In Vitro Free Radical Scavenging Properties and Anti-Inflammatory Activity of Ilex paraguariensis (Maté) and the Ability of Its Major Chemical Markers to Inhibit the Production of Proinflammatory Mediators

Ilex paraguariensis A. St. Hil. (Aquifoliaceae), popularly known as “yerba mate,” has great economic and social significance for the population of Southern Latin America. This study was conducted (1) to investigate the phytochemical composition of four different standardized extracts, (2) to investigate its free radical scavenging properties, and (3) to investigate the anti-inflammatory action of I. paraguariensis and its major chemical markers. The chemical profile was achieved by Folin-Ciocalteu, by LC/DAD, and by LC/MS assays, while the antioxidant and anti-inflammatory properties were investigated, respectively, by DPPH assay and by inhibition of nitric oxide (Griess reaction) and TNF-α (ELISA). Our results demonstrated that the IA (aqueous infusion extract) showed higher amounts of total phenolic contents (266.62 ± 10.85 mg CAE·g−1 DE), the highest amounts of all six chemical markers (theobromine, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, caffeine, and rutin), and stronger antioxidant activity (EC50 = 54.4 ± 5.14 μg · mL−1). The IA extract also showed the lowest inhibition of NOx secretion (50.10 ± 8.97%) as well as inhibition of TNF-α (83.33 ± 4.01%). Regarding the chemical markers, all compounds showed strong inhibition of NOx secretion, especially theobromine, which was 200x more potent than dexamethasone. Furthermore, TNF-α secretion was also significantly decreased by THEO at 0.033 μM (22.15 ± 6.49%), NCA at 1.97 μM (27.46 ± 3.98%), CCA at 0.35 μM (39.76 ± 5.73%), CGA at 0.56 μM (23.58 ± 5.79%), CAF at 0.52 μM (26.45 ± 5.34%), and RUT at 0.16 μM (40.18 ± 3.70%). Our results suggest that I. paraguariensis and its major chemical markers have strong free radical scavenging properties as well as showed important anti-inflammatory activity and that these compounds in a plant extract may work based on several different mechanisms synergistically, resulting in moderating the immune system.


Introduction
Inflammation usually occurs as a protective event to rid the organism of harmful stimuli. Inflammation may also happen in response to processes such as tissue injury, cell death, cancer, ischemia, and degeneration, leading to the activation of intracellular pathways and triggering the kinase cascades and nuclear transcription factors, such as MAPKs and NF-κB [1,2]. Although the development of anti-inflammatory drugs has improved the treatment of several diseases, other inflammatory pathologies such as asthma, atherosclerosis, and sepsis have not shown adequate response to current medications. Moreover, the chronic use of these drugs is reported to cause severe adverse effects, including gastrointestinal, cardiovascular, and renal abnormalities [3,4]. Free radicals are also related to diseases associated with elevated inflammatory signaling. Atherosclerosis, diabetes, and Alzheimer's are related with an altered redox balance. Therefore, the investigation of new anti-inflammatory and free radical scavenging agents remains relevant [5,6].
The in vitro models represent the beginning of the rational research, this complex and intricate process needs a fast and reliable screening to follow to the next level of pharmacological research, and the model using RAW 264.7 cells is widespread in research laboratories worldwide due to its versatility and reproducibility in the results [7,8]. Moreover, the most common inflammatory biomarkers evaluated in this model are the nitric oxide (NOx) and tumoral necrosis factor alpha (TNF-α); both are produced immediately after initial action of the phlogistic agent (LPS) that induced the activation of nuclear transcription factor-kB (NF-κB) [9,10].
In this context, natural products can be an effective alternative in the treatment of inflammation, since medicinal plants are used worldwide as an efficient treatment of many diseases, including inflammation. Also, natural products have produced significant numbers of agents used as drugs or drug leads in recent decades [11].
Ilex paraguariensis A. St. Hil. (Aquifoliaceae) or "yerba mate" has great economic and social significance for the population of Southern Latin America in view of the high consumption of mate, terere, or mate-tea [12]. Yerba mate is also used in popular medicine for liver protection, inflammation, weight reduction, and cholesterol [13]; pharmacological properties all of which are linked to the presence of methylxanthines [14], saponins [15], and phenolic compounds [12,16].
There are several in vitro and in vivo pharmacological studies that show modulation of lipid metabolism and antioxidant, antidiabetic [12,17,18], antiobesity [13], and antiinflammatory properties [18][19][20], as well as central nervous system stimulant and neuroprotective effects [21] for I. paraguariensis extracts. Regarding the anti-inflammatory properties, there is one clinical study that showed this activity for I. paraguariensis extracts [22] and few works that investigated the anti-inflammatory properties of isolated compounds from I. paraguariensis [19]. However, some of these works were conducted using no standardized extract preparations, which may have an impact on the physicochemical stability of the extracts as well as on the reproduction of the results [23,24].
Phytopharmaceutical products can represent an enormous challenge in the search for new anti-inflammatory agents with a high level of uniformity, reproducibility, and stability [23,24]. Moreover, polyphenol compounds, the major phytochemicals present in I. paraguariensis preparations, can regulate immunity by interfering with immune cell regulation and proinflammatory cytokines' synthesis, inactivate NF-κB (nuclear factor kappa-light-chainenhancer of activated B cells), and modulate the mitogenactivated protein kinase (MAPK) and arachidonic acid pathways [25]. Therefore, we aimed in this work to evaluate the in vitro free radical scavenging and the ability of different standardized extracts of I. paraguariensis as well as its major chemical markers to inhibit the production of proinflammatory mediators.

Materials and Methods
2.1. Plant Material. The plant material was purchased from the local market in the city of Florianópolis, in the state of Santa Catarina, Brazil, in 2018. The plant material was botanically characterized in accordance with the Argentine Pharmacopeia [26] and the Brazilian Pharmacopeia [27].

Preparation of Extracts.
The extracts were prepared using two different extraction methods: infusion and turboextraction as previously described [28]. In both techniques, the extracts were produced using a raw material : solvent ratio of 5% (w/v). Crushed leaves of I paraguariensis were subjected to the infusion or turboextraction with water or hydroethanolic solution (20%) for 30 min. The extracts prepared by turboextraction were prepared using an Ultra-TURRAX® (model T25, IKA®, Germany; catalogue number 0003725032), with a S25N-10G stirring stem (IKA®, Germany; catalogue number 0000594000), at a stirring speed of 9500 rpm, for 5 minutes, with water or a hydroethanolic solution (20%). All four extracts were then filtered and dried using a spray dryer. The spray dryer conditions used were Mini Spray Dryer B-290 (Buchi®; catalogue number 044699) with a two-component nozzle and current flow under the following operating conditions: inlet temperature of 160°C, outlet temperature 100°C, 10% pump flow (3 mL/min), 100% aspirator, and atomizer diameter 0.7 mm. The spray-dried extracts were identified as IA (aqueous infusion), IE (hydroethanolic infusion), TA (aqueous turboextraction), and TE (hydroethanolic turboextraction). All procedures were conducted in triplicate.

Chemical Characterization by LC/MS Analysis.
The LC/MS analysis was performed according Feltrin et al. [31]. Briefly, the compounds were identified by highperformance liquid chromatography (Waters® Acquity™ UPLC™; catalogue number 186015018) coupled to a photodiode array detector (PDA, Waters®; catalogue number 186015033) and a high-resolution mass spectrometer (Xevo®G2 QTof, WATERS® model; catalogue number 186006532) equipped with a source of electrospray ionization (ESI) operating in positive and negative mode. The mobile phase was consisted by a gradient of 0.1% of aqueous formic acid (A) and methanol (B) at constant flow rate (0.5 mL/min). This gradient was programmed as follows: initial condition 15-85% (B-A), a linear gradient from 15 to 25% (B) for 2 min, 25-60% (B) for 2-4 min, 60-70% (B) for 4-9 min, and 70-15% (B) for 9-10 min. The mass spectrometer parameters were set as follows: for ESI−, capillary voltage of 2.5 kV, source block temperature of 150°C; desolvation temperature of 500°C, nebulizer nitrogen flow rate of 150 L/h, and desolvation nitrogen gas flow of 1000 L/h; for ESI+, capillary voltage of 3.5 kV, source block temperature of 90°C, desolvation temperature of 400°C, nebulizer nitrogen flow rate of 30 L/h, and desolvation nitrogen gas flow of 900 L/h were set. MS/MS analyses were performed using a collision energy ramp 10-30 eV.

Screening of Biological Activities
2.4.1. Radical Scavenging Assay-DPPH Assay. The radical scavenging assay of the four samples (IA, IE, TA, and, TE) and chemical markers (THEO, NCA, CCA, CGA, CAF, and RUT) was performed by the DPPH (2,2-diphenyl-1picrylhydrazyl, Sigma-Aldrich®; catalogue number D9132) assay, described by Brand-Williams et al. [32] and modified by Zhu et al. [33]. The analysis was performed in triplicate, and the concentration of each sample that reduced 50% of the DPPH concentration (EC 50 ) was expressed as μg·mL -1 for the extracts and μM for the chemical markers.

2.4.2.
In Vitro Cytotoxicity Assay. The cytotoxicity of I. paraguariensis extracts on RAW 264.7 from American Type Culture Collection (ATCC® TIB-71™) was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich®; catalogue number M5655) [34]. Briefly, the cells were grown in appropriate plastic bottles with Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM, Gibco®; catalogue number 11965-092), supplemented with 10% of inactive fetal bovine serum (FBS, Gibco®; catalogue number 12657-029) and 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep, Sigma-Aldrich®; catalogue number P4333), in an incubator humidified at 37°C with 5% CO 2 emissions. Before the experiments, the number of viable cells was determined by the trypan blue exclusion method, performing the counts in a Neubauer chamber. Afterwards, the cells were distributed into 96-well culture microplates (1 × 10 4 cells/well) and incubated with the extracts at different concentrations (3, 10, 30, 100, 300, and 1000 μg·mL -1 ). Dimethyl sulfoxide (DMSO, Sigma-Aldrich®; catalogue number 472301) was used to solubilize the samples at a maximum concentration of 1% per well/treatment, at which concentration at which there is no cytotoxicity to the cells. After the treatment period (24 h), the medium was removed and 100 μL of a MTT solution was added to 0.5 mg·mL -1 in culture medium and incubated for 2 hours. After this period, the medium was removed, the formazan precipitate was dissolved in 100 μL DMSO/well, and the absorbance was measured at 540 nm (ELISA Reader MR96A, Mindray®; catalogue number 104-20-62564). The optical density obtained in the control group-untreated cells (incubated with growth medium only)-was regarded as 100% viable cells. The cytotoxic concentration (CC 10 ), which is the concentration required to reduce cell viability by 90%, was calculated through a Hill concentration-response curve using the software Prism 6.0 (GraphPad Software, La Jolla, CA, USA).

NOx Measurement.
Nitrite accumulation in the culture supernatants was measured as an indicator of NO production based on the Griess reaction [35]. To evaluate which extraction procedure had the highest anti-inflammatory properties, the nitrite production was evaluated in the following groups: The concentrations of TNF-α were determined using a commercially available enzyme-linked immunosorbent assay kit (Mouse TNF-α, PeproTech®, Rocky Hill, NJ, USA; catalogue number 900-T54) according to the manufacturer's instructions. Cytokine level was estimated by interpolation from the standard curve, and the results are expressed in pg/mL.

Statistical
Analysis. The data were presented as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 6 software®. Data were submitted to analysis of variance (ANOVA) followed by Tukey post hoc test (p < 0:05).

Total Phenolic Content (TPC).
The TPC values are shown in Figure 1. The analysis showed that all extracts have similar TPC, except for TES extract, which showed lower TPC (p < 0:05) than all the other preparations.
3.2. UPLC-PDA Analysis. The extracts showed the same fingerprint, but differences in the amounts of chemical markers were detected. Moreover, the UV profiles observed for these compounds were in agreement with those described in the literature [19]. The typical qualitative chromatographic profile of IA and the UV profile of its chemical markers are shown in Figure 2. The quantification of chemical markers is presented in Table 1. The IA was the extract that showed the higher amounts of all seven chemical markers, in a single extract.  Figure 3 and Table 2.

Cytotoxicity
In Vitro Assay. The cytotoxicity effect of Ilex extracts on RAW 264.7 macrophages was determined using the MTT assay. The cells were incubated with different concentrations of the extracts (3-1000 μg·mL -1 ). All extracts showed a CC 10 higher than 50 μg·mL -1 ( Figure 5). Therefore, to ensure that only nontoxic concentrations of the extract would be used, all subsequent experiments were conducted at concentrations below 50 μg·mL -1 (3, 10, and 30 μg·mL -1 ).

Anti-Inflammatory
In Vitro Assay. The antiinflammatory activity was measured by NOx secretion in LPS-treated macrophages. As expected, LPS increase the levels of NOx secretion, while the anti-inflammatory drug dexamethasone (DEX), at 7 μM, reduced the levels of this proinflammatory mediator release (% inhibition: 29:10 ± 3:05) (p < 0:001). NOx secretion was also inhibited by different extracts and ranged from 36.05 to 53.57% ( Figure 6). Considering the results of NOx inhibition by the different I. paraguariensis extracts, its free radical scavenging activities, and its high amounts of chemical markers, it is possible to verify that the most effective results were produced when cells were treated with IA extract. Thus, to 5 Mediators of Inflammation clarify the putative compounds responsible for the antiinflammatory effect detected, the evaluation of THEO (theobromine), NCA (neochlorogenic acid), CCA (cryptochlorogenic acid), CGA (chlorogenic acid), CAF (caffeine), and RUT (rutin) on NOx secretion and TNF-α level was conducted, using the same concentration as that present in the aqueous extract (IA).

Discussion
The chemical profile of I. paraguariensis extracts was investigated by FC assay and UPLC/DAD. TPC analysis showed that IA, IE, and TE extracts possess high amounts of phenolic compounds. Also, UPLC/DAD showed THEO, NCA, CCA, CGA, CAF, and RUT as the major compounds while UPLC/MS analysis confirmed these compounds and the presence of quinic acid, citric acid, caffeoylglucose, feruloylquinic acid, quercetin-glycoside, and kaempferolrhamnoglucoside. All these data are in agreement with the literature data [18,19,20,31,36]. Considering the high amounts of phenolic compounds detected in I. paraguariensis preparations, the antioxidant activity of these extracts and its chemical markers was also investigated by the DPPH assay. This is a free radical scavenging method that offers the first approach for evaluating the antioxidant potential of a compound, extract, or other biological material. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a predefined period, based on the measurement of the scavenging capacity of antioxidants towards it [38]. The extracts exhibited strong free radical scavenging, since IA showed EC 50 at 54:4 ± 5:1 μg/mL. Phenolic compounds have been linked to favorable impacts in healthcare, particularly due to their antioxidant activity [39]. Studies have suggested an inverse correlation between fruit and vegetable consumption and serum levels of inflammatory markers [40]. In addition, it is also evidenced that oxidative stress plays a pathogenic role in chronic inflammatory diseases [41], since the oxygen radical absorbance capacity assay showed that phenolic compounds present in the extracts are a strong antioxidant agent, and more importantly, these metabolites also inhibit cytokines such as TNF-α and IL-6 [42]. Therefore, a considerable number of studies have investigated the antioxidant [20,43] and antiinflammatory properties of I. paraguariensis [18][19][20].
The anti-inflammatory activity of I. paraguariensis was previously described [18][19][20]. Schinella et al. [20] showed that oral administration of the aqueous mate extract significantly reduced the carrageenan-induced edema, an effect that was accompanied by a 43% and 53% reduction of the expression of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Luz et al. [19] showed that a hydroethanolic extract of I. paraguariensis leaves was able to reduce leukocyte migration, exudate concentration, MPO and ADA activities, and NOx levels. The aqueous extract was also beneficial in adjuvant-induced arthritic rats by diminishing both inflammation and oxidative stress [18]. Our study therefore reinforces the anti-inflammatory activity of standardized extract of I. paraguariensis and demonstrates the strong activity of IA on NOx secretion, especially at the lowest dose (3 μg/mL). This anti-inflammatory activity detected for I. paraguariensis extracts can be explained, in part, by the presence of phenolic compounds in the extracts.
In view of previous reports that describe the potential anti-inflammatory properties of hydroxycinnamic acid derivatives [44], we also investigated the effects of chemical markers on NOx secretion. The results reinforce the potential of chlorogenic acid and its structural analogues (NCA; CCA) as anti-inflammatory agents. CGA is one of the most widely available polyphenols among consumers because it exists in most abundant quantities in different foods [45].
It is also important to highlight that theobromine also showed strong inhibition of NOx secretion. This compound is not used in clinical care, but pentoxifylline, a synthetic theobromine derivative, is an immunological agent that is sometimes used to treat pediatric septic shock. Pentoxifylline acts by inhibiting erythrocyte phosphodiesterase, which increases expression of the anti-inflammatory protein adenosine monophosphate protein kinase (AMPK), suppressing neutrophils and proinflammatory cytokines and probably preventing chronic inflammation [46,47].
Nitric oxide is an important signaling molecule, and it is also highly reactive and diffusible. Therefore, it is important to have a strict control and regulation of production under physiologic conditions [48]. This molecule plays many important roles in the immune system, as well as on inflammation conditions. It is produced in high amounts from specialized cells of the immune system, mainly for cells of the innate immune system as macrophages.
TNF-α also plays as an important inductor of inflammation in several pathologies. TNF-α is one of the most important inflammatory cytokines that controls different types of cell functions. This inflammatory cytokine is produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signaling events within cells, leading to necrosis or apoptosis [49]. Therefore, it is evident that the suppression of TNF-α could be beneficial in different inflammatory diseases.
The effect of dietary habit on TNF-α represents an important subject with significant clinical implications, since several researches showed that phenolic-rich food intake was significantly and inversely related to TNF-α and IL-6 secretion [40]. Although the precise mechanisms of this antiinflammatory activity are not fully elucidated, it is has been hypothesized that phenolic compounds exert antiinflammatory activity by inhibiting the synthesis of proinflammatory mediators, modification of eicosanoid synthesis, inhibition of activated immune cells, or inhibition of nitric oxide synthase and cyclooxygenase-2 via its inhibitory effects on nuclear factor NF-κβ [50].
According to previous study described by Correa et al. [18], our data also showed that chlorogenic acid and its structural analogues were effective to suppress the TNF-α secretion levels in LPS-treated RAW 264.7 macrophages. Furthermore, similar potential was detected for THEO, CAF, and RUT, since all these compounds were able to suppress the TNF-α secretion as DEX.
NOx, TNF-α, and reactive oxygen species (ROS) are responsible for the nuclear transcription factor kappa (NF-κB) activation, which leads to the upregulation of many proinflammatory genes [51]. Our results showed that IA and its major chemical markers possess a strong potential to suppress NOx, TNF-α, and ROS production, suggesting that IA

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Mediators of Inflammation extracts and the related major compounds present in a plant extract may work based on several different mechanisms synergistically, resulting in moderating the immune system.

Conclusion
According to the results described herein, the extract prepared by infusion showed the best results in terms of antiinflammatory and antioxidant activities and total phenolic content. In addition, all major chemical markers present in the IA extract showed a strong anti-NOx activity and suppress the TNF-α secretion. Among these chemical markers, theobromine was highlighted, since in comparison with reference anti-inflammatory drug (DEX) it showed the same effect but at concentration 200x lower.

Data Availability
The data that support the findings of this study are available from the corresponding author.

Conflicts of Interest
The authors declare that they have no conflict of interest.

Authors' Contributions
I.V.F. was responsible for data curation, formal analysis, investigation, and roles/writing-original draft. E.F. was responsible for methodology, formal analysis, investigation, and roles/writing-original draft. L.C.T. was responsible for methodology and formal analysis. A.M.C. was responsible 11 Mediators of Inflammation for conceptualization, methodology, formal analysis, funding acquisition, and writing-review and editing. E.M.D. was responsible for conceptualization, methodology, formal analysis, funding acquisition, supervision, and writing-review and editing. F.H.R. was responsible for conceptualization, methodology, formal analysis, funding acquisition, supervision, and writing-review and editing. Ingrid Vicente Farias, Eduarda Fratoni, and Lais Cristina Theindl contributed equally to this work.