Reduction of NADH oxidase, NO synthase, TNFα, and IL-1β mRNA expression levels on lipopolysacharide-stimulated murine macrophages by Zataria Multiflora

Document Type: Original article


1 Zataria multiflora and anti-inflammatory effects

2 Institute of Biotechnology , Shiraz University, Shiraz, Iran

3 Department of Immunology, Autoimmune Disease Research Center and Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.


Zataria multiflora (ZM) is a thyme-like aromatic plant in the Lamiaceae family that grows in central and southern Iran. ZM is extensively used as a flavor ingredient in a wide variety of foods and is used as part of popular traditional folk remedies. In the present study, ZM essential oil (ZMO) was obtained from ZM leaves via hydro-distillation and then analyzed by GC-MS (gas chromatography-mass spectrometry). The anti-inflammatory activity of ZMO was determined via measures of NADH oxidase (NOX), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β mRNA expression in lipopolysaccharide-stimulated murine macrophages using real-time polymerase chain reaction (PCR). GC-MS analysis indicated that the main components in the ZMO were carvacrol (29.4%), thymol (25.7%), p-cymene (11.2%), linalool (9.3%), and γ-terpinene (8.0%). ZMO significantly reduced NOX, iNOS, TNFα, and IL-1β mRNA expression in cells at concentrations of 0.1-1 μg/mL, indicating a capacity for this product to potentially modulate/diminish immune responses. ZMO has anti-oxidant and anti-inflammatory properties and could be potentially used as a safe effective source of natural anti-oxidants in therapy against oxidative damage and a number of inflammatory conditions associated with stress.


1. Zargari A. (Ed.) Medicinal Plants, Sixth Edition. Tehran, Iran: Tehran University Publications. 2001.

2. Iranian Herbal Pharmacopoeia. Tehran: Ministry of Health and Medical Publications, 2002; 51-56.

3. Sajed H, Sahebkar A, Iranshahi MZ. multiflora Boiss (Shirazi thyme) - An ancient condiment with modern pharmaceutical uses. J Ethnopharmacol 2013;145:686-698.

4. Akhondzadeh-Basti A, Misaghi A, Khaschabi D. Growth response and modeling of effects of Z. multiflora essential oil, pH, and temperature on S. typhimurium and S. aureus. LWT-Food Sci Tech 2007;40:973-981.

5. Saei-Dehkordi SS, Tajik H, Moradi M, Khalighi-Sigaroodi F. Chemical composition of essential oils in Z. multiflora Boiss from different parts of Iran and their radical scavenging and antimicrobial activity. Food Chem Toxicol 2010;48:1562-1567.

6. Fazeli MR, Amin G, Attari MM, Ashtiani H, Jamalifar H, Samadi N. Anti-microbial activities of Iranian sumac and avishan-e shirazi (Z. multiflora) against some food-borne bacteria. Food Control 2007;18:646-649.

7. Mohammadi Purfard A, Kavoosi G. Chemical composition, radical scavenging, anti-bacterial and anti-fungal activities of Z. multiflora bioss essential oil and aqueous extract. J Food Saf 2012;32:326-332.

8. Abdollahi M, Hamzehzarghani H, Saharkhiz, MJ. Effects of the essential oil of Z. multiflora, a thyme-like medicinal plant from Iran on the growth and sporulation of A. niger both in vitro and on lime fruits. J Food Saf 2011;31:424-432.

9. Gandomi H, Misaghi A, Akhondzadeh-Basti A, Bokaei S, Khosravi A, Abbasifar A, Javan AJ. Effect of Z. multiflora essential oil on growth and aflatoxin formation by A. flavus in culture media and cheese. Food Chem Toxicol 2009;47:2397-2400.

10. Khosravi AR, Shokri H, Sharifrohani M, Mousavi HE, Moosavi Z. Evaluation of anti-fungal activity of Z. multiflora, Geranium herbarium, and Eucalyptus camaldolensis essential oils on Saprolegnia parasitica-infected rainbow trout eggs. Foodborne Pathogen Dis 2012;9: 674-679.

11. Shahsavari N, Barzegar M, Sahari MA, Naghdi Badi H. An investigation on the anti-oxidant activity of essential oil of Z. multiflora Boiss. in soy bean oil. J Med Plants Res 2008;7:56-68.

12. Sharififar F, Moshafi MH, Mansouri SH, Khodashenas M, Khoshnoodi M. In vitro evaluation of anti-bacterial and anti-oxidant activities of the essential oil and methanol extract of endemic Z. multiflora Boiss. Food Control 2007;18:800-805.

13. Karimian P, Kavoosi G, Saharkhiz MJ. Antioxidant, nitric oxide scavenging and malondialdehyde scavenging activities of essential oil from different chemotypes of Z. multiflora. Nat Prod Res 2012;26:2144-2147.

14. Kavoosi G, Teixeira da Silva JA, Inhibitory effects of Z. multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte. Food Chem Toxicol 2012;50:3079-3085.

15. Kavoosi G, Teixeira da Silva JA, Saharkhiz MJ. Inhibitory effects of Z. multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in lipopolysaccharide-stimulated macrophages. J Pharm Pharmacol 2012;64:1492-1500.

16. British Pharmacopeia. London: British Pharmacopeia, HMSO, 1998;137-138.

17. Adams RP. 2007. Identification of Essential Oil Components by Gas Chromatogra-phy/Mass Spectrometery, Fourth Edition.  Chicago: Allured Publishing Corporation, 2007; 456.

18. Mc Lafferty FW. (Ed.) Wiley Registry of Mass Spectral Data, 9th Edition. Hoboken, NJ: John Wiley and Sons, Inc. 2009; 662.

19. Nouri AM, Thompson C, Cannell H, Symes M, Purkiss S, Amirghofran Z. Profile of epidermal growth factor receptor (EGFr) expression in human malignancies: Effects of exposure to EGF and its biological influence on established human tumor cell lines. Int J Mol Med 2000;6:495-500.

 20. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a house-keeping gene: Analysis of GAPDH mRNA expression in panel of 72 human tissues. Physiol Genomics 2005;21:389-395.

21. Larionov A, Krause A, Miller W. A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 2005;6:62-68.

22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-408.

23. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Rad Biol Med 2010; 48:749-762.

24. Katalinic V, Milos M, Kulisic T, Jukic M. Screening of 70 medicinal plants extracts for anti-oxidant capacity and total phenols. Food Chem 2006;94:550-557.

25. Amirghofran Z, Ahmadi H, Karimi MH. Immunomodulatory activity of the water extract of Thymus vulgaris, T. daenensis, and Z. multiflora on dendritic cells and T-cell responses. J Immunoass Immunoch 2012;33:388-402.

26. Amirghofran Z, Hashemzadeh R, Javidnia K, Golmoghaddam H, Smaeilbeig A. In vitro immunomodulatory effects of extracts from three plants of the Labiatae family and isolation of the active compound(s). J Immunotoxicol 2011;8:265-273.

27. Groemping Y, Rittinger K. Activation and assembly of the NADPH oxidase: A structural perspective. Biochem J 2005;386:401-416.

28. Minakami R, Sumimotoa H. Phagocytosis-coupled activation of the superoxide-producing phagocyte oxidase, a member of the NADPH oxidase (NOX) family. Int J Hematol 2006;84: 193-198.

29. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiol Rev 2007;87:245-313.

 30. Kleniewska P, Piechota A, Skibska B, Gorąca A. The NADPH oxidase family and its inhibitors. Arch Immunol Ther Ex 2012;60:277-294.

31. Zielonka J, Zielonka M, Sikora A, Adamus J, Joseph J, Hardy M, Ouari O, Dranka BP, Kalyanaraman B. Global profiling of reactive oxygen and nitrogen species in biological systems: High-throughput real-time analyses. J Biol Chem 2012;287:2984-2995.

32. Kroncke KD, Fehsel K, Kolb BV. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol 1998;113:147-156.

33. Branes PJ, Karin M. Nuclear factor-kB: A pivotal transcription factor in chronic inflammatory diseases.  New Eng J Med 1996;336:1066-1071.

34. Peranzoni E, Marigo I, Dolcetti L, Ugel S, Sonda N, Taschin E, Mantelli B, Bronte V, Zanovello P. Role of arginine metabolism in immunity and immunopathology. Immunobiology 2007;212;795-812.

35. Aristatile B, Al-Assaf AH, Pugalendi KV. Carvacrol suppresses the expression of inflammatory marker genes in D-galactosamine-hepatotoxic rats. Asian Pacific J Trop Med 2013;6:205-211.

36. Stebbings R, Eastwood D, Poole S, Thorpe R. After TGN1412: Recent developments in cytokine release assays.  J Immunotoxicol 2013;10:75-82

37. Zha LY, Mao LM, Lu XC, Deng H, Ye JF, Chu XW, Sun SX, Luo HJ. Anti-inflammatory effect of Soya saponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-kB-mediated nitric oxide synthase expression. Bioorgan Med Chem 2011;21:2415-2418.

38. Hsu HY, Wen MH. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of IL-1 gene expression. J Biol Chem 2002;277:22131-2239.

39. Ma Q, Kinneer K, Ye J, Chen BJ. Inhibition of nuclear factor-kB by phenolic anti-oxidants: Interplay between antioxidant signaling and inflammatory cytokine expression. Mol Pharmacol 2003;64:211-219.