4.6.4. Biochemical Assays

Sample

Blood samples were subjected to centrifugation (10 min at 1400× *g*) for plasma separation, which was used for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity determination and urea and creatinine concentration.

#### Hepatic Function Assays

Alanine Aminotransferase (ALT) Activity

Determined through the reaction between pyruvate, formed by the transfer of amino groups from alanine to ketoglutarate, catalyzed by ALT, and NADH, forming L-lactate and NAD+, catalyzed by lactate dehydrogenase (LDH). ALT catalytic concentration (U/L) was determined from the rate of consumption of NADH, measured by spectrophotometry (λ = 340 nm) [44,45].

Aspartate Aminotransferase (AST) Activity

Determined through the reaction between oxaloacetate, formed by the transfer of amino groups from aspartate to ketoglutarate, catalyzed by AST and NADH, forming Lmalate and NAD+, catalyzed by malate dehydrogenase (MDH). AST catalytic concentration (U/L) was determined from the rate of NADH consumption, measured by spectrophotometry (λ = 340 nm) [45,46].

#### Kidney Function Assays

#### Creatinine

Serum creatinine was determined by its reaction with picric acid (Jaffe reaction), which forms a yellowish-red chromogen whose intensity, measured by spectrophotometry (λ = 510 nm), is proportional to its concentration (mg/dL) [47].

Urea

Urea concentration was measured in serum, based on the reaction between ammonia, formed by hydrolysis of urea in an aqueous medium, with alpha-ketoglutarate and NADH, catalyzed by glutamate dehydrogenase (GLDH), which forms glutamate and NAD+. NADH consumption, measured by spectrophotometry (λ = 340 nm), is proportional to urea concentration [48].

### 4.6.5. Histopathological Analysis

The heart, liver, stomach, and kidneys were weighed, fixed in buffered formalin (10%), and embedded in paraffin. Sections of 5 µm (thick) were then obtained, which were subjected to an alcohol-xylene series and mounted on slides. Staining was performed with hematoxylin and eosin (H&E), and the examination was made under an optical microscope (Nikon Eclipse E200).

#### *4.7. Antinociceptive Activity*

#### 4.7.1. Acetic-Acid-Induced Peritonitis

Based on the Koster et al. (1959) [49] model, cavity inflammation was induced by ip injection of ACA (0.6% *v*/*v*) in mice (*n* = 7/group) pretreated (60 min) with saline added 4% DMSO (Control); MnE increasing doses (200, 400, and 800 mg/kg) or indomethacin (INDO, 10 mg/kg). Nociception was then measured by the number of writhes manifested between 10 to 30 min after ACA administration.

After euthanasia by cervical dislocation under anesthesia, 3 mL of saline was injected into the peritoneal cavity of the animals. Peritoneal wash was collected, and protein concentration (mg/mL) was measured by Lory's (1951) method [50], as an indirect marker of plasma leakage. A group that was not given ACA (white) was added to determine baseline protein concentrations.

#### 4.7.2. Formalin Test (FT)

To assess the participation of neural and/or inflammatory processes, biphasic nociception was induced by plantar injection (sc; right hind paw) of 20 µL of formalin (0.92%) [51] of mice pretreated (60 min) with saline added 4% DMSO (Control), MnE (400 mg/kg), or morphine (4 mg/kg). Morphine was administrated 30 min before the noxious stimulus.

Nociception was measured through the time expended licking the formalin-injected paw, being evaluated in two phases: phase I (0–5 min), neuropathic nociception, which is triggered by direct stimulation of sensory terminals by formalin; and phase II (15–30 min) is inflammatory nociception, generated by the consequent inflammatory process [22].

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

To verify possible effects on consciousness or mobility that could compromise the animal's performance submitted to formalin test, control and MnE groups were evaluated in the open field, as described above, 5 min before exposure to the noxious stimulus.

#### *4.8. Statistical Analysis*

Data are presented by the mean ± standard error of the mean (SEM). The Shapiro–Wilk method was applied to evaluate the data distribution. Difference between groups with Gaussian distribution was evaluated by Student's *t*-test, one-way ANOVA, followed by Dunnett's test, or one-way RM-ANOVA, followed by Holm Sidak's test. Groups with a non-Gaussian distribution were evaluated using Mann–Whitney's test; Kruskal–Wallis's test followed by Dunn's test or Friedman's test. We considered statistically significant the differences with *p* < 0.05.

#### **5. Conclusions**

In summary, our results present the chromatographic profile of MnE (ethanolic extract of *M. nobilis*), identifying 44 secondary metabolites, classified as phenolic acids, flavonoids, and hydrolyzed tannins. We demonstrate that its LD<sup>50</sup> for acute oral administration is above 2000 mg/kg, being classified as a low-toxicity xenobiotic. It also shows its activity in reducing nociception, as well as inhibiting plasma extravasation, both associated with inflammatory processes, which probably involves the ability of its secondary metabolites, such as ellagic acid, gallic acid, methyl gallate, *p*-coumaric acid, quercetin 3-O-xyloside, quercetin, and kaempferol, to reduce the synthesis and/or release of inflammatory enzymes and mediators, such as COX-2, iNOS, cytokines, prostanoids, and NO, in addition to increasing the production of anti-inflammatory cytokines and presenting antioxidant activity. We hypothesize, therefore, that the MnE antinociceptive and plasma leakage inhibition properties are the product of the synergistic action of its constituents, still considering

that several of the substances annotated in the MnE still need to have their properties elucidated, as well as other mechanisms to be explored, which should compose future investigations. The present study, therefore, highlights the potential of this Amazonian species for the development of therapeutic agents devoted to the treatment of painful and inflammatory conditions.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ph16050689/s1, Figure S1: Histological evaluation of the stomach, kidney, liver, and heart of rats acutely treated with MnE limit dose.

**Author Contributions:** Conceptualization, E.A.F.-J. and C.Y.Y.e.S.; formal analysis, E.A.F.-J., F.M.S.C., J.C.C.S., C.A.B.A., W.L.P., M.C.M., C.Y.Y.e.S., M.N.d.S. and C.F.M.; investigation, E.A.F.-J., F.M.S.C., J.C.C.S., C.A.B.A., W.L.P., M.C.M., C.Y.Y.e.S., M.N.d.S. and C.F.M.; methodology, E.A.F.-J., F.M.S.C., B.C.d.C., E.K.S.C. and J.C.C.S.; supervision, E.A.F.-J., and C.Y.Y.e.S.; writing—original draft, E.A.F.-J., F.M.S.C., B.C.d.C., E.K.S.C., and J.C.C.S.; writing—review and editing, J.C.C.S., C.A.B.A., C.Y.Y.e.S.; C.F.M. and E.A.F.-J. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Research Pro-Rectory of the Federal University of Pará (PROPESP, UFPA, Brazil) provided a research grant and the article publication fee; the Fundação Amazônia de Amparo a Estudos e Pesquisas provided a research grant (EDITAL 01/2023–PROPESP and PRO463-2017).

**Institutional Review Board Statement:** The study was approved by the Federal University of Pará Ethics Committee (code: 9568260617), being carried out following the guidelines of the Care and Use of Laboratory Animals Guide (2011).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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