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Serjania marginata Casar. hydroalcoholic extract reduced cytokine and inammatory parameters in experimental models of inammation and infection in mice

This study investigated the antimycobacterial, anti-inammatory and antihyperalgesic effects of EESM in in vitro and in vivo models. EESM (0.98–1000 µg/ml) was evaluated in in vitro against Mycobacterium tuberculosis, M. bovis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis. The EESM oral administration (p.o.) (30, 100 and 300 mg/kg) and dexamethasone subcutaneous injection (s.c.) (1 mg/kg) were tested against the carrageenan-induced inammatory paw edema and pleurisy in Swiss mice. The EESM (30 and 100 mg/kg, p.o.) and dexamethasone (1 mg/kg, s.c.) were tested against the CFA-induced paw inammation and M. bovis (bacillus Calmette-Guerin - BCG)-induced pleurisy in C57bL6 mice. The minimum inhibitory concentration (MIC) of EESM in the presence of M. tuberculosis was 62.4 µg/ml. The values of MIC of EESM in the presence S. epidermidis, K. pneumoniae were 1000 µg/mL while EESM did not interfere with against P. aeruginosa growth. EESM signicantly inhibited paw edema/mechanical hyperalgesia in carrageenan induced paw inammation and leukocytes migration/proteins exudation in carrageenan-induced pleurisy model. In the BCG-induced pleurisy model, the daily treatment for 7 days, with EESM inhibited the levels of IL-1β in blood and in pleural exudate. The EESM did not alter the mycobacterial growth in the cell culture from pleural lavage, spleen and liver samples collected from BCG-treated animals. The EESM signicantly inhibited the persistent edema and mechanical hyperalgesia induced by CFA. This study conrms the EESM anti-inammatory property and showed that EESM has high potency in inducing inhibition of mycobaterial growth


Introduction
The immune and in ammatory process respond promptly to the presence of infectious pathogens. If the infectious pathogens persist, the body develops an adaptative immune and in ammatory response (Frizinsky et al., 2019, Doddam et al., 2019. For the discovery of potential anti-in ammatory/antiinfective agents, the infectious pathogens or PAMPs (Pathogen Associated Molecular Patterns) could be used in experimental models. The in ammatory response to live infectious agents and PAMPs depends on several components, such as leukocytes, receptor pattern recognition receptors (PRR), the complement system, pro-in ammatory mediators, cytokines, among others (Frizinsky et  The alternative strategies are needed to eliminate the pathogens and also harmful in ammation (Mookherjee et al., 2020). The plant candidate as alternative source is Serjania marginata Casar. (Sapindaceae) found in Brazil, Paraguay, Bolívia and Argentina (Acevedo-Rodríguez, 1990; Ferrucci, 1991; Flora do Brasil 2020, 2020). The traditional knowlegde calls this plant as "cipó-uva" or "cipó-timbó" (Corrêa, 1984;Heredia-Vieira et al., 2015) or blancuzco (Ferrucci, 1991) and the leaves, prepared in the form of juice, are used to treat stomachache by the Guarani Indians (Bourdy et al., 2004).
Gastroprotective activity of S. marginata leaves aqueous (Ota et al., 2019) and hydroalcoholic extracts (Périco et al., 2015) were scienti cally shown. Périco and colleagues (2015) also showed that hydroalcoholic extract from leaves of S. marginata was antimicrobial, antidiarrheal, and (anti)mutagenic. Moreira et al. (2019) demonstrated that S. marginata leaves aqueous extract was not toxic after a single exposure, however if it was used after prolonged periods it affected some parameters. Périco et al. (2015) showed that after 14 days of administration hydroalcoholic extract also affected some parameters such as induce gastric lesions.
The present work was motivated since the description of in vitro antimicrobial property of S. marginata was related by Périco et al., 2015, and by Serjania sp are used against other infections and in ammatory conditions in folk medicine (Bourdy et al., 2004;De Lima et al., 2006;Arruda et al., 2009). This study investigated the anti-in ammatory and anti-mycobacterial activity of hydroalcoholic extract from leaves of S. marginata (EESM) in several models contributing to the pharmacological knowledge of this plant.

Vegetal material and extract preparation
The in vitro antimycobacterial activity was performed according to Palomino et al. (2002). The EESM was dissolved in 5% of DMSO in a solution of pure water and the minimum inhibitory concentration (MIC) values for EESM was assayed at range of 0.98 -250 μg/ml and for isoniazid between 0.004 -1 μg/ml in the presence of the M. tuberculosis strain H37Rv (ATCC27294).

Animals, control drugs' administration, EESM dilution and doses for in vivo experiments
Female and male Swiss (28 -32 g) and C57BL/6 mice (25 -30 g) were provided by the central biotherium at the Federal University of Grande Dourados (UFGD). Animals were kept in polypropylene boxes in a sectorial Biotherium at Faculty of Health Sciences at UFGD with the following conditions: 22 °C, in the presence of a 12 h light/dark cycle, with food and water ad libitum. The EESM was dissolved in saline solution (0.9 %) and the dose was calculated according to mice weight. Vehicle solution is the saline solution since it was used to dissolve the EESM. The EESM oral administration (p.o.) doses used in the carrageenan-induced paw in ammation and pleurisy were 30, 100 and 300 mg/kg to verify if the EESM presents a dose-response pro le. As the doses of 30 and 100 mg/kg of EESM were effective against in ammatory parameters, these doses were tested also against BCG-induced pleurisy and CFAinduced in ammatory reaction. To verify the inhibitory e cacy of the commercial drugs, the dose of 1 mg/kg (by subcutaneous route -s.c.) of dexamethasone was used as control group in in vivo models while the dose of 25 mg/kg (p.o.) of isoniazid was used in BCG model.

Paw edema, mechanical and cold hyperalgesia evaluation in carrageenan induced paw in ammation
The male Swiss mice were distributed in ve experimental groups (n = 6) and one hour before the carrageenan injection, they were treated (p.o.) with 30, 100 and 300 mg/kg of EESM, with the vehicle and dexamethasone (1 mg/kg, s.c.). Subsequently, the animals received a subcutaneous injection (100 μL) of a solution containing 300 μg of carrageenan (Winter et al., 1962) dissolved in sterile 0.9 % saline in the right hind paw. The contralateral paw, which was used as a control, received 100 μL of saline. Paw edema was analyzed using a plethysmometer (Panlab, Spain) at 1, 2 and 4 h after injection. In addition, mechanical hyperalgesia was assessed using an electronic version of the von Frey test (Analgesimeter von frey, InSight) and cold sensitivity was analyzed with the acetone drop test (Decosterd and Woolf, 2000) at 3 and 4 h after carrageenan injection.

Leukocyte migration and protein analysis in pleural exudate collected from carrageenan induced pleurisy
Female Swiss mice were distributed in ve experimental groups (n = 6) and one hour before the pleurisy, they were treated (p.o.) with 30, 100 and 300 mg/kg of EESM, with the vehicle and dexamethasone (1 mg/kg, s.c.). Subsequently, with the aid of an adapted needle, 100 μL of carrageenan (containing 100 μg) was injected into the right side of the animals' chest cavity to induce in ammation (Vinegar et al., 1973). Four hours after the injection of carrageenan, the animals were euthanized (100 mg/kg of ketamine and Groups of six male C57BL/6 mice were treated with vehicle (control group), EESM (30 and 100 mg/kg, p.o.), and dexamethasone 1 mg/kg (s.c., the positive control group) every day, once a day for 22 days. After the rst treatment, the animals received 30 μL of Freund's complete adjuvant (CFA) (oil suspension containing inactive M.tuberculosis) via intraplantar injection in the right paw. Mechanical and cold sensitivity and paw edema were measured 6, 11, 16 and 22 days after the injection of CFA.

Statistical analysis
The data are presented as the mean ± standard error (SEM). The determination of signi cant differences among groups was made via one-way analysis of variance (ANOVA) and the Newman-Keuls test was chosen as a post hoc (GraphPad Prism Software). The percentage of inhibition was calculated from the control group. Differences were considered to be signi cant when p < 0.05.

Effects of EESM on M. tuberculosis and other microorganisms in vitro
The MIC value of EESM in the presence of the M. tuberculosis strain was 62.4 µg/ml, while the MIC value of isoniazid was 0.030 µg/ml. EESM was effective against S. epidermidis, K. pneumoniae in 1000 µg/ml and was not effective against P. aeruginosa.

Effects of EESM on paw edema induced by carrageenan
The paw edema was signi cantly inhibited at doses of 100 and 300 mg/kg of EESM after 2 h of carrageenan injection with inhibition of 38 % and 44 %, respectively (p < 0.05) (Figures 1B). After 4 h, all doses showed signi cant differences from the control group with inhibition of 36 %, 33 % and 58 % in doses 30, 100 (p < 0.05) and 300 mg/kg (p < 0.01). The dexamethasone group showed signi cant inhibition at 1, 2 and 4 h.
Dexamethasone was different from all treated groups when the comparison among groups was perfomed ( Figures 1A-C). The groups treated with doses of 100 and 300 mg/kg differed from the control and from the group treated with 30 mg/kg of EESM ( Figure 1B) and in gure 1C the groups treated with doses of 30 and 100 mg/kg differed from the control and from group treated with 300 mg/kg of EESM.
In the carrageenan induced mechanical hyperalgesia, 100 % inhibition was observed in two evaluations at the dose of 300 mg/kg and the dexamethasone group (p < 0.001) (Figures 2A and 2B). The dose of 100 mg/kg also demonstrated prevention of hyperalgesia development, with 100 % (p < 0.001) and 96 % (p < 0.01) e cacy, in 3 and 4 h after carrageenan injection, respectively (Figures 2A and 2B). The dose of 30 mg/kg was not effective for the prevention of carrageenan paw hyperalgesia. The control and the group treated with 30 mg/kg of EESM differed signi cantly from the other treated groups (Figures 2A and  2B). The groups treated with doses of 100, 300 mg/kg and dexamethasone did not differ among themselves (Figure 2A) and in gure 2B the groups treated with doses of 300 mg/kg and dexamethasone differed from the other treated groups.
In the cold nociceptive response, no doses showed a signi cant difference from control group, however the dexamethasone group obtained an inhibition of 52 % and 49 % (p < 0.05) in 3 and 4 h, respectively ( Figures 2C and 2D).
The statistical comparison among groups showed that control and the group treated with 30 mg/kg of EESM differed from the other groups in Figure 3A while only control group differed from others groups in Figure 3B.

Effects of EESM on Leukocyte migration, Il-1β dosage and CFU growth in BCG-induced pleurisy model
After 7 days of oral treatment, EESM (30 and 100 mg/kg) did not decrease the leukocyte migration into the pleural spaces after intrathoracic injection of BCG, however isoniazid reduced signi cantly the leukocyte invasion to the pleura ( Figure 4A). In pleural exudates, the reduction of IL-1β levels were signi cant with inhibition of 72 % and 76 % with the treatment of 30 and 100 mg/kg EESM (p.o.) and 76 % isoniazid (25 mg/kg, p.o.) ( Figure 4B). In serum, the reduction of IL-1β levels were signi cant with inhibition 50 % and 59 % with the treatment of 30 and 100 mg/kg EESM (p.o.) and 67 % isoniazid (25 mg/kg, p.o.) ( Figure 4C). The statistical comparison among groups showed that only the control group differed from the other groups in Figures 4B and 4C.
During the cell culture of the pleural lavage, spleen and liver samples collected from BCG treated animals, the EESM (30 or 100 mg/kg) did not alter the mycobacteria growth (results not shown) and the UFC count did not differ from control group (results not shown) until 60 days. The negative control group samples presented mycobacterial growth in all cultivated plaques, with a mean of 54 CFU after 60 days of culture, while the isoniazid group samples did not show any growth after 60 days (results not shown).

Effects of EESM on CFA induced paw in ammation
A single daily oral treatment with EESM for 6 days at doses of 30 and 100 mg/kg and dexamethasone (1 mg/kg, s.c.) reduced mechanical hyperalgesia in 100 % respectively ( Figure 5A). After 11 and 16 days from CFA injection, no signi cant inhibitions in mechanical response were observed with 30 mg/kg of EESM, however a signi cant inhibition was detected with dexamethasone ( Figure 5B and 5C). At 22 days after CFA, the inhibition observed with 30 mg/kg of EESM was 100 % ( Figure 5D). The groups treated with EESM (30 and 100 mg/kg) did not differ statistically among themselves at days 6, 11 and 16, however they were different from the dexamethasone group on 11 and 16 days (Figures 5A-C). The group treated with 100 mg/kg differed from the group treated with 30 mg/kg at 22 days after CFA injection ( Figure 5D).
The dose of 100 mg/kg, but not the dose of 30 mg/kg of EESM and dexamethasone groups showed a signi cant difference in paw edema on days 6 and 11 after the administration of CFA with the inhibition of 21 % and 47 %, respectively ( Figures 6A and 6B). On days 16 and 22, the doses of 30, 100 mg/kg of EESM and dexamethasone groups were different from the control groups with inhibitions of 33 %, 50 % and 65 % on day 16 and 57 %, 67 % and 87 % on day 22, and 57 %, 67 % and 87 % on day 22, respectively ( Figures 6C and 6D). The treatment with 100 mg/kg did not statistically differ from dexamethasone group on days 6, 11 and 16, however on day 22 these groups were statistically different ( Figure 6). Both treatments with EESM were not statistically different to dexamethasone group on day 16, nevertheless on day 22 both treatments with EESM differed from dexamethasone group.
In cold hyperalgesia induced by CFA, the treatment with EESM at dose of 100 mg/kg was statistically different from the control group, with the inhibition 52 % showing similarity inhibition to dexamethasone group on day 6. The treatments with both doses of EESM did not differ from the control group on days 11, 16 and 22.

Discussion
Species of the Serjania genus have been used in folk medicine for their anti-in ammatory potential in Brazil and in Mexico (Salinas-Sánchez et al., 2017) while S. marginata leaves are squeezed for juice by the Bolivian people as an analgesic agent for stomach (Bourdy et al., 2004). The present study was the rst one to show the anti-in ammatory, analgesic and potential antibiotic properties of EESM in in ammatory/infection/hyperalgesic models. The data of the present work corroborate with the traditional folk use of S. marginata as an analgesic and expand the information regarding its use.
Some in ammatory models used in this present research involve carrageenan and CFA. The carrageenan was used in two acute in ammatory models to analyze the e cacy and potency of anti-in ammatory agents until 48 h (Posadas et al., 2004). When the carrageenan is administered into the mice paw the edema, mechanical and cold hyperalgesia can be measured (Santos el al., 2020) and when it is injected by intrapleural route the leukocyte and protein can be measured in pleural exudate . In the present study the EESM showed to be effective against acute edema (2, and 4 h, Figure 1B and 1C), mechanical hyperalgesia (3 and 4 h, Figures 2A and 2B), however the cold hyperalgesia ( Figures 2C and  2D) was not inhibited in carrageenan paw in ammatory model. In the carrageenan induced-pleurisy model, both leukocyte migration and protein exudation were inhibited by EESM (Figures 3A and 3B). In this model, the EESM induced a dose dependent inhibition ( Figures 1B, 1C, 2A, 2B and 3A) in which the e cacy response to EESM differed signi cantly when its dose was increased. In Figure 2B the classical dose-response effect could be detected since each dose of EESM signi cantly differed in e cacy among themselves. As far as we know, only the research of Périco et al. (2015) showed the myeloperoxidase activity inhibition by EESM in ulcer models. The present study showed the anti-in ammatory and mechanical anti-hyperalgesic (analgesic) response of EESM occurs a dose-dependent manner.
The major compounds such as avonoids, proanthocyanidins and saponins have already been described in EESM (Heredia-Vieira et  Since the EESM activity seems to be more selective against M. tuberculosis than to other microorganisms the in vivo EESM potential against mycobacteria was performed. The EESM was tested in BCG-induced pleurisy since it is not possible to work with M. tuberculosis in vivo in our University. Another reason to work with BCG as a mycobacteria model, is that its immunization schedule is applied against tuberculosis at birth in various countries. The licensed vaccine with attenuated BCG is performed in health care and several new vaccines are in clinical phases (Pérez et al., 2020). BCG was inoculated in the interpleural space, inducing an infectious and in ammatory mycobacterial response con rmed by the results of the control groups compared to the naive group ( Figure 4). The results of leukocyte migration into the pleural space, IL-1β levels in serum and in pleural exudate in the negative control group signi cantly differed from those of the naïve group after 7 days. In BCG pleurisy model, oral doses of 30 and 100 mg/kg of EESM did not interfere with leukocyte migration to the pleural exudate however it induced a signi cant inhibition on the interleukin-1 beta (IL-1β) levels in serum and pleural exudate. The increase in IL-1β levels after 7 days in BCG model may be correlated with the mycobacterial growth, as showed previously by our group . Bourigault et al. (2013) indicated that mice de cient in interleukin (IL) showed reduction in bacterial load 35 days' post infection. Tumor necrosis factor alpha (TNF-α) and IL-1β are produced by immune cells that are involved in pulmonary tuberculosis (Jayaraman et al., 2014;Weiss et al., 2019). These results showed that doses of EESM controlled the BCG-in ammation.
An aliquot of precipitate sample from pleural exudate and also spleen and liver macerate from all BCGinjected animals were platted in Middlebrook 7H9/Ogawa Kudu culture medium for analysis of BCG growth. The BCG culture was found positive for Ziehl-Neelsen stain con rming that this microorganism is a Mycobacterium sp. The isoniazid inhibited signi cantly all the aspects of in vivo in ammatory response ( Figure 4) Figure 5D). These results demonstrated that chronic anti-in ammatory ability of EESM even also in the persistent CFA model of in ammation.

Conclusion
The present study was the rst one to show the anti-in ammatory, anti-hyperalgesic and potential antibiotic properties of EESM in in ammatory/infection/hyperalgesic in acute and persistent models. EESM has high potency in inducing inhibition of mycobaterial growth and low potency or no effects in relation to other microorganisms in in vitro. EESM chemical composition showed the presence of several classes of compounds such as avonoids, proanthocyanidins and saponins could be responsible for the EESM anti-in ammatory activity.

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