2014; 57(3):407-412.

Isa NM, Abdelwahab SI, Mohan S, Abdul AB, Sukari MA, Taha MME, Syam S, Narrima P, Cheah SC, Ahmad S, Mustafa MR. In vitro anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A, a chalcone isolated from Boesenbergia rotunda (L.) (fingerroot). Braz J Med

Biol Res. 2012; 45(6):524-30.

Moon-ai W, Niyomploy P, Boonsombat R, Sangvanich P, Karnchanatat A. A Superoxide Dismutase Purified from the Rhizome of Curcuma aeruginosa Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line. Appl Biochem Biotechnol. 2012; 66(8):2138-55.

Mustafa I, Chin NL, Fakurazi S, Palanisamy A. Comparison of Phytochemicals, Antioxidant and Anti-Inflammatory Properties of Sun-, Oven- and Freeze-Dried Ginger Extracts. Foods. 2019; 8(10):456.

Wany A, Kumari A, Gupta KJ. Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress. Plant Cell Environ. 2017; 40(12):3002-17.

Chandrasekaran C, Sundarajan K, Edwin J, Gururaja G, Mundkinajeddu D, Agarwal A. Immune-stimulatory and anti-inflammatory activities of Curcuma longa extract and its polysaccharide fraction. Pharmacogn Res. 2013; 5(2):71.

Pan M, Wu J, Ho C, Badmaev V. Effects of water extract of Curcuma longa (L.) roots on immunity and telomerase function. J Complement Integr Med. 2017; 14(3): 20150107.

Kawasaki K, Okuda-Hanafusa C, Aoyagi M, Taoka K, Yamamoto N, Muroyama K, Murosaki S, Yamamoto Y. Inhibitory effect of the compounds from the water extract of Curcuma longa on the production of PGE2 and NO in a macrophage cell line stimulated by LPS. Biosci Biotechnol

Biochem. 2018; 82(12):2109-17.

Figueira LW, de Oliveira JR, Camargo SEA, de Oliveira LD. Curcuma longa L. (turmeric), Rosmarinus officinalis L. (rosemary), and Thymus vulgaris L. (thyme) extracts aid murine macrophages (RAW 264.7) to fight Streptococcus mutans during in vitro infection. Arch Microbiol. 2020;


Figueira LW, de Oliveira JR, Netto AA, S Zamarioli L dos, Marcucci MC, Camargo SE, de Oliveira LD. Curcuma longaL. helps macrophages to control opportunistic microorganisms during host-microbe interactions. Future Microbiol. 2020; 15(13):1237-48.

Kim DW, Lee SM, Woo HS, Park JY, Ko BS, Heo JD, Ryu YB, Lee WS. Chemical constituents and anti-inflammatory activity of the aerial parts of Curcuma longa. J Funct Foods. 2016; 26:485-93.

Okuda-Hanafusa C, Uchio R, Fuwa A, Kawasaki K, Muroyama K, Yamamoto Y, Murosaki S. Turmeronol A and turmeronol B from Curcuma longa prevent inflammatory mediator production by lipopolysaccharide-stimulated RAW264.7 macrophages, partially via reduced NF-κB signaling.

Food Funct. 2019; 10(9):5779-88.

Yuandani, Nugraha S, Laila L, Satria D. Immunomodulatory effects of standardized extract of Curcuma mangga val. on cytokines, antibody and delayed-type hypersensitivity response in Wistar rats. Res Pharm Sci. 2021; 16(1):16.

Lee TK, Trinh TA, Lee SR, Kim S, So HM, Moon E, Hwang GS, Kang KS, Kim JH, Yamabe N, Kim KH. Bioactivitybased analysis and chemical characterization of antiinflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated RAW 264.7 cells. Bioorganic Chem.

; 82:26-32.

Tungcharoen P, Wattanapiromsakul C, Tansakul P, Nakamura S, Matsuda H, Tewtrakul S. Antiinflammation constituents from Curcuma zedoaroides. Phytother Res. 2018; 32(11):2312-20.

Yao F, Huang Y, Wang Y, He X. Anti-inflammatory diarylheptanoids and phenolics from the rhizomes of kencur (Kaempferia galanga L.). Ind Crops Prod. 2018; 125:454-61.

Tungcharoen P, Wattanapiromsakul C, Tansakul P, Nakamura S, Matsuda H, Tewtrakul S. Anti‐inflammatory effect of isopimarane diterpenoids from Kaempferia galanga. Phytother Res. 2020; 34(3):612-23.

Putra ED, Nazliniwaty, Syafruddin. Component Analysis of White Ginger (Zingiber officinale Roscoe) Extract and Red Ginger (Zingiber officinale Rubra) Extract. Trop J Nat Prod Res. 2021; 5(9):1634-7.

Saanin SN, Wahyudianingsih R, Afni M, Afifah E, Maesaroh M, Widowati W. Suppression of pro-inflammatory cytokines and mediators production by ginger (Zingiber officinaleRoscoe) ethanolic extract and gingerol in lipopolysaccharide-induced RAW 264.7 murine macrophage

cells. Indian J Nat Prod Resour. 2020; 11(4):260-6.

Arunporn Itharat. Accelerated Stability Study on AntiAllergic, Anti-inflammatory Activities and Phytochemical Contents of the Ethanolic Extract of Zingiber officinaleRoscoe. Sci Technol Asia. 2020; 25:8696.

Li F, Wang Y, Parkin KL, Nitteranon V, Liang J, Yang W. Isolation of quinone reductase (QR) inducing agents from ginger rhizome and their in vitro anti-inflammatory activity. Food Res Int. 2011; 44(6):1597-603.

Li F, Nitteranon V, Tang X, Liang J, Zhang G, Parkin KL. In vitro antioxidant and anti-inflammatory activities of 1-dehydro-[6]-gingerdione, 6-shogaol, 6-dehydroshogaol and hexahydrocurcumin. Food Chem. 2012; 135(2):332-7.

Lee HY, Park SH, Lee M, Kim HJ, Ryu SY, Kim ND, Hwang BY, Hong JT, Han SB, Kim Y. 1-Dehydro-[10]-gingerdione from ginger inhibits IKKβ activity for NF-κB activation and suppresses NF-κB-regulated expression of inflammatorygenes: Anti-inflammatory action of dehydrogingerdione. Br J Pharmacol. 2012; 167(1):128-40.

Huang SH, Lee CH, Wang HM, Chang YW, Lin CY, Chen CY, Chen YH. 6-Dehydrogingerdione Restrains Lipopolysaccharide-Induced Inflammatory Responses in RAW 264.7 Macrophages. J Agric Food Chem. 2014; 62(37):9171-9.

Yang X, Wei S, Lu X, Qiao X, Simal-Gandara J, Capanoglu E, Wozniak L, Zou L, Cao H, Xiao J, Tang X, Li N. A neutral polysaccharide with a triple helix structure from ginger: Characterization and immunomodulatory activity. Food Chem. 2021; 350:129261.

Pinho BR, Sousa C, Valentão P, Andrade PB, Sambhara S. Is Nitric Oxide Decrease Observed with Naphthoquinones in LPS Stimulated RAW 264.7 Macrophages a Beneficial Property? PLoS ONE. 2011; 6(8):e24098.

Liu Y, Su WW, Wang S, Li PB. Naringin inhibits chemokine production in an LPS-induced RAW 264.7 macrophage cell line. Mol Med Rep. 2012; 6(6):1343-50.

Muangnoi C, Chingsuwanrote P, Praengamthanachoti P, Svasti S, Tuntipopipat S. Moringa oleifera Pod Inhibits Inflammatory Mediator Production by LipopolysaccharideStimulated RAW 264.7 Murine Macrophage Cell Lines. Inflammation. 2012; 35(2):445-55.

Somensi N, Rabelo TK, Guimarães AG, Quintans-Junior LJ, de Souza Araújo AA, Moreira JCF, Daniel PG. Carvacrol suppresses LPS-induced pro-inflammatory activation in RAW 264.7 macrophages through ERK1/2 and NF-kB pathway. Int Immunopharmacol. 2019; 75:105743.

Vargas-Maya NI, Padilla-Vaca F, Romero-González OE, Rosales-Castillo EAS, Rangel-Serrano Á, Arias-Negrete S, Franco B. Refinement of the Griess method for measuring nitrite in biological samples. J Microbiol Methods. 2021; 187:106260.

Ramos-Payan R, Aguilar-Medina M, Estrada-Parra S, Gonzalez-y-Merchand JA, Favila-Castillo L, Monroy-Ostria A, Estrada-Garcia ICE. Quantification of Cytokine Gene Expression Using an Economical Real-Time Polymerase Chain Reaction Method Based on SYBRR Green I. Scand J

Immunol. 2003; 57(5):439-45.

Joffe AM, Bakalar MH, Fletcher DA. Macrophage phagocytosis assay with reconstituted target particles. Nat Protoc. 2020; 15(7):2230-46.

Wang J, Zhang H, Wang H, Wang J, Sun-Waterhouse D, Waterhouse GIN, Changyang M, Wenyi K. An immunomodulatory polysaccharide from blackberry seeds and its action on RAW 264.7 cells via activation of NF-κB/MAPK pathways. Food Agric Immunol. 2020; 31(1):575-86.

Yuan T, Zhang C, Qiu C, Xia G, Wang F, Lin B, Hua L, Chen L. Chemical constituents from Curcuma longa L. and their inhibitory effects of nitric oxide production. Nat Prod Res. 2018; 32(16):1887-92.

Cheng X, Li H, Wu P, Xu L, Xue J, Wei X. Two new bisabolane-type sesquiterpenoids from the cooking liquid of Curcuma longa rhizomes. Phytochem Lett. 2019; 29:169-72.

Huang Y, Xue C, He W, Zhao X. Inhibition effect of Zedoary turmeric oil on Listeria monocytogenes and Staphylococcus aureus growth and exotoxin proteins production. J Med Microbiol. 2019; 68(4):657-66.