1. Introduction
n addition to inflammation, free radicals like Nitric Oxide (NO) are the most important pathophysiological cause of inflammatory diseases such as peritonitis [1]. Free radicals cause extensive damage to the body’s macromolecules, which are neutralized by the anti-oxidant system [2]. So, researchers started an investigation for new anti-inflammatory and anti-oxidant drugs due to the need for effective drugs with fewer side effects than chemical drugs [3]. 
Over thousands of years, many natural products have been used to treat various diseases, regardless of their antagonistic and synergistic effects [4]. Nigella Sativa (NS) and Silybum Marianum (SM) are promising herbal medicine in Asia used for their anti-inflammatory properties [5]. Other studies have shown that SM has anti-inflammatory and anti-oxidant properties and is not even toxic in large quantities [6, 7, 8, 9, 10]; however, the effect of its compound is unclear. Thus, in this study, we evaluated the anti-inflammatory effect of Nigella sativa and Silybum Marianum extracts and their combination of inflammatory diseases like thioglycollate peritoneal inflammation.
2. Materials and Methods
Dried SM was extracted in a Soxhlet system using n-hexane and methanol as solvent. Also, NS seeds were extracted with 95% ethanol. Then, they were filtered, and the solvent was removed in a rotary evaporator [11, 12]. The safety profile of extracted was performed on spleen lymphocytes and acute toxicity assay according to the Organization for Economic Co-operation and Development (OECD) guidelines using the MTT assay.
Experiments were performed using male Balb/C mice (18-22 gr). The control group received DMSO 30% and PBS as a vehicle [13]. The positive control group gave dexamethasone 0.15 mg/kg intraperitoneal [14]. The SM and NS group were gavaged orally for 14 days. According to a previous study with 2000 mg/kg [15, 16], the mixture group gavage by the SM+NS extracts was equally observed through the study [17]. 
To induce the peritonitis model, on day 10 of treatment, a 1 ml sterile solution of thioglycollate medium (3% w/v in PBS) was injected intraperitoneally [18]. Finally, 12-14 hours after the last treatment, the mice were killed, and blood was collected from the heart to measure the total anti-oxidant power and NO concentration.
The FRAP method was based on that of Benzie and Strain [19]. Briefly, FRAP reagent (included: 300 mmol/L acetate buffer, 10 mmol/L TPTZ/HCL solution, and 20 mmol/L ferric chlorides) was added to 100 µl of diluted serum. After incubation, time absorbance was measured directly at 620 nm. The NO was measured by the Griess reaction [20]. Briefly, 100 μl diluted serum of treated mice was incubated with 100 µL of Griess reagent; finally, nitrite levels were measured at 620 nm after incubation (Figure 1, 2 & 3). 3. Results
Ethanolic Extract of NS (NEE) and Methanolic extract of SM (SME) exhibited no cytotoxic effects on spleen lymphocytes. Also, the acute toxicity test showed no significant changes in weight and physical appearance of the mice. These extract and their mixture decrease NO level significantly (P=0.000) in the treated group compared to the control group. Also, the mixture group increased significantly total anti-oxidant power (P=0.015) compared to the control and each extract individually. 
However, the anti-inflammatory effect of NEE and SME was shown in previous studies [21]. No changes in the weight of treated mice, with a mentioned dosage of extracts and no apparent toxicity on splenic lymphocytes, were seen. So results reveal the non-toxic nature of extract at the tested dosage consistent and, the inhibitory effects were not due to toxicity and cell killing [22, 23]. In parallel with our study, the SME and the NEE had no acute and chronic toxicity on laboratory animals, even higher doses [24, 25]. 
4. Discussion and Conclusion
It seems that the significant increase in anti-oxidant power of the combination group against each of these extracts and the control group indicates the synergistic effect of all flavonoids in the anti-oxidant capacity of the extract [26, 27]. In confirmation, NS oil and its fractions (neutral lipids, glycolipids, and phospholipids) showed anti-oxidant activity related to their entire content [28]. In parallel, the anti-oxidant power of NS oil and SM seeds oil were reported equally [27]. 
Shahin et al. Showed that NS extract has more anti-oxidant effect than SM [29]. This difference can be due to the different solvents used in the extraction and confirming the role of alcoholic solvents in anti-oxidant power. According to our data, each mentioned extract and their combination has significantly decreased NO production release.
Similarly, a study showed suppression of NO production by aqueous extract of NS in stimulated peritoneal macrophages [23]. The reduction of NO production in the inflammatory model of rheumatoid arthritis in mice can confirm our results [30]. Another study showed that the extract of SM and silibinin (one of its main ingredients) reduced the NO production by blocking p38, MAPK, or NF-κB signaling pathways in RAW 264.7 cells and peritoneal macrophages, which have been in line with its anti-inflammatory effects [31].

Ethical Considerations
Compliance with ethical guidelines
All experimental process was performed following the guidelines according to the Animal Ethics Committee of Arak University of Medical Sciences (Code: IR.ARAKMU.REC.1397.359).

Funding
This article is taken from a research project with the code 3196 and it was funded by the Vice Chancellor for Research and Technology of Arak University of Medical Sciences.

Authors' contributions
All authors met the writing standards based on the International Committee of the Journal of Medical Journalists (ICMJE). 

Conflicts of interest
The authors declared no conflicts of interest.

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


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