Sains Malaysiana 48(8)(2019): 1737–1744

http://dx.doi.org/10.17576/jsm-2019-4808-20

 

Inhibitory Effects of Gynura procumbens Ethanolic Extract on Nitric Oxide Production and Inducible Nitric Oxide Synthase (iNOS) Protein Expression in Macrophages

(Kesan Perencatan Ekstrak Etanol Gynura procumbens terhadap Penghasilan Nitrik Oksida dan Ekspresi Protein Sintase Nitrik Oksida Teraruh (iNOS) di dalam Makrofaj)

 

TAN JIAH NING1, SYARATUL DALINA YUSOFF1, ZAKIAH JUBRI2, FHATAHEYA BUANG1, TAN ZE SONG1, AMEERAH BUDIONO1, IBRAHIM JANTAN3, ROZA DIANITA4, ENDANG KUMOLOSASI1, NORAZRINA AZMI1 & NORSYAHIDA MOHD FAUZI1*

 

1Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

 

2Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

3School of Pharmacy, Taylor's University, Lakeside Campus, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia

 

4School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM Pulau Pinang, Malaysia

 

Received: 14 September 2018/Accepted: 31 May 2019

 

ABSTRACT

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders.

 

Keywords: Gynura procumbens; inducible nitric oxide synthase; inflammation; lipopolysaccharide; macrophage; nitric oxide

ABSTRAK

Penghasilan nitrik oksida (NO) yang berlebihan oleh sintase nitrik oksida teraruh (iNOS) mungkin boleh dikaitkan dengan radang akut dan kronik. Makrofaj sebagai sel yang penting dalam sistem keimunan inat berupaya dirangsang dan menyebabkan aktivasi iNOS dan penghasilan NO yang berlebihan. Gynura procumbens ialah tumbuhan perubatan tempatan yang digunakan secara tradisi untuk merawat pelbagai jenis penyakit termasuk radang namun mekanisme aktiviti anti-radang oleh tumbuhan ini masih sukar difahami. Kajian ini dijalankan untuk mengkaji kesan terapeutik anti-radang oleh ekstrak etanol Gynura procumbens terhadap penghasilan NO dan ekspresi protein iNOS dalam makrofaj RAW 264.7 yang dirangsang oleh lipopolisakarida (LPS). Kebolehhidupan sel makrofaj RAW 264.7 oleh rawatan ekstrak etanol Gynura procumbens ditentukan dengan asai MTT. Reagen Griess digunakan untuk menentukan penghasilan NO diikuti rawatan ekstrak etanol Gynura procumbens atau dengan gabungan rangsangan LPS. Ekspresi protein iNOS dikaji dengan pemblotan western. Di bawah rawatan ekstrak etanol Gynura procumbens selama 24 jam menggunakan kepekatan 3.9 μg/mL hingga 500 μg/mL, makrofaj RAW 264.7 menunjukkan kebolehhidupan sel lebih daripada 90%. Perencatan paras penghasilan NO yang signifikan telah dikenal pasti dalam sel RAW 264.7 yang dirangsang oleh LPS bersama pra-rawatan 250 μg/mL ekstrak etanol Gynura procumbens (p<0.05) manakala semua kepekatan ekstrak etanol Gynura procumbens yang dipilih menunjukkan tiada perubahan yang signifikan dalam penghasilan NO tanpa perangsangan LPS. Pra-rawatan 250 μg/mL ekstrak etanol Gynura procumbens juga mendemonstrasikan perencatan ekspresi protein iNOS yang signifikan dalam sel RAW 264.7 yang dirangsang oleh LPS (p<0.05). Kesimpulannya, kajian ini menunjukkan bahawa ekstrak etanol Gynura procumbens mempamerkan potensi anti-radang melalui perencatan penghasilan NO dan ekspresi protein iNOS dalam makrofaj yang dirangsang oleh LPS, menunjukkan bahawa tumbuhan ini boleh dikaji secara lebih lanjut tentang kegunaannya yang bermanfaat dalam gangguan keradangan.

 

Kata kunci: Gynura procumbens; lipopolisakarida; makrofaj; nitrik oksida; radang; sintase nitrik oksida teraruh

REFERENCES

Akowuah, G.A., Sadikun, A. & Mariam, A. 2002. Flavonoid identification and hypoglycaemic studies of the butanol fraction from Gynura procumbens. Pharmaceutical Biology 40(6): 405-410.

Aktan, F. 2004. iNOS-mediated nitric oxide production and its regulation. Life Sciences 75(6): 639-653.

Aldridge, C., Razzak, A., Babcock, T.A., Helton, W.S. & Espat, N.J. 2008. Lipopolysaccharide-stimulated RAW 264.7 macrophage inducible nitric oxide synthase and nitric oxide production is decreased by an Omega-3 fatty acid lipid emulsion. The Journal of Surgical Research 149(2): 296-302.

Alexander, C. & Rietschel, E.T. 2001. Bacterial lipopolysaccharides and innate immunity. Journal of Endotoxin Research 7(3): 167-202.

Anderson, K.V. 2000. Toll signaling pathways in the innate immune response. Current Opinion in Immunology 12(1): 13-19.

Bobryshev, Y.V., Ivanova, E.A., Chistiakov, D.A., Nikiforov, N.G. & Orekhov, A.N. 2016. Macrophages and their role in atherosclerosis: Pathophysiology and transcriptome analysis. BioMed Research International 2016: 9582430.

Byeon, S.E., Lee, J., Kim, J.H., Yang, W.S., Kwak, Y.S., Kim, S.Y., Choung, E.S., Rhee, M.H. & Cho, J.Y. 2012. Molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from Korean red ginseng. Mediators of Inflammation 2012: 732860.

Dianita, R., Jantan, I., Amran, A.Z. & Jalil, J. 2015. Protective effects of Labisia pumilavar. alata on biochemical and histopathological alterations of cardiac muscle cells in isoproterenol-induced myocardial infarction rats. Molecules (20): 4746-4763.

Fujiwara, N. & Kobayashi, K. 2005. Macrophages in inflammation. Current Drug Targets: Inflammation and Allergy 4(3): 281- 286.

Hämäläinen, M., Nieminen, R., Vuorela, P., Heinonen, M. & Moilanen, E. 2007. Anti-inflammatory effects of flavonoids: Genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and NF-kappaB activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-kappaB activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages. Mediators of Inflammation 2007: 45673.

Harikrishnan, H., Jantan, I., Haque, M.A. & Kumolosasi, E. 2018. Anti-inflammatory effects of Phyllanthus amarus Schum. & Thonn. through inhibition of NF-κB, MAPK, and PI3K-Akt signaling pathways in LPS-induced human macrophages. BMC Complementary and Alternative Medicine 18(1): 224.

Hwang, S.J., Kim, Y.W., Park, Y., Lee, H.J. & Kim, K.W. 2014. Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflammation Research 63(1): 81-90.

Iskander, M.N., Song, Y., Coupar, I.M. & Jiratchariyakul, W. 2002. Anti-inflammatory screening of the medicinal plant Gynura procumbens. Plant Foods for Human Nutrition 57(3-4): 233-244.

Jacobs, A.T. & Ignarro, L.J. 2001. Lipopolysaccharide-induced expression of interferon-β mediates the timing of inducible nitric-oxide synthase induction in RAW 264.7 macrophages. The Journal of Biological Chemistry 276(51): 47950-47957.

Joo, T., Sowndhararajan, K., Hong, S., Lee, J., Park, S.Y., Kim, S. & Jhoo, J.W. 2014. Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumilaL. Saudi Journal of Biological Sciences 21(5): 427- 435.

Kaewseejan, N., Puangpronp, D. & Nakornriab, M. 2012. Evaluation of phytochemical composition and antibacterial property of Gynura procumbensextract. Asian Journal of Plant Sciences 11(2): 77-82.

Kamaruzaman, K.A. & Noor, M.M. 2017. Gynura procumbens leaf improves blood glucose level, restores fertility and libido of diabetic-induced male rats. Sains Malaysiana46(9): 1471-1477.

Kim, H.J., Tsoyi, K., Heo, J.M., Kang, Y.J., Park, M.K., Lee, Y.S., Lee, J.H., Seo, H.G., Yun-Choi, H.S. & Chang, K.C. 2007. Regulation of lipopolysaccharide-induced inducible nitric-oxide synthase expression through the nuclear factor-kappaB pathway and interferon-beta/tyrosine kinase 2/Janus tyrosine kinase 2-signal transducer and activator of transcription-1 signaling cascad. The Journal of Pharmacology and Experimental Therapeutics 320(2): 782-789.

Kim, J., Lee, C.W., Kim, E.K., Lee, S.J., Park, N.H., Kim, H.S., Kim, H.K., Char, K., Jang, Y.P. & Kime, J.W. 2011. Inhibition effect of Gynura procumbens extract on UV-B-induced matrix-metalloproteinase expression in human dermal fibroblasts. Journal of Ethnopharmacology137(1): 427-433.

Kim, M.S. & Kim, S.H. 2011. Inhibitory effect of astragalin on expression of lipopolysaccharide-induced inflammatory mediators through NF-κB in macrophages. Archives of Pharmacal Research 34(12): 2101-2107.

Kopincová, J., Púzserová, A. & Bernátová, I. 2012. L-NAME in the cardiovascular system - Nitric oxide synthase activator? Pharmacological Reports 64(3): 511-520.

Korhonen, R., Lahti, A., Kankaanranta, H. & Moilanen, E. 2005. Nitric oxide production and signaling in inflammation. Current Drug Targets: Inflammation and Allergy 4(4): 471- 479.

Kraaij, M.D., Vereyken, E.J.F., Leenen, P.J.M., van den Bosch, T.P.P., Rezaee, F., Betjes, M.G.H., Baan, C.C. & Rowshani, A.T. 2014. Human monocytes produce interferon-gamma upon stimulation with LPS. Cytokine 67(1): 7-12.

Li, F., Wang, W., Cao, Y., Liang, D., Zhang, W., Zhang, Z., Jiang, H., Guo, M. & Zhang, N. 2014. Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells. Journal of Surgical Research 192(2): 573-581.

Li, X.J., Mu, Y.M., Li, T.T., Yang, Y.L., Zhang, M.T., Li, Y.S., Zhang, W.K., He-Bin, T. & Shang, H.C. 2015. Gynura procumbens reverses acute and chronic ethanol-induced liver steatosis through MAPK/SREBP-1c-dependent and -independent pathways. Journal of Agricultural and Food Chemistry 63(38): 8460-8471.

Li, Y.H., Yan, Z.Q., Brauner, A. & Tullus, K. 2002. Activation of macrophage nuclear factor-κB and induct ion of inducible nitric oxide synthase by LPS. Respiratory Research 3(1): 23.

Łuszczki, J.J., Jaskólska, A., Dworzański, W. & Zółkowska, D. 2011. 7-nitroindazole, but not NG-nitro-L-arginine, enhances the anticonvulsant activity of pregabalin in the mouse maximal electroshock-induced seizure model. Pharmacological Reports: PR 63(1): 169-175.

Ma, Z., Piao, T., Wang, Y. & Liu, J. 2015. Astragalin inhibits IL-1β-induced inflammatory mediators production in human osteoarthritis chondrocyte by inhibiting NF-κB and MAPK activation. International Immunopharmacology 25(1): 83-87.

Mustafizur Rahman, A.F.M. & Al Asad, M.S. 2013. Chemical and biological investigations of the leaves of Gynura procumbens. International Journal of Biosciences 3(4): 36-43.

Nicholas, C., Batra, S., Vargo, M.A., Voss, O.H., Gavrilin, M.A., Wewers, M.D., Guttridge, D.C., Grotewold, E. & Doseff, A.I. 2007. Apigenin blocks lipopolysaccharide-induced lethality in vivo and proinflammatory cytokines expression by inactivating NF-kappa B through the suppression of p65 phosphorylation. Journal of Immunology 179(10): 7121- 7127.

Rosidah, Y., Sadikun, A.M. & Asmawi, M. 2008. Antioxidant potential of Gynura procumbens. Pharmaceutical Biology 46(9): 616-625.

Salim, T., Sershen, C.L. & May, E.E. 2016. Investigating the role of TNF-α and IFN-γ activation on the dynamics of iNOS gene expression in LPS stimulated macrophages. Plos ONE 11(6): e0153289.

Shao, D., Dunlop, W.D., Lui, E.M.K. & Bernards, M.A. 2008. Immunostimulatory and anti-inflammatory polysaccharides from Tripterygium wilfordii: Comparison with organic extracts. Pharmaceutical Biology 46(1-2): 8-15.

Shi, Q., Cao, J., Fang, L., Zhao, H., Liu, Z., Ran, J., Zheng, X., Li X., Zhou, Y., Di, Ge., Zhang, H., Wang, L., Rana, Y. & Fua, J. 2014. Geniposide suppresses LPS-induced nitric oxide, PGE2 and inflammatory cytokine by downregulating NF- κB, MAPK and AP-1 signaling pathways in macrophages. International Immunopharmacology20(2): 298-306.

Soufli, I., Toumi, R., Rafa, H. & Touil-Boukoffa, C. 2016. Overview of cytokines and nitric oxide involvement in immuno-pathogenesis of inflammatory bowel diseases. World Journal of Gastrointestinal Pharmacology and Therapeutics 7(3): 353-360.

Sunarwidhi, A.L., Sudarsono, S. & Nugroho, A.E. 2014. Hypoglycemic effect of combination of Azadirachta indicaA. Juss. and Gynura procumbens(Lour.) Merr. ethanolic extracts standardized by rutin and quercetin in alloxan-induced hyperglycemic rats. Advanced Pharmaceutical Bulletin 4(2): 613-618.

Tabas, I. & Bornfeldt, K.E. 2016. Macrophage phenotype and function in different stages of atherosclerosis. Circulation Research 118(4): 653-667.

Tripathi, P., Tripathi, P., Kashyap, L. & Singh, V. 2007. The role of nitric oxide in inflammatory reactions. FEMS Immunology & Medical Microbiology 51(3): 443-452.

Wong, S.K., Lee, M.S.J., Sudi, S., Hassan, R.M., Lee, P.C., Embi, N. & Sidek, H. 2015. Anti-malarial and anti-inflammatory effects of Gynura procumbensare mediated by kaempferol via inhibition of glycogen synthase kinase-3β (GSK3β). Sains Malaysiana44(10): 1489-1500.

Wu, C.C., Lii, C.K., Liu, K.L., Chen, P.Y. & Hsieh, S.L. 2013. Antiinflammatory activity of gynura bicolor (紅鳳菜 Hóng Fèng Cài) ether extract through inhibits nuclear factor Kappa B activation. Journal of Traditional and Complementary Medicine 3(1): 48-52.

Yuandani, Jantan, I. & Husain, K. 2017. 4,5,4’-Trihydroxychalcone, 8,8’-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol and rutin from Gynura segetum inhibit phagocytosis, lymphocyte proliferation, cytokine release and nitric oxide production from phagocytic cells. BMC Complementary and Alternative Medicine 17(1): 211.

 

*Corresponding author; email: drnorsyahida@ukm.edu.my

 

 

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