*Article Margaritaria nobilis* **L.f. (Phyllanthaceae) Ethanolic Extract: Low Acute Oral Toxicity and Antinociceptive Activity**

**Fabiana Menezes S. Camara <sup>1</sup> , Brenda Costa da Conceição 1,2, Eloise Karoline S. Cardoso 1,2 , Johan Carlos C. Santiago <sup>3</sup> , Carlos Alberto B. Albuquerque <sup>3</sup> , Washington L. Pereira <sup>4</sup> , Marta C. Monteiro <sup>2</sup> , Consuelo Y. Yoshioka e Silva 2,3, Milton Nascimento da Silva 2,3 , Cristiane F. Maia 1,2 and Eneas A. Fontes-Junior 1,2,\***


**Abstract:** *Margaritaria nobilis* L.f. (Phyllanthaceae), a native Brazilian tree occurring mainly in the Amazon, is used in folk medicine for the treatment of abscesses (bark) and cancer-like symptoms (leaves). The present study evaluates the safety of its acute oral administration and its effects on nociception and plasma leakage. The chemical constitution of the leaf's ethanolic extract is determined by ultra-performance liquid chromatography–high-resolution mass spectrometry (LC-MS. Its acute oral toxicity is evaluated in female rats at a dose of 2000 mg/kg, evaluating the occurrence of deaths and Hippocratic, behavioral, hematological, biochemical, and histopathological changes, as well as food and water consumption and weight gain. Antinociceptive activity is evaluated in male mice with acetic-acid-induced peritonitis (APT) and formalin (FT) tests. An open field (OF) test is performed to verify possible interferences in the animals' consciousness or locomotion. LC-MS analysis shows the presence of 44 compounds classified as phenolic acid derivatives, flavonoids and O-glycosylated derivatives, and hydrolyzable tannins. No deaths or significant behavioral, histological, or biochemical changes are observed in the toxicity assessment. In nociception tests, *M. nobilis* extract significantly reduces abdominal contortions in APT, demonstrating selectivity for inflammatory components (FT second phase), not interfering in neuropathic components (FT first phase) or consciousness and locomotion levels in OF. Additionally, *M. nobilis* extract inhibits plasma acetic-acid-induced leakage. These data demonstrate the low toxicity of *M. nobilis* ethanolic extract, as well as its effectiveness in modulating inflammatory nociception and plasma leakage, possibly related to the flavonoids and tannins present in its composition.

**Keywords:** *Margaritaria nobilis*; natural products; medicinal plants; toxicity; nociception; pain; antinociceptive

#### **1. Introduction**

Treatment of inflammation and pain management is still a major challenge for global public health. Although they are physiological processes, they are closely linked to the genesis and evolution of numerous pathologies [1]. Therefore, adequately treating inflammation and pain can positively impact patients' clinical improvement and quality of life. This reality clarifies that analgesic and anti-inflammatory drugs are among the most prescribed and used in many countries, except for those where traditional medicine and medicinal plants are widely included in national public health policies [2].

**Citation:** Camara, F.M.S.; da Conceição, B.C.; Cardoso, E.K.S.; Santiago, J.C.C.; Albuquerque, C.A.B.; Pereira, W.L.; Monteiro, M.C.; Yoshioka e Silva, C.Y.; da Silva, M.N.; Maia, C.F.; et al. *Margaritaria nobilis* L.f. (Phyllanthaceae) Ethanolic Extract: Low Acute Oral Toxicity and Antinociceptive Activity. *Pharmaceuticals* **2023**, *16*, 689. https://doi.org/10.3390/ph16050689

Academic Editor: Diana Roxana Pelinescu

Received: 6 March 2023 Revised: 14 April 2023 Accepted: 20 April 2023 Published: 3 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Orthodox therapy, with steroidal (SAIDs) or non-steroidal (NSAIDs) anti-inflammatory drugs, opioids, antidepressants, antiepileptics, and sedatives, although efficient in many cases, find limitations in a significant variety of diseases, mainly when the risks outweigh the observed benefits [3]. In acute conditions, the need for high doses, which affect the stomach, liver, blood, and kidneys, is the main concern, in addition to respiratory and CNS depression, with possible cardiac changes as well [2,4]. Chronic conditions, however, which require prolonged or even continuous use of these medications, are the most challenging. In addition to worsening the adverse reactions already mentioned, they are associated with the induction of metabolic and immunological disorders, tolerance, dependence, and important behavioral impairments [3,4].

Therefore, the search for innovative therapeutic strategies, ranging from herbal medicines to new, safer, and more effective drugs, becomes imperative. In this sense, the medicinal culture of traditional populations proves to be an important source of information about plant species with pharmacological potential. It also provides clues about the safety of their use, reaffirming the value of ethnopharmacological knowledge [5].

Plant species *Margaritaria nobilis* L.f. (synonyms: *Phyllanthus antillanus* (A. Juss.) Müll. Arg.; *P. nobilis* (L.f.) Müll. Arg.; *P. ibonensis* Rusby; *Bradleia sinica* Müll. Arg.; *Cicca antillana* A.Juss.; *C. pavoniana* Baill.; *C. chinensis* Baill.; *C. surinamensis* Miq.; *C. sinica* Baill.; *Diasperus antillanus* (A. Juss.) Kuntze; *Margaritaria adelioides* Rich. ex Baill.; *M. alternifolia* L.; among others), popularly known as 'botãozinho' (little button), 'figueirinha' (little fig tree), 'cabelode-cotia' (agouti hair), 'café-bravo' (wild coffee) and 'fruto-de-jacamin' (jacamin fruit), for a long time was considered part of the genus Phyllanthus (family: Phyllanthaceae). Recently, however, it was reclassified based on phylogenetic studies, being now placed in the genus Margaritaria (family: Phyllanthaceae). This tree is native to Brazilian territory and distributed in all of the region's states, emphasizing the Amazon [6]. In traditional medicine, its bark is cited for treating abscesses, fruits as a tonic, and leaves for treating cancer-like symptoms [7].

There are few studies regarding the chemical constitution of *M. nobilis*, such as the study by Moraes et al. [8], which addresses the phytochemical profile and leishmanicidal activity, and the study recently published with comprehensive characterization, indicating the presence of derivatives of phenolic acids, flavonoids, and hydrolyzable tannins [7]. In the genus *Margaritaria*, the presence of phenolic derivatives, glycosylated flavonoids (*M. discoidea*), and alkaloids (*M. indica*) has been described, together with their potential as antioxidant, antinociceptive, and anti-inflammatory agents [9–13]. The present study, therefore, aims to investigate the safety of acute oral administration of *M. nobilis* leaves ethanolic extract (MnE), considering behavioral, anatomical, histological, and biochemical aspects. It also seeks to elucidate its properties on nociception and inflammatory components.

#### **2. Results**

#### *2.1. MnE Phytochemical Composition*

Through the LC-MS technique, 44 chemical compounds of the sample under study were annotated. The chromatogram of total MnE ions can be seen in Figure 1; these compounds were classified into three major groups: phenolic acids, flavonoids, and hydrolyzable tannins, which can be seen in Table 1 with information on their *m/z* ratio, retention time, and their fragmentation patterns. Most of the annotated compounds are hydrolyzable tannins, a subclass of ellagitannins.

**Figure 1.** Total ion current chromatograms of *Margaritaria nobilis* ethanolic extract (MnE). The blue dashed lines indicate the retention time (RT) in which the compounds were recorded, being numbered in correspondence with Table 1. **Figure 1.** Total ion current chromatograms of *Margaritaria nobilis* ethanolic extract (MnE). The blue dashed lines indicate the retention time (RT) in which the compounds were recorded, being numbered in correspondence with Table 1.


#### **Table 1.** Compounds identified by LC-HRMS-ToF in *Margaritaria nobilis* ethanolic extract (MnE). **Table 1.** Compounds identified by LC-HRMS-ToF in *Margaritaria nobilis* ethanolic extract (MnE).

22 8.73 Excoecariphenol C Isomer C37H30O24 857.1077 3.3 23 8.73 Tetragalloyl-glucose C34H28O22 787.0977 2.2

 8.87 Kaempferol 3-*O*-xylosyl-glucoside C26H28O15 579.1376 4.0 8.87 Quercetin 3-*O*-glucoside Isomer II C21H20O12 463.0898 4.5 8.93 Di-*O*-Methyl ellagic acid *O*-glucoside C22H20O13 491.0852 5.3 9.41 Quercetin 3-*O*-rhamnosyl-xyloside C26H28O15 579.1350 0.0 9.61 Quercetin 3-*O*-xyloside C20H18O11 433.0765 1.4 9.70 Kaempferol 3-*O*-glucoside Isomer I C21H20O11 447.0935 1.8


**Table 1.** *Cont.*

#### *2.2. MnE Acute Oral Toxicity 2.2. MnE Acute Oral Toxicity*

2.2.1. MnE Does Not Change Hippocratic Signs, Motor Behavior, or Emotionality, or Cause Deaths 2.2.1. MnE Does Not Change Hippocratic Signs, Motor Behavior, or Emotionality, or Cause Deaths

No significant changes in Hippocratic markers were observed in the four hours (15, 30, 60, and 240 min) following oral administration of MnE (2000 mg/kg), as well as in the 14 subsequent days. Similarly, oral administration of the extract did not alter motor parameters, such as total ambulation (6.72 ± 1.48 m; *p* = 0.960; Figure 2A) and locomotion speed (0.023 ± 0.005 m/s; *p* = 0.932; Figure 2B), or emotionality, such as freezing time (220.78 ± 14.93 s; *p* = 0.619; Figure 2C) and locomotion in the central area (0.29 ± 0.10 m; *p* = 0.928; Figure 2D) in the open field test, when compared to control animals (total locomotion = 6.84 ± 1.50 m; speed = 0.023 ± 0.005 m/s; freezing = 232.35 ± 16.39 s; central locomotion = 0.27 ± 0.11 m). No deaths were observed during the entire toxicity test period. No significant changes in Hippocratic markers were observed in the four hours (15, 30, 60, and 240 min) following oral administration of MnE (2000 mg/kg), as well as in the 14 subsequent days. Similarly, oral administration of the extract did not alter motor parameters, such as total ambulation (6.72 ± 1.48 m; *p* = 0.960; Figure 2A) and locomotion speed (0.023 ± 0.005 m/s; *p* = 0.932; Figure 2B), or emotionality, such as freezing time (220.78 ± 14.93 s; *p* = 0.619; Figure 2C) and locomotion in the central area (0.29 ± 0.10 m; *p* = 0.928; Figure 2D) in the open field test, when compared to control animals (total locomotion = 6.84 ± 1.50 m; speed = 0.023 ± 0.005 m/s; freezing = 232,35 ± 16.39 s; central locomotion = 0.27 ± 0.11 m). No deaths were observed during the entire toxicity test period.

**Figure 2.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) acute oral administration (2000 mg/kg) on rats' behavior in open field test, considering (**A**) total locomotion, (**B**) average locomotion speed, (**C**) freezing time, and (**D**) central locomotion. Data are presented as mean ± SEM (*n* = 5/group). Student's *t*-test. **Figure 2.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) acute oral administration (2000 mg/kg) on rats' behavior in open field test, considering (**A**) total locomotion, (**B**) average locomotion speed, (**C**) freezing time, and (**D**) central locomotion. Data are presented as mean ± SEM (*n* = 5/group). Student's *t*-test.

#### 2.2.2. MnE Does Not Alter Water or Feed Consumption, Weight Gain, or Organ Histology 2.2.2. MnE Does Not Alter Water or Feed Consumption, Weight Gain, or Organ Histology

Animals treated with MnE (2000 mg/kg; v.o.) showed feed and water consumption patterns equivalent to those observed in the control group (Figure 3A,B), as well as weight gain. No changes in morphology, color, or size were observed during macroscopic analysis of the heart, liver, stomach, and kidneys. Animals treated with MnE (2000 mg/kg; v.o.) showed feed and water consumption patterns equivalent to those observed in the control group (Figure 3A,B), as well as weight gain. No changes in morphology, color, or size were observed during macroscopic analysis of the heart, liver, stomach, and kidneys.

**Figure 3.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) on daily (**A**) feed and (**B**) water intake in the 14 days following the acute limit dose (2000 mg/kg) administration and (**C**) on the relative organ-body weights on the 14th day. Data expressed as mean ± SEM (*n* = 5/group). (**A**) Friedman test; (**B**) one-way RM-ANOVA; (**C**) Student *t*-test (\* *p* < 0.05 vs. control group). **Figure 3.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) on daily (**A**) feed and (**B**) water intake in the 14 days following the acute limit dose (2000 mg/kg) administration and (**C**) on the relative organ-body weights on the 14th day. Data expressed as mean ± SEM (*n* = 5/group). (**A**) Friedman test; (**B**) one-way RM-ANOVA; (**C**) Student *t*-test (\* *p* < 0.05 vs. control group).

The stomach (1.427 ± 0.024 g/100 g b.w.; *p* = 0,00316) and kidney (L—0.610 ± 0.015 g/100 g b.w.; *p* = 0,00362; R—0.614 ± 0.015 g/100 g b.w.; *p* = 0,00303) relative weights of the animals treated with MnE were higher than the control group (stomach—1.256 ± 0.033 g/100 g b.w.; L kidney—0.521 ± 0.016 g/100 g b.w.; R kidney—0.519 ± 0.017 g/100 g b.w.). The heart and liver have relative weights equivalent to controls (Figure 3C). The histological evaluation did not reveal alterations indicative of toxicity, with histological normality being identified in all kidney and heart samples. Some animals (two controls and three treated) had mild chronic superficial gastritis not associated with acute treatment. The glandular and non-glandular regions of the stomach showed histological normality. Liver tissue, in general, was preserved, with histological normality. In some animals treated with MnE, the discreet presence of cytoplasmic vacuoles was observed but without repercussions for cell function (Figure S1). The stomach (1.427 ± 0.024 g/100 g b.w.; *p* = 0,00316) and kidney (L—0.610 ± 0.015 g/100 g b.w.; *p* = 0,00362; R—0.614 ± 0.015 g/100 g b.w.; *p* = 0,00303) relative weights of the animals treated with MnE were higher than the control group (stomach—1.256 ± 0.033 g/100 g b.w.; L kidney—0.521 ± 0.016 g/100 g b.w.; R kidney—0.519 ± 0.017 g/100 g b.w.). The heart and liver have relative weights equivalent to controls (Figure 3C). The histological evaluation did not reveal alterations indicative of toxicity, with histological normality being identified in all kidney and heart samples. Some animals (two controls and three treated) had mild chronic superficial gastritis not associated with acute treatment. The glandular and nonglandular regions of the stomach showed histological normality. Liver tissue, in general, was preserved, with histological normality. In some animals treated with MnE, the discreet presence of cytoplasmic vacuoles was observed but without repercussions for cell function (Figure S1).

#### 2.2.3. MnE Does Not Interfere with the WBC or Liver and Kidney Function Markers 2.2.3. MnE Does Not Interfere with the WBC or Liver and Kidney Function Markers

MnE limit dose (2000 mg/kg; v.o.) administration did not change the leukogram of the animals (Table 2). Both groups had normocytic and normochromic red blood cells and no changes were observed in platelets. In addition, no changes were observed in the liver (AST and ALT; Figure 4A) and renal (urea and creatinine; Figure 4B) function markers, which showed plasma concentration equivalent to the control group. MnE limit dose (2000 mg/kg; v.o.) administration did not change the leukogram of the animals (Table 2). Both groups had normocytic and normochromic red blood cells and no changes were observed in platelets. In addition, no changes were observed in the liver (AST and ALT; Figure 4A) and renal (urea and creatinine; Figure 4B) function markers, which showed plasma concentration equivalent to the control group.

**Table 2.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) acute oral administration (2000 mg/kg) on differential leukocyte count. Data presented as mean ± SEM (*n* = 4–5/group). Student's *t*test. **Table 2.** Effects of *Margaritaria nobilis* leaf's ethanolic extract (MnE) acute oral administration (2000 mg/kg) on differential leukocyte count. Data presented as mean ± SEM (*n* = 4–5/group). Student's *t*-test.


**Figure 4.** Effects of acute oral administration of *Margaritaria nobilis* ethanolic extract (MnE) (2000 mg/kg) on (**A**) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; and (**B**) urea and creatinine serum concentrations. Data expressed as mean ± SEM (*n* = 5/group). Student's *t*-test. **Figure 4.** Effects of acute oral administration of *Margaritaria nobilis* ethanolic extract (MnE) (2000 mg/kg) on (**A**) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; and (**B**) urea and creatinine serum concentrations. Data expressed as mean ± SEM (*n* = 5/group). Student's *t*-test.

#### *2.3. MnE Antinociceptive and Antiinflamatory Activity 2.3. MnE Antinociceptive and Antiinflamatory Activity*

#### 2.3.1. MnE Reduces ACA-Induced Abdominal Writhing and Plasma Leakage 2.3.1. MnE Reduces ACA-Induced Abdominal Writhing and Plasma Leakage

Intraperitoneal administration of ACA (0.6%) produced 67.57 ± 3.33 writhes in the observation interval. The lowest MnE dose (200 mg/kg, v.o.), despite reducing the average of writhing (48.33 ± 10.54), did not present a statistically significant effect (*p* = 0.219). Doses of 400 and 800 mg/kg, on the other hand, reduced acetic-acid-induced writhing by 64.73% (23.83 ± 8.14; *p* = 0.001) and 72.87% (18.33 ± 8.76; *p* < 0.001), respectively, showing an effect like the indomethacin (10 mg/kg; 19.14 ± 2.56) (Figure 5A) and with an estimated median effective dose (ED50) of 338.298 mg/kg (r2 = 0.882) (Figure 5B). Intraperitoneal administration of ACA (0.6%) produced 67.57 ± 3.33 writhes in the observation interval. The lowest MnE dose (200 mg/kg, v.o.), despite reducing the average of writhing (48.33 ± 10.54), did not present a statistically significant effect (*p* = 0.219). Doses of 400 and 800 mg/kg, on the other hand, reduced acetic-acid-induced writhing by 64.73% (23.83 ± 8.14; *p* = 0.001) and 72.87% (18.33± 8.76;*p* < 0.001), respectively, showing an effect like the indomethacin (10 mg/kg; 19.14 ± 2.56) (Figure 5A) and with an estimated median effective dose (ED50) of 338.298 mg/kg (r<sup>2</sup> = 0.882) (Figure 5B).

Regarding proteins concentration in peritoneal fluid, the ACA (0.6%; ip.) administration increased it by around 144% (0.996 ± 0.065 mg/mL; *p* = 0.01) compared to the white group (0.408 ± 0.02 mg/mL), which did not receive ACA. Treatment with MnE at 200 mg/kg reduced the mean protein concentration (0.540 ± 0.105 mg/mL), but its effect was not statistically significant (*p* = 0.087) when compared to the ACA group. The dose of 400 mg/kg reversed the ACA-induced damage by 59.43% (0.404 ± 0.044 mg/mL; *p* = 0.003), showing an effect like indomethacin (10 mg/kg; 0.624 ± 0.044 mg/mL). At 800 mg/kg, MnE reduced the elevation in peritoneal fluid protein levels by 63.33% (0.365 ± 0.019 mg/mL; *p* < 0.001), showing an effect superior (*p* = 0.022) to that produced by the standard drug (Figure 5C).

test).

(Figure 5C).

= 0.031) (Figure 6A).

**Figure 5.** Effect of *Margaritaria nobilis* ethanolic extract (MnE) (200, 400, and 800 mg/kg; v.o.) on (**A**) acetic acid (0.6%)-induced abdominal writhing, with (**B**) identification of its median effective dose (ED50) by linearregression and (**C**) plasma leakage. Data expressed as mean ± SEM (A: *n* = 6–7/group; B: 5–6/group). ++ *p* < 0.01 vs. WHITE; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p*< 0.001 vs. ACA; (ANOVA, Dunnett's **Figure 5.** Effect of *Margaritaria nobilis* ethanolic extract (MnE) (200, 400, and 800 mg/kg; v.o.) on (**A**) acetic acid (0.6%)-induced abdominal writhing, with (**B**) identification of its median effective dose (ED50) by linear regression and (**C**) plasma leakage. Data expressed as mean ± SEM (A: *n* = 6–7/group; B: 5–6/group). ++ *p* < 0.01 vs. WHITE; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p*< 0.001 vs. ACA; (ANOVA, Dunnett's test).

2.3.2. MnE Reduces the Nociception in the Inflammatory Phase of the Formalin Test

Regarding proteins concentration in peritoneal fluid, the ACA (0.6%; ip.) administration increased it by around 144% (0.996 ± 0.065 mg/mL; *p* = 0.01) compared to the white group (0.408 ± 0.02 mg/mL), which did not receive ACA. Treatment with MnE at 200 mg/kg reduced the mean protein concentration (0.540 ± 0.105 mg/mL), but its effect was Treatment with MnE (400 mg/kg; v.o.; 82.33 ± 14.96 s) did not interfere with formalininduced licking time (83.33 ± 14.87 s; *p* = 0.998) during the neuropathic phase. In the inflammatory phase, on the other hand, MnE (400 mg/kg; v.o.; 129.17 ± 12.29 s) significantly reduced the licking time of the animals compared to the control group (171.67 ± 14.85 s; *p* = 0.031) (Figure 6A).

not statistically significant (*p* = 0.087) when compared to the ACA group. The dose of 400 mg/kg reversed the ACA-induced damage by 59.43% (0.404 ± 0.044 mg/mL; *p* = 0.003), showing an effect like indomethacin (10 mg/kg; 0.624 ± 0.044 mg/mL). At 800 mg/kg, MnE reduced the elevation in peritoneal fluid protein levels by 63.33% (0.365 ± 0.019 mg/mL; *p* Additionally, the treatment with MnE (400 mg/kg; 19.06 ± 3.15 m) did not interfere with the locomotor capacity or exploration of the animals, when compared to the control group (17.69 ± 2.27 m; *p* = 0.809), excluding possible interferences in antinociceptive tests, such as sedation or motor impairment (Figure 6B).

induced licking time (83.33 ± 14.87 s; *p* = 0.998) during the neuropathic phase. In the in-

Treatment with MnE (400 mg/kg; v.o.; 82.33 ± 14.96 s) did not interfere with formalin-

< 0.001), showing an effect superior (*p* = 0.022) to that produced by the standard drug

2.3.2. MnE Reduces the Nociception in the Inflammatory Phase of the Formalin Test

**Figure 6.** Effect of *Margaritaria nobilis* ethanolic extract (MnE) (200, 400, and 800 mg/kg; v.o.) on (A) neuropathic and inflammatory nociception in formalin test (**B**) and total locomotion in the open field. Data expressed as mean ± SEM (A: *n* = 6/group; B: *n* = 7/group). \* *p* < 0.05, \*\*\* *p*< 0.001 vs. ACA; ((**A**) ANOVA, Dunnett's test; (**B**) Student *t*-test). **Figure 6.** Effect of *Margaritaria nobilis* ethanolic extract (MnE) (200, 400, and 800 mg/kg; v.o.) on (**A**) neuropathic and inflammatory nociception in formalin test (**B**) and total locomotion in the open field. Data expressed as mean ± SEM (A: *n* = 6/group; B: *n* = 7/group). \* *p* < 0.05, \*\*\* *p* < 0.001 vs. ACA; ((**A**) ANOVA, Dunnett's test; (**B**) Student *t*-test).

#### Additionally, the treatment with MnE (400 mg/kg; 19.06 ± 3.15 m) did not interfere **3. Discussion**

with the locomotor capacity or exploration of the animals, when compared to the control group (17.69 ± 2.27 m; *p* = 0.809), excluding possible interferences in antinociceptive tests, such as sedation or motor impairment (Figure 6B). **3. Discussion** The present study demonstrates for the first time that MnE (ethanolic extract of *M. nobilis* leaves) has low toxicity when orally administered in an acute pattern, as well as its ability to modulate nociception, possibly by peripheral mechanisms related to inflammation, and to inhibit plasma leakage. Such effects are probably related to its secondary metabolites identified by our group, such as flavonoids, hydrolyzed tannins, and phenolic acids. We described the annotation of 44 phytoconstituents (Table 1) present in the MnE [7]. On this aspect, the metabolic annotation has expedited the clarification of the chemical-pharmacological signature of complex matrices, without ignoring the unequivocal identification that may allow bioactivity of greater magnitudes [14]. Thus, because some The present study demonstrates for the first time that MnE (ethanolic extract of *M. nobilis* leaves) has low toxicity when orally administered in an acute pattern, as well as its ability to modulate nociception, possibly by peripheral mechanisms related to inflammation, and to inhibit plasma leakage. Such effects are probably related to its secondary metabolites identified by our group, such as flavonoids, hydrolyzed tannins, and phenolic acids. We described the annotation of 44 phytoconstituents (Table 1) present in the MnE [7]. On this aspect, the metabolic annotation has expedited the clarification of the chemical-pharmacological signature of complex matrices, without ignoring the unequivocal identification that may allow bioactivity of greater magnitudes [14]. Thus, because some of these compounds are structural scaffolds for promising pharmacological segments, we contend that they may explain the observed activities interactively, such as gallic acid derivatives (1), quercetin O-glycosylated derivatives (40), and similar compounds (10, 14, 15, 21, 26, 28, 29, and 41), kaempferol (42) and its glycosylated derivatives, ellagitannins (analogs of 5–7, 17, 31, 38, and 48), and gallotannins (analogs of 8, 23, and 32).

of these compounds are structural scaffolds for promising pharmacological segments, we contend that they may explain the observed activities interactively, such as gallic acid derivatives (1), quercetin O-glycosylated derivatives (40), and similar compounds (10, 14, 15, 21, 26, 28, 29, and 41), kaempferol (42) and its glycosylated derivatives, ellagitannins (an-However, research on the efficacy of natural products with therapeutic potential must be preceded by an investigation of the safety of their administration [15]. Secondary metabolites present in numerous genera are extremely important for their biological benefits, but their toxic capacity becomes a limiting factor for the use because of their risk [16].

alogs of 5–7, 17, 31, 38, and 48), and gallotannins (analogs of 8, 23, and 32).

In this sense, the present study is based on OECD guideline 425 and resolution nº 90 of the Brazilian health surveillance agency (ANVISA), which deals with the tests necessary for the registration of herbal medicines [17,18]. A limit dose (2000 mg/kg) of MnE was then administered orally, with subsequent evaluation of behavioral, physiological, anatomical, histological, and biochemical markers of toxicity.

Considering the lethality parameter, the limit dose administration did not cause immediate deaths over the 14 days of observation, suggesting the relative safety of the natural product. This observation is reinforced by the lack of physiological or behavioral changes that would indicate toxicity. Animals orally treated with MnE did not show changes in nutritional behavior or Hippocratic signs, nor in locomotion and emotionality parameters, evaluated in the open field. Such observations indicate that the extract, even in high doses, does not interfere with vital physiological functions, such as feeding, hydration, and weight gain, or alter the animals' consciousness, mobility, and emotionality [19,20].

Even at therapeutic levels, several analgesic or anti-inflammatory drugs promote significant changes in the heart, stomach, liver, and/or kidneys, a feature that often limits the dose or duration of treatment [21,22]. The macroscopic and histological evaluation of these organs showed the absence of lesions when using the limit dose. These findings were reinforced by the absence of changes in biochemical markers, such as ALT, the most specific indicator of hepatocellular injury, AST, which can reveal lesions in the liver but also in other tissues, and urea and creatinine, markers of renal injury [15]. Similarly, the administration of the MnE limit dose did not promote hematological alterations. Given these findings and following OECD guidelines [17], MnE can be classified as a low-toxicity xenobiotic, with an estimated LD<sup>50</sup> greater than 2000 mg/kg. Based on this classification, the evaluation of bioactive properties started with a dose of 200 mg/kg, corresponding to 10% of the dose tested in the toxicity assay, being adjusted based on the response, also testing doses of 400 and 800 mg/kg.

Initially, the MnE antinociceptive property was evaluated in the acetic-acid-induced peritonitis model, a classic test with high sensitivity, useful for screening drugs with antinociceptive potential. Intraperitoneal administration of acetic acid triggers local nonselective activation of cation channels, peritoneal membrane irritation, mast cells, and macrophages activation, the elevation of proinflammatory enzyme (nitric oxide synthase, cyclooxygenase, etc.) and mediator (cytokines, prostanoids, leukotrienes, bradykinin, serotonin, nitric oxide, etc.) levels [23]. The installed inflammatory process activates visceral nociceptors, causing primary hyperalgesia and intense nociception, manifested in the form of abdominal writhing [24,25]. Due to its characteristics, acetic-acid-induced peritonitis is sensitive to drugs capable to modulate inflammatory mechanisms of nociception, such as NSAIDs, although its nociception is also inhibited by other types of agents, such as opioids and antispasmodics [23,26]. Our trials demonstrated that MnE, at doses of 400 and 800 mg/kg, reduced by ~65% and ~73%, respectively, the acetic-acid-induced inflammatory nociception, with an estimated ED<sup>50</sup> of 338.298 mg/kg, suggesting a potential for inflammatory pathways modulation, especially prostaglandins and nitric oxide.

A second consequence of acetic-acid-induced acute peritonitis is vasodilatation and increased vascular permeability, related to mediators such as histamine, prostaglandins, and nitric oxide. This condition favors the migration of leukocytes and plasma proteins to the peritoneal cavity [27]. In fact, in our tests, a great increase (~144%) in protein concentration was observed in the peritoneal fluid of animals administered with acetic acid. Treatment with MnE, mainly at doses of 400 and 800 mg/kg, completely inhibited plasma extravasation, promoting peritoneal protein concentrations equivalent to those of animals that were not injected with acetic acid (white group), reinforcing its ability to modulate inflammatory mechanisms such as vascular relaxation, diapedesis, and plasma leakage, which is associated with the elevation of interleukins, prostaglandins, and nitric oxide, potential targets of MnE action [28].

Next, the formalin test was carried out to evaluate MnE influences on peripheral and central components of nociception, since the intraplantar injection of formalin (2.5%) induces a characteristic biphasic nociceptive process. In the first 5 min, the nociception observed is due to direct stimulation of sensory terminals, corresponding to the neuropathic component of the test. In this phase, the modulation of nociception has been linked mainly to drugs capable of interfering with neural mechanisms that conduct the noxious stimulus, such as opioids, anticonvulsants, and antidepressants, among others [23,27,29]. MnE did not promote alterations in the neuropathic component of formalin-induced nociception, indicating that its constituent probably does not act on nervous components. A 10-minute hiatus is observed in the formalin test, which is followed by a new period (15–30 min) of nociceptive manifestation, now caused by an inflammatory process triggered by formalin aggression, which involves prostaglandins, histamine, serotonin, and NO and plasma leakage [29,30]. MnE (400 mg/kg) significantly reduced formalin-induced inflammatory nociception.

Some of the secondary metabolites annotated by our group in the sample under study, such as ellagic acid, *p*-coumaric acid, and quercetin 3-O-xyloside, have been attributed to antioxidant activity, with the ability to scavenge reactive oxygen species. Ellagic acid is also capable of positively modulating Nrf2, a key mediator in the regulation of oxidative balance [30–33]. Oxidative stress and inflammation are closely related, as the overproduction of reactive oxygen and nitrogen species intrinsically participates in the cellular and humoral response, also influencing the inflammatory mechanisms of pain [30]. Furthermore, ellagic acid, gallic acid, methyl gallate, *p*-coumaric acid, quercetin 3-O-xyloside, quercetin, and kaempferol have also demonstrated important anti-inflammatory activity, related to the suppression of synthesis and release of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-17, TNFα, INFγ) and elevation of anti-inflammatory cytokines (e.g., IL-4, IL-10) and to the reduction in expression and activity of pro-inflammatory enzymes (e.g., COX-2, iNOS) and mediators (e.g., PGE2, NO), in addition to the ability to modulate nuclear factor kB (NF-kB), a key element in the inflammatory response regulation, attributed to *p*-coumaric acid [30,32–40].

Such observations, in association with our findings, reinforce the thesis that the reduction in acetic-acid- and formalin-induced nociception and acetic-acid-induced leakage promised by MnE is due to the modulation of pain inflammatory components, possibly a product of the synergistic action of its metabolites. We cannot, however, rule out other accessory mechanisms, such as antihistamine, antispasmodic or adrenergic activity, which should be explored in future essays. The other compounds annotated by our group, which lack studies on their biological properties, should also be explored. The present study, by exposing the phytochemical profile of MnE, annotating 44 constituent substances, and demonstrating its low acute toxicity and antinociceptive potential, paves the way for new studies on the medicinal use of *M. nobilis*, expanding knowledge about the safety of its use, considering repeated administrations in a subchronic or chronic pattern, its antioxidant and anti-inflammatory properties, and underlying mechanisms—both of its extracts and the molecules identified.

### **4. Materials and Methods**

## *4.1. Chemicals and Reagents*

In the present study, all reagents were of analytical grade. As an extraction solvent, 99% absolute ethanol was used (Exodus Scientific, São Paulo, SP, Brazil). Ultrapure water, acetonitrile, and concentrated formic acid solution, obtained from Merck (Darmstadt, Germany), were used to prepare the mobile phase and solubilize the sample for LC-MS analyses. The dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), ALT, AST, urea, and creatinine assay kits was from VIDA (Belo Horizonte, MG, Brazil). The acetic acid was from (ACA) and formaldehyde from (Vetec Química Fina, Rio de Janeiro, RJ, Brazil), the indomethacin from (Sigma-Aldrich, St. Louis, MO, USA) and morphine sulfate from (Cristália, Rio de Janeiro, RJ, Brazil).

#### *4.2. Plant Collection and Extract Preparation Protocol*

Leaf samples of *M. nobilis* (~1 kg) were collected at 1◦0200800 S and 46◦4904100 W (forest region of Bragança-PA, Brazil), identified by botanist Nascimento, E.A.P. (Brazilian Company of Agricultural Research—EMBRAPA), and a voucher specimen was deposited in its IAN herbarium (registration nº 191496).

Botanical material was washed, dried in a circulating oven (45 ◦C) until constant weight, and then pulverized in a ball mill (Fritsch, Idar-Oberstein, Germany). The semi-fine powder (60–100 µm) obtained was subjected to two sequential 24 h cycles of extraction with ethanol (99%; 4 L for each 1.0 kg of powder). The extract was concentrated in a rotary evaporator (Büchi, Flawil, Germany) and dried in an oven (40 ◦C) until constant weight.

#### *4.3. Phytochemical Analysis*

Metabolic characterization of MnE was obtained using a liquid chromatography system coupled to a time-of-flight mass spectrometer and triple quadrupole analyzer (model UPLC-QTf Xevo G2-S™, Waters, Milford, MA, USA). The chromatographic separation method was a 30 min gradient elution (0–2 min, 10–20% B; 2–30 min, 20–50% B). A 2 µL aliquot of the extract (3 mg.mL−<sup>1</sup> ) was eluted on a BEH C18 column (Waters, Wexford, Ireland; 50 × 2.1 mm i.d., particle size 1.7 µm) at 40 ◦C and under a flow of 0.3 mL/mL of ultrapure water (solvent A) and acetonitrile (solvent B) + 0.1% (*v*/*v*) formic acid. The analyzed ionization mode was negative with a mass range of 100 to 1200 *m/z* and a scanning interval of 0.1 s. The source was maintained at 120 ◦C and the gas flow was adjusted to 50 L.h−<sup>1</sup> . The desolvation gas was set to 800 L.h−<sup>1</sup> and 450 ◦C. The capillary voltage was set at 2.0 kV with cone voltage at 80 V. Data acquisition and processing was performed by MassLynx software licensed from Waters®.

#### *4.4. Animals*

Female Wistar rats (150–200 g) and male mice (25–30 g) were provided by the Federal University of Pará (UFPA) vivarium. They were kept in a standardized environment, with exhaustion, acclimatization (22 ± 1 ◦C) and light cycle (light 6 a.m. to 6 p.m.), in polypropylene cages (39 × 32 × 16 cm; up to five/box). Water and food were available ad libitum [39]. The experimental protocols were approved by the UFPA ethics committee (protocol nº 9568260617) and performed in a sound-attenuated laboratory under lowintensity light (12 lux), between 12:00 a.m. and 5:00 p.m.

#### *4.5. Drug Solutions and Administration*

MnE was solubilized in saline solution added with 4% DMSO. Standard drugs were dissolved in 0.9% saline. ACA and formaldehyde were dissolved in distilled water. Solutions were prepared with an administration pattern according to body weight (mice: 0.1 mL/10 g; rats: 0.1 mL/100 g). The oral (v.o.; gavage—MnE and indomethacin), subcutaneous (sc.—morphine), intraperitoneal (ip.—ACA), and intraplantar (formaldehyde) routes were adopted.

#### *4.6. Acute Oral Toxicity*

#### 4.6.1. Treatment and Hippocratic Screening

The toxicity study was conducted by Guidelines for Testing Chemicals nº 425 of the Organization for Economic Cooperation and Development (OECD) and resolution 90 of the Brazilian health surveillance agency [18,41]. Therefore, female rats (five/group) were treated with 0.9% saline added with 4% DMSO (control) or a limit dose (2000 mg/kg) of MnE.

Manifestation of signs of toxicity and death was then evaluated, as described by Malone (1977) [42], at intervals of 0, 15, 30, 60, and 240 min after administration, and daily thereafter for 14 days. Animals' weights, as well as the consumption of water and feed, were recorded daily too. The rats were euthanized by cervical dislocation under anesthesia at the end of the evaluation period. Blood samples were then collected by a ventricular

puncture for hematological and biochemical assays. Heart, liver, stomach, and kidneys were collected for relative weight and macroscopic and histological characteristics evaluations.

#### 4.6.2. Open Field Test (OF)

The open field test was performed 4 h after MnE limit dose (2000 mg/kg) administration to evaluate possible harmful effects on mobility and emotionality. For that, animals were positioned individually in the center of a square arena (width 100 × depth 100 × height 40 cm), the spontaneous locomotion being evaluated for 5 min [19]. The experiment was recorded by a cam positioned above the arena. Total locomotion, locomotion speed, freezing time, and locomotion in the center of the arena were evaluated using ANY-maze® (Stoelting Co., Wood Dale, IL, USA) software.

#### 4.6.3. Blood Count

Differential leukocyte counts and evaluation of possible abnormalities in red blood cells, leukocytes, and platelets were performed using the blood smear technique. Therefore, smears were prepared immediately after blood collection by the wedge method [43]. The evaluation was performed using an optical microscope (Nikon Eclipse E200, Melville, NY, USA).
