Nrf2/HO-1 Mediated Antioxidant Activities, Cytotoxicity Analysis and LCESI/ MS Profiling of Eulophia nuda L.

Author(s): Vikas Nanekar, Varsha Shriram*, Tushar Khare, Vinay Kumar*.

Journal Name: The Natural Products Journal

Volume 10 , Issue 1 , 2020

Become EABM
Become Reviewer

Graphical Abstract:


Background: Eulophia nuda, is a highly medicinal orchid with strong antioxidant and anticancer potentials in traditional systems of medicine. But few reports are available on the scientific validation. The aim of the study was to investigate phytochemical constituents, antioxidant and cytotoxic efficacies of extracts of Eulophia nuda, and the underlying mechanisms-of-action via upregulation of nuclear transcription factor-erythroid-2 related factor (Nrf2) and hemeoxygenase-1 (HO-1) pathways.

Methods: Petroleum Ether (PEE), Ethyl Acetate (EAE), Methanol (ME) and Aqueous Methanol (AqME) extracts of shade dried tubers were obtained and concentrated in vacuo. Total phenols, flavonoids, condensed tannins, ascorbic acid and carotenoids were estimated from the extracts using standard methods. Antioxidant activities of extracts were determined by total antioxidant, FRAP, ABTS, DPPH, OH, H2O2, NO, O2 ·- radical scavenging assays. Cytotoxicity of EAE and ME were assessed against MCF7 cells in vitro. LC-ESI/MS profiling of EAE was carried out. Quantitative Real-Time (qRT) PCR was used for the expression analysis of Nrf2 and HO1 genes in EAE-treated MCF7 cells.

Results: In vitro models confirmed strong dose-dependent antioxidant and free-radical scavenging potencies of E. nuda tuber extracts. Overall antioxidant efficacies were in the order EAE > ME > AqME > PEE. EAE showed striking cytotoxicity followed by ME (0.86% and 5.17% cell survival at 1000 µg ml-1, respectively). LC-ESI/MS profiling of most potent extract EAE revealed 37 identified compounds including catechin, taxifolin, tocopherol, trigallic acid and chlorogenic acid, all known for their strong antioxidant/anticancer properties. Expression levels of Nrf2 and HO1 genes were up-regulated in MCF7 cells beyond 50 μg ml-1 extract concentration with > 2-fold increase at 200 µg ml-1 EAE.

Conclusion: The data demonstrated that E. nuda extracts possess strong free radical scavenging and antioxidant efficacies and the mechanism of action may be via inducing Nrf2 and HO-1.

Keywords: Eulophia nuda, antioxidants, Nrf2, hemeoxygenase, cytotoxicity, LC-ESI/MS, total phenols, total flavonoids.

Nanekar, V.; Shriram, V.; Kumar, V.; Kishor, P.B.K. Asymbiotic in vitro seed germination and seedling development of Eulophia nuda Lindl. An endangered medicinal orchid. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci., 2014, 84, 837-846.
Shriram, V.; Nanekar, V.; Kumar, V.; Kishor, P.B.K. In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl. Indian J. Exp. Biol., 2014, 52, 1112-1121.
Kumar, V.; Lemos, M.; Sharma, M.; Shriram, V. Antioxidant and DNA damage protecting activities of Eulophia nuda Lindl. Free Radic. Antioxid., 2013, 3, 55-60.
Singh, A. Duggal, S. Medicinal orchids - An overview. Ethnobot. Leafl., 2009, 13, 399-412.
Jagdale, S.P.; Shimpi, S.; Chachad, D. Pharmacological studies of Salep. J. Herb. Med. Toxicol., 2009, 3, 153-156.
Hossain, M.M. Therapeutic orchids: Traditional uses and recent advances. An overview. Fitoterapia, 2011, 82, 102-140.
Schuster, R.; Zeindl, L.; Holzer, W.; Khumpirapang, N.; Okonogi, S.; Viernstein, H.; Mueller, M. Eulophia macrobulbon - an orchid with significant anti-inflammatory and antioxidant effect and anticancerogenic potential exerted by its root extract. Phytomedicine, 2017, 24, 157-165.
Narkhede, A.; Nirmal, P.; Tupe, R.; Kulkarni, O.; Harsulkar, A.; Jagtap, S. In vitro antioxidant, antiglycation and α- amylase inhibitory potential of Eulophia ochreata L. J. Pharm. Res., 2012, 5, 2532-2537.
Khansari, N.; Shakiba, Y.; Mahmoudi, M. Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent Pat. Inflamm. Allergy Drug Discov., 2009, 3, 73-80.
Kumar, H.; Kim, I.S.; More, S.V.; Kim, B.W.; Choi, D.K. Natural product-derived pharmacological modulators of Nrf2/ARE pathway for chronic diseases. Nat. Prod. Rep., 2014, 31, 109-139.
Dinkova-Kostova, A.T.; Talalay, P. Direct and indirect antioxidant properties of inducers of cytoprotective proteins. Mol. Nutr. Food Res., 2008, 52, S128-S138.
Kim, J.K.; Jang, H.D. Nrf2-mediated HO-1 induction coupled with the ERK signaling pathway contributes to indirect antioxidant capacity of caffeic acid phenethyl ester in HepG2 cells. Int. J. Mol. Sci., 2014, 15, 12149-12156.
Motohashi, H.; Yamamoto, M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol. Med., 2004, 10, 549-557.
Magesh, S.; Chen, Y.; Hu, L. Small molecule modulators of Keap1-Nrf2-ARE pathway as potential preventive and therapeutic agents. Med. Res. Rev., 2012, 32, 687-726.
Son, Y.; Lee, J.H.; Chung, H.T.; Pae, H.O. Therapeutic roles of heme oxygenase-1 in metabolic diseases: Curcumin and resveratrol analogues as possible inducers of heme oxygenase-1. Oxid. Med. Cell. Longev., 2013, 2013639541
Yin, W.; Li, Y. Curcumin upregulate expression of HO-1 and Nrf-2 in SHSY5Y cells. In: Proceedings of the 4th Int. Conf. Bioinform.Biomed. Eng; , 2010; pp. 1-4.
Gul, M.Z.; Bhakshu, L.M.; Ahmad, F.; Kondapi, A.K.; Qureshi, I.A.; Ghazi, I.A. Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays. BMC Complement. Altern. Med., 2011, 11, 64.
Kalita, P.; Tapan, B.K.; Pal, T.K.; Kalita, R. Estimation of total flavonoids content and antioxidant activities of methanolic whole plant extract of Biophytum sensitivum Linn. J. Drug Deliv. Ther., 2013, 3, 33-37.
Rhimi, W.; Ben Salem, I.; Immediato, D.; Saidi, M.; Boulila, A.; Cafarchia, C. Chemical composition, antibacterial and antifungal activities of crude Dittrichia viscosa (L.) greuter leaf extracts. Molecules, 2017, 22, 942.
Kapur, A.; Hasković, A.; Čopra-Janićijević, A.; Klepo, L.; Topčagić, A.; Tahirović, I.; Sofić, E. Spectrophotometric analysis of total ascorbic acid content in various fruits and vegetables. Bullet. Chemist Technol. Bosn. Herz., 2012, 38, 39-42.
Jensen, A. Chlorophylls and carotenoids. In: Hellebust, J.A.,Craige, I.S. (Eds.),Handbook of Phycological Methods. Physiological and Biochemical Methods; Cambridge University Press, 1978; pp. 59-70.
Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal. Biochem., 1999, 269, 337-341.
Benzie, I.F.; Strain, J.J. Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. In: Methods Enzymol. 299.; 15-27; Academic Press, 1999.
Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med., 1999, 26, 1231-1237.
Braca, A.; Sortino, C.; Politi, M.; Morelli, I.; Mendez, J. Antioxidant activity of flavonoids from Licania licaniaeflora. J. Ethnopharmacol., 2002, 79, 379-381.
Kunchandy, E.; Rao, M.N.A. Oxygen radical scavenging activity of curcumin. Int. J. Pharm., 1990, 58, 237-240.
Cetinkaya, Y.; Gocer, H.; Menzek, A.; Gulcin, I. Synthesis and antioxidant properties of (3,4 dihydroxyphenyl)(2,3,4- trihydroxyphenyl) methanone and its derivatives. Archiv. der Pharmazie,, 2012, 345, 323-334.
Garratt, D.C. The quantitative analysis of drugs; Vol. 3. pp. 456-458.Chapman and Hall Ltd. Japan;. , 1964.
Fontana, M.; Mosca, L.; Rosei, M.A. Interaction of enkephalines with oxyradicals. Biochem. Pharmacol., 2001, 61, 1253-1257.
Nile, S.H.; Park, S.W. Chromatographic analysis, antioxidant, anti-inflammatory, and xanthine oxidase inhibitory activities of ginger extracts and its reference compounds. Ind. Crops Prod., 2015, 70, 238-244.
Wang, Y.; Gao, Y.; Ding, H.; Liu, S.; Han, X.; Gui, J.; Liu, D. Subcritical ethanol extraction of flavonoids from Moringa oleifera leaf and evaluation of antioxidant activity. Food Chem., 2017, 218, 152-158.
Uluata, S.; McClements, D.J.; Decker, E.A. How the multiple antioxidant properties of ascorbic acid affect lipid oxidation in oil-in-water emulsions. J. Agric. Food Chem., 2015, 63, 1819-1824.
Chinsam, M.; Finnie, J.F.; Van Staden, J. Anti-inflammatory, antioxidant, anti-cholinesterase activity and mutagenicity of South African medicinal orchids. S. Afr. J. Bot., 2014, 91, 88-98.
Paudel, M.R.; Chand, M.B.; Pant, B. Cytotoxic activity of antioxidant-riched Dendrobium longicornu. Pharmacogn. J., 2017, 9, 499-503.
During, A.; Debouche, C.; Raas, T.; Larondelle, Y. Among plant lignans, pinoresinol has the strongest antiinflammatory properties in human intestinal caco-2 cells-3. J. Nutr., 2012, 142, 1798-1805.
Jarial, R.; Thakur, S.; Sakinah, M.; Zularisam, A.W.; Sharad, A.; Kanwar, S.S.; Singh, L. Potent anticancer, antioxidant and antibacterial activities of isolated flavonoids from Asplenium nidus. J. King. Saud. Univ., 2016, 30, 185-192.
Topal, F.; Nar, M.; Gocer, H.; Kalin, P.; Kocyigit, U.M.; Gülçin, İ.; Alwasel, S.H. Antioxidant activity of taxifolin: An activity-structure relationship. J. Enzyme Inhib. Med. Chem., 2016, 31, 674-683.
Kim, H.S.; Quon, M.J.; Kim, J.A. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol.; epigallocatechin 3-gallate. Redox Biol., 2014, 32, 187-195.
MacLeod, A.K.; McMahon, M.; Plummer, S.M.; Higgins, L.G.; Penning, T.M.; Igarashi, K.; Hayes, J.D. Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: Demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds. Carcinogenesis, 2009, 30, 1571-1580.
Yan, M.; Li, G.; Petiwala, S.M.; Householter, E.; Johnson, J.J. Standardized rosemary (Rosmarinus officinalis) extract induces Nrf2/sestrin-2 pathway in colon cancer cells. J. Funct. Foods, 2015, 13, 137-147.
Valdés, A.; García-Cañas, V.; Koçak, E.; Simó, C.; Cifuentes, A. Foodomics study on the effects of extracellular production of hydrogen peroxide by rosemary polyphenols on the anti-proliferative activity of rosemary polyphenols against HT-29 cells. Electrophoresis, 2016, 37, 1795-1804.
Bajpai, V.K.; Alam, M.B.; Quan, K.T.; Kwon, K.R.; Ju, M.K.; Choi, H.J.; Lee, J.S.; Yoon, J.I.; Majumder, R.; Rather, I.A.; Kim, K. Antioxidant efficacy and the upregulation of Nrf2-mediated HO-1 expression by (+)-lariciresinol, a lignan isolated from Rubia philippinensis, through the activation of p38. Sci. Rep., 2017, 7, 46035.
Wu, K.C.; McDonald, P.R.; Liu, J.; Klaassen, C.D. Screening of natural compounds as activators of the keap1-Nrf2 pathway. Planta Med., 2014, 80, 97-104.
Mena, P.; Calani, L.; Dall’Asta, C.; Galaverna, G.; García-Viguera, C.; Bruni, R.; Crozier, A.; Del Rio, D. Rapid and comprehensive evaluation of (poly) phenolic compounds in pomegranate (Punica granatum L.) juice by UHPLC-MS. Molecules, 2012, 17, 14821-14840.
Tuchinda, P.; Udchachon, J.; Khumtaveeporn, K.; Taylor, W.C.; Engelhardt, L.M.; White, A.H. Phenanthrenes of Eulophia nuda. Phytochemistry, 1988, 27, 3267-3271.
Nishimura, H.; Nonaka, G.; Nishioka, I. Tannins and related compounds. XX. Two new ellagitannins containing a proto-quercitol core from Quercus stenophylla Makino. ‎. Chem. Pharm. Bull., 1984, 32, 1750-1753.
Abu-Reidah, I.M.; Ali-Shtayeh, M.S.; Jamous, R.M.; Arráez-Román, D.; Segura-Carretero, A. HPLC–DAD–ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem., 2015, 166, 179-191.
Ye, M.; Yang, W.Z.; Liu, K.D.; Qiao, X.; Li, B.J.; Cheng, J.; Feng, J.; Guo, D.A.; Zhao, Y.Y. Characterization of flavonoids in Millettia nitida var. hirsutissima by HPLC/DAD/ESI-MS. J. Pharm. Anal., 2012, 2, 35-42.
Shabana, M.M.; El Sayed, A.M.; Yousif, M.F.; El Sayed, A.M.; Sleem, A.A. Bioactive constituents from Harpephyllum caffrum Bernh. and Rhus coriaria L. Pharmacogn. Mag., 2011, 7, 298-306.
Fröhlich, B.; Niemetz, R.; Gross, G.G. Gallotannin biosynthesis: Two new galloyltransferases from Rhus typhina leaves preferentially acylating hexa- and heptagalloylglucoses. Planta, 2002, 216, 168-172.
Francescato, L.N.; Debenedetti, S.L.; Schwanz, T.G.; Bassani, V.L.; Henriques, A.T. Identification of phenolic compounds in Equisetum giganteum by LC-ESI-MS/MS and a new approach to total flavonoid quantification. Talanta, 2013, 105, 192-203.
Tuchinda, P.; Udchachon, J.; Khumtayeeporn, K.; Taylor, W.C. Benzylated phenanthrenes from Eulophi nuda. Phytochemistry, 1989, 28, 2463-2466.
Tohma, H.; Gülçin, İ.; Bursal, E.; Gören, A.C.; Alwasel, S.H.; Köksal, E. Antioxidant activity and phenolic compounds of ginger (Zingiber officinale Rosc.) determined by HPLC-MS/MS. J. Food Meas. Charact., 2017, 11, 556-566.
Köksal, E.; Tohma, H.; Kılıç, Ö.; Alan, Y.; Aras, A.; Gülçin, İ.; Bursal, E. Assessment of antimicrobial and antioxidant activities of Nepeta trachonitica: Analysis of its phenolic compounds using HPLC-MS/MS. Sci. Pharm., 2017, 85, 24.
Rodríguez-Pérez, C.; Quirantes-Piné, R.; Amessis-Ouchemoukh, N.; Madani, K.; Segura-Carretero, A.; Fernández-Gutierrez, A. A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves. J. Pharm. Biomed. Anal., 2013, 77, 167-174.
Nyau, V.; Prakash, S.; Rodrigues, J.; Farrant, J. HPLC-PDA-ESI-MS identification of polyphenolic phytochemicals in different market classes of common beans (Phaseolus vulgaris L.). Int. J. Biochem. Res. Rev., 2015, 8, 1-11.
Savić, I.M.; Nikolić, V.D.; Savić, I.M.; Nikolić, L.B.; Jović, M.D.; Jović, M.D. The qualitative analysis of the green tea extract using ESI-MS method. Savremene Tehnologije, 2014, 3, 30-37.
Chen, H.J.; Inbaraj, B.S.; Chen, B.H. Determination of phenolic acids and flavonoids in Taraxacum formosanum Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization technique. Int. J. Mol. Sci., 2011, 13, 260-285.
Lin, L.Z.; Mukhopadhyay, S.; Robbins, R.J.; Harnly, J.M. Identification and quantification of flavonoids of Mexican oregano (Lippia graveolens) by LC-DAD-ESI/MS analysis. J. Food Compos. Anal., 2007, 20, 361-369.
Zhang, X.; Sandhu, A.; Edirisinghe, I.; Burton-Freeman, B. An exploratory study of red raspberry (Rubus idaeus L.) (poly) phenols/metabolites in human biological samples. Food Funct., 2018, 9, 806-818.
Regazzoni, L.; Arlandini, E.; Garzon, D.; Santagati, N.A.; Beretta, G.; Maffei, F.R. A rapid profiling of gallotannins and flavonoids of the aqueous extract of Rhus coriaria L. by flow injection analysis with high-resolution mass spectrometry assisted with database searching. J. Pharm. Biomed. Anal., 2013, 72, 202-207.
Landi, N.; Pacifico, S.; Piccolella, S.; Di Giuseppe, A.M.; Mezzacapo, M.C.; Ragucci, S.; Iannuzzi, F.; Zarrelli, A.; Di Maro, A. Valle Agricola lentil.; an unknown lentil (Lens culinaris Medik.) seed from Southern Italy as a novel antioxidant and prebiotic source. Food Funct., 2015, 6, 3155-3164.
Van Loo, P.; De Bruyn, A.; Verzele, M. On the liquid chromatography and identification of the flavonoids present in the “Sumach Tannic Acid” extracted from Rhus coriaria. Chromatographia, 1988, 25, 15-20.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Page: [69 - 79]
Pages: 11
DOI: 10.2174/2210315509666190215101646
Price: $25

Article Metrics

PDF: 15