Antimicrobial Activity of Extracts from the Humiria balsamifera (Aubl)

Humiria balsamifera (Aubl), commonly known as “mirim”, is a plant of the Humiriaceae family, which consists of 39 species divided between eight genera: Duckesia, Endopleura, Humiria, Humiriastrum, Hylocara, Sacoglottis, Schistostemon, and Vantenea. This study aimed to characterize H. balsamifera extracts by LC-MS/MS and evaluate their antimicrobial potential through in vitro and in vivo assays. The leaves and stem bark of H. balsamifera were collected and dried at room temperature and then ground in a knife mill. The extracts were prepared with organic solvents in order to increase the polarity index (hexane, ethyl acetate, and methanol). The antimicrobial effects of these extracts were evaluated against the following bacterial strains: Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella enterica Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 6538. The best activity was observed in the ethyl acetate (EALE = 780 µg/mL), methanol (MLE = 780 µg/mL), and hexane (HLE = 1560 µg/mL) leaf extracts against S. aureus. Considering the results for both antimicrobial and antibiofilm activities, the EALE extract was chosen to proceed to the infection assays, which used Tenebrio molitor larvae. The EALE treatment was able to extend the average lifespan of the larvae (6.5 days) in comparison to S. aureus-infected larvae (1 day). Next, the samples were characterized by High-Performance Liquid Chromatography coupled to a mass spectrometer, allowing the identification of 11 substances, including seven flavonoids, substances whose antimicrobial activity is already well-reported in the literature. The number of bioactive compounds found in the chemical composition of H. balsamifera emphasizes its significance in both traditional medicine and scientific research that studies new treatments based on substances from the Brazilian flora.


Introduction
Microorganisms are naturally well-spread out in the environment, and they can easily reach surfaces people come into contact with, including food products, whether at the harvest, slaughter, processing, or even packaging. Once in contact with the food, they start their growth process by consuming nutrients and causing the product to deteriorate [1][2][3].
Bacteria, fungi, viruses, and protozoa are the main microorganisms responsible for food contamination, infecting humans through the consumption of beef, fish, poultry, eggs, unhygienic fruits and fresh produce, causing a variety of diseases [4,5]. The World Health Organization estimates that one in 10 people worldwide become ill after consuming contaminated food and about 420,000 people die each year, resulting in the loss of 33 million healthy life years (DALYs) [2,6].
Bacteria represent an added concern for health and food safety organizations, especially those able to grow at low temperatures and resist a wide range of temperature variations [7,8]. Bacterial pathogens such as Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus, among others, are responsible for several global foodborne outbreaks and cause life-threatening illnesses such as diarrheal diseases [5,[9][10][11][12]. Another problem in fighting bacteria is their ability to develop resistance to conventional antimicrobials. These pathogens can use various strategies to inhibit the effects of antimicrobials, such as the production of inactivating enzymes, reduction of outer membrane permeability, efflux system, and blocking or altering the target site of antibiotics, further motivating the research focused on finding alternative ways to combat them [13,14].
In the search for new effective substances against resistant pathogens, several secondary metabolites from plants and endophytic microorganisms have shown promise [15,16]. Most of the drugs used in general today were developed based on ethnopharmacological knowledge [17][18][19], indicating that the chemistry of natural products is a big ally in the development of therapeutic agents [20,21].
The plant species Humiria balsamifera (Aubl), popularly known as "mirim", presents interesting biological activities. The literature reports, most of all, anti-inflammatory [22,23], antimalarial [24], antioxidant [25,26], and antifungal activity [27], highlighting the therapeutic potential of this plant. Some substances isolated from this species so far have already been reported as well, such as bergenin, arjunolic acid, friedelin, lupeol, phytol, caryophyllene oxide, epoxide humulene, and trans-isolongifolanone, among others [24]. However, the antibacterial and antibiofilm activities of its derived products have not been extensively examined. Thus, this work aims to characterize and evaluate the effectiveness of H. balsamifera extracts in terms of the antimicrobial and antibiofilm activities against foodborne pathogens (Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella enterica Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 6538). The in vivo antimicrobial action of the most active extract was analyzed using a method based on the infection of Tenebrio molitor larvae.

Antimicrobial Activity Evaluation
The antimicrobial activity of Humiria balsamifera (Aubl) leaf and stem bark extracts was evaluated by the determination of their minimum inhibitory concentrations (MIC) against four foodborne bacteria species: E. coli, L. monocytogenes, S. enterica Typhimurium, and S. aureus (Table 1). Stem bark extracts did not exhibit antimicrobial action at any of the concentrations tested (MIC > 1250 µg/mL). However, the leaf extracts successfully inhibited S. aureus, with MIC = 780 µg/mL (EALE and MLE) and 1560 µg/mL (HLE). The EALE and the MLE also inhibited L. monocytogenes (MIC = 3120 µg/mL). The leaf extracts presented no action against the Gram-negative bacteria tested in this study.

Evaluation of the In Vivo Activity of the Ethyl Acetate Leaf Extract of Humiria balsamifera (Aubl)
To evaluate the antimicrobial efficacy of the ethyl acetate leaf extract (EALE), the selected method was an alternative infection model based on the S. aureus ability to infect T. molitor larvae ( Figure 2). The group infected with a lethal dose of S. aureus with no

Evaluation of the In Vivo Activity of the Ethyl Acetate Leaf Extract of Humiria balsamifera (Aubl)
To evaluate the antimicrobial efficacy of the ethyl acetate leaf extract (EALE), the selected method was an alternative infection model based on the S. aureus ability to infect T. molitor larvae (Figure 2). The group infected with a lethal dose of S. aureus with no treatment presented an average lifespan of 1 day. In contrast, the uninfected larvae inoculated with the extract or its vehicle did not show a decrease in their lifespan. The EALE treatment was able to extend the average lifespan of the larvae (6.5 days), and by the end of the evaluation period, 50% of all larvae in this group were still alive.

Chemical Characterization of Humiria balsamifera (Aubl) Leaf and Stem Bark Extracts
Analyses of the leaf and stem bark extracts of Humiria balsamifera (Aubl) by HPLC-ESI-IT/MS in negative-ion mode identified 11 molecular ions (Tables 2-4). Their structures were proposed ( Figure 3) based on the fragments originated from the molecular ion by multi-stage mass spectrometry (MS n ). The mass spectrometry ionization source was the electrospray (ESI). The ESI source may not have ionized the compounds like steroids and triterpenes. It was possible to identify only phenolic compounds in the extracts. From the 11 identified substances, seven were flavonoids (gallocatechin, kaempferol 3-neohesperidoside, sophoricoside, quercetin 3-arabinoside, quercetin-O-rhamnoside, kaempferol-dirhamnoside, and vitexin-dirhamnoside); three were coumarins (bergenin and two derivatives: galloylbergenin and hydroxybenzoyl bergenin); and one was an oligosaccharide (maltotetraose). Table 2. Identification of the substances present in the ethyl acetate stem bar extract of Humiria balsamifera (Aubl).

Discussion
This research aimed to characterize and evaluate the antimicrobial potential of the extracts of Humiria balsamifera (Aubl), also known as "mirim". This species belongs to the Humiriaceae family, and its tea is used in many Brazilian regions for its anti-inflammatory action, especially for treating uterine inflammation [22,23].
The substances present in the chemical composition of H. balsamifera tell a lot about the species. Flavonoids, according to the characterization presented in this study, are the most abundant class of compounds. These substances exhibit high bioactive potential and present anti-ulcer, antioxidant, anti-inflammatory, anti-allergic, antitumor, antiviral, antifungal, and antidiabetic activities [34][35][36][37].
Antimicrobial tests with flavonoids have received increasing attention in recent years, since these compounds are synthesized by plants in response to various types of stress, including microbial infections [38][39][40]. Researchers are also interested in how flavonoids are able to exhibit antibacterial activity through mechanisms different from conventional drugs, hindering the development of microbial resistance [41,42].
During this study, the H. balsamifera extracts were subjected to three tests for the evaluation of their antimicrobial potential against different bacteria strains. By the end of the in vitro tests, the extracts which presented the best results were selected for the in vivo anti-infective assay using T. molitor larvae. Our results showed that the H. balsamifera ethyl acetate leaf extract (EALE) showed efficacy against S. aureus, one of the most resistant pathogens in existence, in all three tests (MIC, antibiofilm potential, and the tests in vivo). The efficacy of this extract is believed to be due to the flavonoids present in its composition. Flavonoids are well-known in the literature, as other polyphenols, to be able to inhibit microbial growth through several mechanisms, such as the inhibition of ATP synthesis in the electron transport chain, inhibition of nucleic acid synthesis, inhibition of the efflux pump, inhibition of biofilm formation, inhibition of virulence factors, inhibition of quorum sensing, membrane disruption, inhibitors of bacterial toxins, and inhibition of cell envelope synthesis [41,43,44].
Bergenin, identified in the three extracts analyzed, has already been reported in H. balsamifera [24] and other two species from the Humiriaceae family: Endopleura uchi and Sacoglottis gabonensis [45,46]. This isocoumarin and its derivatives, such as the identified flavonoids, can be directly related to the antimicrobial activity of the extracts against S. aureus. A recent study showed that six synthetic derivatives of bergenin obtained by Williamson synthesis inhibited S. aureus growth, especially 8,10-dihexyl-bergenin and 8,10-didecyl-bergenin, which presented the most promising MIC value: 3.12 µg/mL [47].

Botanical Material
The leaves and stem bark of Humiria balsamifera (Aubl) were collected during the rainy season in Contrato Village, located in the municipality of Morros-MA (2 •

Preparation of the Extracts
The leaves were dried at room temperature, while the stem barks were dried at 40-50 • C in an oven for 24 h; after which, they were ground in a knife mill separately. The crude extracts were prepared from the ground materials by cold percolation, and each extraction was performed twice for each solvent within a period of 5 days, following the sequence hexane, ethyl acetate, and methanol. The extracts obtained were filtered and concentrated on a rotary evaporator [54].

Test Microorganisms
The microbial strains used in this work were obtained from the Microbial Culture Collection of CEUMA University. It used the following strains: Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella enterica Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 6538.

Minimum Inhibitory Concentration (MIC)
The antimicrobial activity of the extracts was evaluated by the determination of the minimum inhibitory concentration (MIC). The MIC was determined by the broth microdilution method. Sterile 96-well plates were prepared with 150 µL of Müeller Hinton broth (MHB) and 50 µL of the extract following the serial dilution method. After the dilutions (1250 µg/mL−2.0 µg/mL for the stem bark extracts and 6250 µg/mL-1.0 µg/mL for the leaf extracts), 10 µL of the microbial suspension were added to the plates until a 0.5 McFarland standard was reached, and they were then incubated in a lab oven at 37 • C for 24 h. The test was performed in duplicate. Once the incubation period ended, 20 µL of resazurin (Sigma-Aldrich, St. Louis, MO, USA; 0.03%) were added, and the readings were executed after 40 min of incubation at 37 • C. Alterations in color, from blue to pink, were considered an indication of microbial growth. The MIC was defined as the lowest concentration able to prevent microbial growth [55].

Antibiofilm Test
To evaluate the antibiofilm activity, a sample of 10 µL of S. aureus suspension (prepared as described in the MIC section) was mixed with 140 µL of MHB and 50 µL of EACH to reach subinhibitory concentrations

Infection Model Using Tenebrio molitor Larvae
The assessment of the antimicrobial effect in vivo used larvae of the insect T. molitor (Tenebrionidae). Larvae of approximately 100 mg were randomized into groups with a minimum of 10 individuals. Before inoculation, the cuticles were cleaned with 70% alcohol. An aliquot of 10 µL of the microbial suspension (1.0 × 10 11 CFU/mL) was injected in the membrane region between the penultimate and antepenultimate rings of the larvae, which were then incubated at 37 • C. After 2 h, the larvae groups received 10 µL of each extract at different concentrations. Viability was assessed daily by the absence of movement. Larvae inoculated with the microorganism and treated with PBS were used as the negative control, while the noninfected larvae were selected as the positive control. Death of all larvae or transition into pulp form in the experimental group determined the end of the experiment [55].

Extracts Characterization by HPLC-ESI-MS and FIA-ESI-IT/MS
For the HPLC-ESI-IT/MS/MS and FIA-ESI-IT/MS n analyses, a clean-up step was performed to remove any contaminants; the solution was purified by solid-phase extraction (SPE) using Phenomenex Strata C18 cartridges (500 mg of stationary phase) that were previously activated with 5 mL of MeOH and equilibrated with 5 mL of MeOH:H 2 O (1:1, v/v). The compounds were eluted from the cartridges using 1 mL of MeOH:H 2 O (1:1, v/v) with a final volume of 5 mL. The samples were then filtered using a 0.22-µm PTFE filter and dried. The extract was diluted to 10 µg/mL in the HPLC solvent, and then, aliquots of 20 µL were injected directly into the LC-ESI-IT/MS [56].
The analysis was carried out on an online LC-ESI-IT-MS in a LCQ Fleet Ion Trap Mass Spectrometer, Thermo Scientific ® (Waltham, MA, EUA). A Kinetex ® (Torrance, CA, USA) C18 LC column (2.1 × 100 mm, 100 Å, and 5 µm) was used to separate the components. The analysis was executed using water with formic acid 0.1% (A) and acetonitrile + formic acid 0.1% (B), with formic acid 0.1% added in gradient boosting, going from 10% to 100% in 6 min with a flow rate of 0.4 mL/min. The sample was injected into the HPLC system, where it was analyzed online by ESI-MS in the negative ion mode with a UV detector. Mass spectrometry was performed in an LCQ Fleet Ion Trap LC/MS n -Thermo Scientific ® (Waltham, MA, EUA) [56].
A FIA-ESI-IT-MS n flow injection analysis was performed in an LTQ XL™ linear ion trap mass spectrometer with an ESI ion source (electrospray ionization) in negative mode (Thermo, San Jose, CA, USA), using a stainless-steel capillary tube at 280 • C, 5.00 kV, capillary voltage of −90 V, −100 V tubular lens, and a flow rate of 5 µL min −1 . A full-scan analysis was conducted at 100-1000 m/z. Multiple-stage fragmentations by electrospray ionization/multi-stage mass spectrometry (ESI-MSn) were performed using collisioninduced dissociation (CID) by helium for ionic activation. The first step was a full-scan MS to acquire data from the ions at the selected m/z. The second step was a tandem mass spectrometry (MS/MS) in data-dependent acquisition mode on [M−H] − molecules of the compounds of interest at a collision energy of 30% and activation time of 30 ms. The product ions were subjected to further fragmentation under the same conditions, until no more fragments were observed. The identification of different compounds in the chromatographic profile of the hydroalcoholic extract was carried out by comparing their retention times and UV spectra with the literature data [56].

Statistical Analysis
All tests were performed in at least two independent assays in quadruplicate. The statistical analyses were performed using the software GraphPad Prism version 5.01 (Graph-Pad Software Inc., La Jolla, CA, USA). Data were analyzed by two-way analysis of variance Plants 2021, 10, 1479 9 of 11 (ANOVA), followed by Tukey's test. A p-value of < 0.05 was considered statistically significant.

Conclusions
The number of bioactive compounds found in the chemical composition of H. balsamifera emphasized its significance in both traditional medicine and scientific research of studies with new treatments based on substances from the Brazilian flora. This highlighted the importance of this study, since the analyses by HPLC-ESI-MS and FIA-ESI-IT/MS identified 11 substances, 10 of which had not yet been reported for H. balsamifera, improving the literature regarding its composition. Additionally, the antimicrobial activity of the ethyl acetate leaf extract (EALE) against S. aureus was, for the first time, described using three different strategies (MIC, antibiofilm activity, and tests in vivo), highlighting all the potential of this plant against one of the most resistant bacteria of the present day, which encourages further studies on natural bioactive metabolites that can be isolated from H. balsamifera.