1. Introduction
nflammatory diseases are associated with protein denaturation, Nitric Oxide (NO), and free radicals overproduction [4, 3]. Therefore, it is necessary to use antioxidant and anti-inflammatory agents to prevent oxidative and inflammatory stress. Today, medicinal plants and their aromatic compounds as natural resources have attracted many researchers’ attention.
Tanacetum parthenium contains antioxidant and anti-inflammatory substances such as sesquiterpene lactones and flavonoids. The studies have shown that T. Parthenium has a role in reducing pain and inflammation in arthritis [12, 13]. Also, Satureja Montana contains various biological compounds such as phenols, alcohols, rosmarinic acid, and ursolic acid triterpenoids [3]. This plant’s leaves and aerial parts have antioxidant, anti-inflammatory, and hepatoprotective properties [14, 15].
Considering the beneficial effects of T. Parthenium and S. Montana and the lack of total antioxidant capacity and anti-inflammatory effects of their combination, we decided to combine these two plants’ anti-inflammatory and antioxidant effects on induced acute inflammation in BALB/c mice.
2. Materials and Methods
The aerial parts of the dried plants were pulverized separately. Percolation and soxhlet methods, respectively, then performed the extraction of T. Parthenium and S. Montana. The extracts were concentrated using a rotary evaporator, and the alcoholic solvent was separated [16, 17]. According to the OECD (Organization for Economic Co-operation and Development) guidelines, the extracts’ acute toxicity was assessed. 
Then, the mice were treated with the extracts and their mixture by gavage for 14 days [19, 20, 21]. The control group received DMSO (as a vehicle) by gavage [24], and the positive control group received dexamethasone intraperitoneally (i.p.) as an anti-inflammatory drug [22, 23]. During the treatment, the weight of the mice was measured. On the 10th day of treatment, mice were injected intraperitoneally with 1 ml of sterile 3% (w/v) Brewer-modified thioglycollate solution. 12 to 14 hours after the last gavage, the mice were killed under ethics principles, and the blood was isolated from the mice’s hearts [25]. FRAP and Griess’s analyses performed total antioxidant capacity and serum nitric oxide concentration, respectively [26, 27]. The results were analyzed in SPSS software using one-way ANOVA.  
3. Results
The results indicated no change in the skin, fur, and breaths in the mice. The mean weight in the treatment groups with T. Parthenium and S. Montana and their combination in the 1, 5, 10, and 14 days during the gavage period showed that the mice receiving the extracts had a similar weight pattern and clinical symptoms to the control group.
Also, the results indicated that the highest antioxidant effect is related to the group of composition of the extracts compared to the control group (P=0.006). Also, the FRAP in receiving S. Montana extract increased significantly compared to the control group (P=0.021). On the other hand, the group of T. parthenium extract, despite the increase in FRAP level compared to the control group, did not show a significant increase (P=0.01). The results also showed nitric oxide levels in receiving groups of T. parthenium extract (P=0.034), S. Montana (P=0.04), and the mixture of the extract (P=0.003) decreased significantly compared to the control group (Figure 1, 2 & 3).  4. Discussion and Conclusion
The present study’s findings show that the combination of alcoholic extracts of T. Parthenium and S. Montana causes a significant increase in antioxidant potential and inhibition of serum-free radicals. In this study, the nitric oxide concentration was also evaluated as an inflammatory mediator. The results indicated that the concentration of nitric oxide decreased significantly compared to the control group. 
Many researchers have suggested that the major constituents of Satureja species are phenolic monoterpenes, which are often present with γ-terpinene, paracetamol, linalool, oleanolic acid, and ursolic acid and this group of phenolic compounds has antioxidant properties [33, 34]. In one study investigating the neuroprotective effect of oleanolic acid, as an active ingredient of S. Montana, in a model of Alzheimer’s disease, it was observed that oleanolic acid reduced NO release from BV2-stimulated microglial cell line LPS. In this study, NO production by oleanolic acid was more inhibited than iNOS gene expression, indicating a possible effect of triterpene oleanolic acid on the post-translational process [37]. 
Also, Previous studies, consistent with other studies, indicate that T. Parthenium and its active ingredients reduce NO concentration by inhibiting the expression and synthesis of the iNOS [31, 32]. However, there are differences in the amount and type of these compounds that have different reasons. In general, the constituent compounds can change according to the geographical area of growth, plant age, method of extraction, and the genetic differences of the plants.
The results show that these two plants’ extract’ combined administration effectively reduces inflammation and increases total antioxidant capacity. The findings indicated synergistic effects of the combination of these two plants. Awareness of these changes’ mechanism of action requires further investigation, which we hope to achieve in future studies.

Ethical Considerations
Compliance with ethical guidelines
The study was approved by the Research Ethics Committee of Arak University of Medical Sciences (AUMS) (Code: IR.ARAKMU.REC.1398.021).

Funding
The Vice-Chancellor for Research and Technology of Arak University of Medical Sciences has financially supported the present study.

Authors' contributions
The authors had the writing standards based on the International Committee of Medical Journal Publishers (ICMJE).

Conflicts of interest
The authors declare no conflict of interest.

Acknowledgements
The authors would like to thank the Deputy and Council for Research of Arak University of Medical Sciences for their support.



References

1. Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease-a double-edged sword. Neuron. 2002; 35(3):419-32. [DOI:10.1016/S0896-6273(02)00794-8]
2. Vishal V, Ganesh S, Mukesh G, Ranjan B. A review on some plants having anti-inflammatory activity. J Phytopharmacol. 2014; 3(3):214-21. http://www.phytopharmajournal.com/Vol3_Issue3_09.pdf
3. Vitanza L, Maccelli A, Marazzato M, Scazzocchio F, Comanducci A, Fornarini S, et al. Satureja montana L. essential oil and its antimicrobial activity alone or in combination with gentamicin. Microbial Pathogenesis. 2019; 126:323-31. [DOI:10.1016/j.micpath.2018.11.025]
4. Prashanth S, Pooja S, Suchetha K, Vidya V. Radical scavenging and antioxidant activities of ethanolic and aqueous extract from the leaves of Feverfew (Tanacetum parthenium L.) and asynthetic compound parthenolide. J pharmacogn phytochem. 2015; 4(1): 223-7. https://www.researchgate.net/profile/Priya-Priya-6/publication/320346118_
5. Hariharan P, Subburaju T. Medicinal plants and its standardization-A global and industrial overview. Glob J Med Plant Res. 2012; 1(1):10-3. http://www.aensiweb.com/old/GJMPR/2012/10-13.pdf
6. Kulkarni KM, Patil LS, Khanvilkar VV, Kadam VJ. Fingerprinting techniques in herbal standardization. Indo Am J Pharm. 2014; 4(2):1049-62. https://www.semanticscholar.org/paper/
7. World Health Organization. WHO monographs on selected medicinal plants. Geneva: World Health Organization; 2006. https://apps.who.int/iris/handle/10665/42052
8. Morteza-Semnani K, Saeedi M, Mahdavi MR, Rahimi F. Antimicrobial effects of methanolic extracts of some species of Stachys and Phlomis. J Maz Univ Med Sci. 2007; 17(57):57-66. https://vlibrary.emro.who.int/imemr/antimicrobial-effects-of-methanolic-extracts-of-some-species-of-stachys-and-phlomis-2/
9. Kulisic T, Radonic A, Katalinic V, Milos M. Use of different methods for testing antioxidative activity of oregano essential oil. Food Chemistry. 2004; 85(4):633-40. [DOI:10.1016/j.foodchem.2003.07.024]
10. Singh G, Maurya S, DeLampasona M, Catalan CA. A comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. Food Chem Toxicol. 2007; 45(9):1650-61. [DOI:10.1016/j.fct.2007.02.031]
11. Cretnik L, Kotnik P, Škerget M, Knez Ž. Separation of Parthenolide from Tanacetum Parthenium. Maribor: University of Maribor; 2000. https://www.researchgate.net/profile/Petra-Kotnik/publication/228763462_
12. Parvin N, Ansari Samani R, Shahinfard N, Reissi S, Alibabaie Z, A Asgari A. Effect of alcoholic extract of Tanacetum parthenium on acute pain in rat. J Inflamm Dis. 2012; 16(1):15-21. http://journal.qums.ac.ir/article-1-1236-en.html
13. Pareek A, Suthar M, Rathore GS, Bansal V. Feverfew (Tanacetum parthenium L.): A systematic review. Pharmacogn Rev. 2011; 5(9):103. [DOI:10.4103/0973-7847.79105]
14. Escudero J, López JC, Rabanal RM, Valverde S. Secondary metabolites from Satureja species. New triterpenoid from Satureja acinos. J NatProd. 1985; 48(1):128-31. [DOI:10.1021/np50037a025]
15. Kim SH, Hong JH, Lee YC. Ursolic acid, a potential PPARγ agonist, suppresses ovalbumin-induced airway inflammation and Penh by down-regulating IL-5, IL-13, and IL-17 in a mouse model of allergic asthma. Eur J Pharmacol. 2013; 701(1-3):131-43. [DOI:10.1016/j.ejphar.2012.11.033]
16. Mahmoodzadeh Y, Mazani M, Rezagholizadeh L. Hepatoprotective effect of methanolic Tanacetum parthenium extract on CCl4-induced liver damage in rats. TToxicol Rep. 2017; 4:455-62. [DOI:10.1016/j.toxrep.2017.08.003]
17. Amiri H. The in vitro antioxidative properties of the essential oils and methanol extracts of Satureja macrosiphonia Bornm. Nat Prod Res. 2011; 25(3):232-43. [DOI:10.1080/14786410903374694]
18. Malik F, Singh J, Khajuria A, Suri KA, Satti NK, Singh S, et al. A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice. Life Sci. 2007; 80(16):1525-38. [DOI:10.1016/j.lfs.2007.01.029]
19. Subha D, Geetha N. Evaluation of acute toxicity of the methanolic extract of Tanacetum parthenium L. in albino wistar rats. J Sci Innov Res. 2017; 6(3):113-5. http://www.jsirjournal.com/Vol6_Issue3_07.pdf
20. Pooja S, Prashanth S, Suchetha K, Vidya V, Krishna B. Evaluation of acute and sub acute toxicity of the leaf extract of Tanacetum parthenium (Asteraceae) and synthetic parthenolide. World J Pharm Pharm Sci. 2016;5(8):703-13. https://www.researchgate.net/profile/Pooja-Shivappa/publication/316643904_
21. Hajhashemi V, Ghannadi A, Pezeshkian SK. Antinociceptive and anti-inflammatory effects of Satureja hortensis L. extracts and essential oil. J Ethnopharmacol. 2002; 82(2-3):83-7. [DOI:10.1016/S0378-8741(02)00137-X]
22. Biagiotti S, Menotta M, Orazi S, Spapperi C, Brundu S, Fraternale A, Bianchi M, Rossi L, Chessa L, Magnani M. Dexamethasone improves redox state in ataxia telangiectasia cells by promoting an NRF2-mediated antioxidant response. FEBS J. 2016; 283(21):3962-78. [DOI:10.1111/febs.13901]
23. Xu T, Qiao J, Zhao L, He G, Li K, Wang J, Tian Y, Wang H. Effect of dexamethasone on acute respiratory distress syndrome induced by the H5N1 virus in mice. Eur Respir J. 2009; 33(4):852-60.[DOI:10.1183/09031936.00130507]
24. Noel PR, Barnett KC, Davies RE, Jolly DW, Leahy JS, Mawdesley-Thomas LE, et al. The toxicity of Dimethyl Sulphoxide (DMSO) for the dog, pig, rat and rabbit. Toxicology. 1975; 3(2):143-69. [DOI:10.1016/0300-483X(75)90081-5]
25. Hoover-Plow J, Gong Y, Shchurin A, Busuttil S, Schneeman T, Hart E. Strain and model dependent differences in inflammatory cell recruitment in mice. Inflamm Res. 2008; 57(10):457-63. [DOI:10.1007/s00011-008-7062-5]
26. Nencini C, Cavallo F, Capasso A, Franchi GG, Giorgio G, Micheli L. Evaluation of antioxidative properties of Allium species growing wild in Italy. Phytother Res. 2007; 21(9):874-8. [DOI:10.1002/ptr.2168]
27. Phizackerley P, Al-Dabbagh S. The estimation of nitrate and nitrite in saliva and urine. Anal Biochem. 1983; 131(1):242-5. [DOI:10.1016/0003-2697(83)90161-6]
28. Rezaei F, Jamei R, Heidari R. Evaluation of the phytochemical and antioxidant potential of aerial parts of Iranian tanacetum parthenium. Pharm Sci. 2017; 23(2):136. [DOI:10.15171/PS.2017.20]
29. Andonova M, Urumova V, Dimitrova D, Slavov E, Dzhelebov P, Chaprazov T, et al. Evaluation of nuclear factor kappa beta, nitric oxide and blood neutrophil/lymphocyte ratio as biomarkers of inflammatory response and complementary therapy in dogs with experimental skin Pseudomonas aerugi-nosa infection. Adv Anim Vet Sci. 2015; 3(3):174-82. [DOI:10.14737/journal.aavs/2015/3.3.174.182]
30. Pal SK, Shukla Y. Herbal medicine: current status and the future. Asian Pac J Cancer. 2003; 4(4):281-8. https://iranjournals.nlai.ir/handle/123456789/31035
31. Oh Y-C, Jeong YH, Cho W-K, Ha J-H, Gu MJ, Ma JY. Anti-inflammatory and analgesic effects of pyeongwisan on LPS-stimulated murine macrophages and mouse models of acetic acid-induced writhing response and xylene-induced ear edema. Int J Mol Sci. 2015; 16(1):1232-51. [DOI:10.3390/ijms16011232]
32. Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008; 83(1):14.[DOI:10.1002/0471142735.im1401s83]
33. Polatoğlu K, Karakoç ÖC, Gören N. Phytotoxic, DPPH scavenging, insecticidal activities and essential oil composition of Achillea vermicularis, A. teretifolia and proposed chemotypes of A. biebersteinii (Asteraceae). Ind Crops Prod. 2013; 51:35-45. [DOI:10.1016/j.indcrop.2013.08.052]
34. Miguel MG. Antioxidant and anti-inflammatory activities of essential oils: A short review. Molecules. 2010; 15(12):9252-87. [DOI:10.3390/molecules15129252]
35. Innovare I. Antioxidant Polyphenolic Constituents Of Satureja Montana L. Growing in Egypt. 6(4):578-81. https://www.researchgate.net/publication/262030381_Antioxidant_polyphenolic_constituents_of_satureja_montana_L_Growing_in_Egypt
36. Gutierrez RMP, Navarro YTG. Antioxidant and hepatoprotective effects of the methanol extract of the leaves of Satureja macrostema. Pharmacogn Mag. 2010; 6(22):125. [DOI:10.4103/0973-1296.62901]
37. Castellano JM, Garcia-Rodriguez S, Espinosa JM, Millan-Linares MC, Rada M, Perona JS. Oleanolic acid exerts a neuroprotective effect against microglial cell activation by modulating cytokine release and antioxidant defense systems. Biomolecules. 2019; 9(11):683. [DOI:10.3390/biom9110683]