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Unlocking the Potential of Brazilian Plant Terpenes to Combat

Antimicrobial Resistance
Published as part of ACS Bio & Med Chem Au special issue “Juneteenth 2025”.
Danae K. R. Bardaji, Nagela B. S. Silva, Renata R. Miranda, Carlos Henrique G. Martins,
Michael A. Savka, and André O. Hudson*

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ABSTRACT: The group of bacteria known as ESKAPE: Enter-

ococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Downloaded via 77.65.81.53 on June 3, 2025 at 16:45:51 (UTC).

Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter

spp. are well recognized for their high virulence and pathogenicity,
employing diverse modalities and mechanisms to resist multiple
classes of clinically relevant antibiotics. Their capacity to evade
treatment presents a major public health challenge, highlighting
the urgent need for novel antibiotics to address the growing
resistance crisis. The plant kingdom presents a promising avenue
to this fight. Plants are naturally endowed with the genomic and
proteomic machinery to synthesize a wide arsenal of secondary metabolites, including terpenes and terpenoids, which have
demonstrated potent antimicrobial properties both as standalone agents and as synergists or enhancers of existing antibiotics. These
plant-derived compounds often operate through mechanisms distinct from those of conventional antibiotics, offering a potentially
effective solution against antibiotic-resistant bacteria. Brazil, home to some of the richest biodiversity on the planet, boasts 46,000
recorded plant species, with 250 new species identified annually. This review delves into the methods of preparing and isolating
terpenes and terpenoids from plants, explores the techniques used to assess their antibacterial activity, and highlights ongoing
research using Brazilian plants to target ESKAPE pathogens. This compilation of knowledge aims to establish a pipeline for
evaluating the antibacterial potential of terpenes and terpenoids, contributing to efforts addressing the growing threat of
antimicrobial resistance.
KEYWORDS: Brazilian Medicinal Plants, Terpenes, Terpenoids, Extraction Methods, Antimicrobial Activity, ESKAPE Pathogens,
Antibiotic Resistance, Essential Oils, Secondary Metabolites, Phytochemical Exploration

1. INTRODUCTION The World Health Organization (WHO) has recognized

antimicrobial resistance as a major threat to global health, food
Antibiotics have been instrumental in treating bacterial
security, and sustainable development, affecting people of all
infections since their introduction. However, unlike most
ages, in every country.5 Currently, this crisis is responsible for
medications, which maintain their effectiveness over time, the 700,000 deaths annually, a number projected to exceed 10
inevitable rise of resistance has severely undermined the million per year by 2050 which will surpass the current death
efficacy of current antibiotics used in clinical settings.1 Two toll from cancer.6 This growing crisis necessitates a concerted
primary factors hinder the research and development of new effort involving political, financial, and research investments, as
antibiotics.2 First, antibiotics are prescribed for shorter it endangers global health by undermining the ability to
durations compared with medications for chronic conditions, effectively treat infectious diseases.7 The increasing number of
which can be taken for years or even decades. In contrast, a antimicrobial-resistant pathogens, particularly those associated
typical antibiotic course lasts only a few weeks, resulting in
fewer prescriptions and lower financial incentives.3 Second, Received: March 3, 2025
although most newly approved drugs are broadly prescribed, Revised: May 3, 2025
new antibiotics are often reserved for treating only the most Accepted: May 6, 2025
severe bacterial infections. These challenges have intensified
the antibiotic resistance crisis, occurring at a time when
antibiotic discovery and development are in decline.4
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Figure 1. Structures of the terpenes. Terpenes are hydrocarbons with significant structural variations, including cyclic and acyclic skeletons, with a
backbone composed of five-carbon isoprene units (C5H8). Terpenes are classified according to the number of the isoprenoid units as
monoterpenes, diterpenes, triterpenes, tetraterpenes, or polyterpenes.

with nosocomial infections, imposes a substantial strain on promising area of research.16 The chemical diversity of
healthcare systems. This issue contributes to elevated mortality terpenes has resulted in the identification of more than
and morbidity, rising treatment expenses, diagnostic chal- 40,000 structural variants, with certain classes already utilized
lenges, and a growing lack of confidence in current therapeutic as pharmaceutical agents.16 Brazil’s rich and diverse flora
options.8 remains largely either unexplored or underexplored, and each
In recent decades, the term “ESKAPE” has emerged to year new compounds are identified that could potentially be
describe a particularly concerning group of pathogens based on developed into new drugs or serve as lead compounds
data from surveillance studies and the American Society for employed by synthetic chemists or computational biologists
Infectious Diseases. The ESKAPE pathogens, comprising to facilitate rational design.17
Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumo- This review focuses on terpenes, a key class of secondary
niae, Acinetobacter baumannii, Pseudomonas aeruginosa, and metabolites that have been extensively studied for their
Enterobacter species, are both Gram-positive and Gram- antimicrobial properties, particularly against antibiotic-resistant
negative bacteria that are not only responsible for most strains. The data presented here are derived from Brazilian
hospital-acquired infections but also serve as models for research groups and summarize the preparation methods and
understanding pathogenesis, virulence, transmission, and in vitro antimicrobial activity of these compounds, emphasizing
resistance.9−11 their potential as novel antimicrobial agents.
Through genetic mutations and the uptake of mobile genetic
elements, these bacteria have acquired resistance to multiple 2. SECONDARY METABOLITES
antibiotic classes, including those considered last-line treat- Plant secondary metabolites are natural products synthesized
ments such as oxazolidinones, macrolides, fluoroquinolones, by plants that, while not essential for their survival, provide
tetracyclines, β-lactams, carbapenems, and glycopeptides.9 crucial benefits in combating biotic and abiotic stresses.18
Since the 1990s, the rate of new antibiotic development has These metabolites serve various ecological and physiological
significantly decreased. Between 2017 and 2019, the US Food functions, such as acting as defense compounds against
and Drug Administration (FDA) approved just 11 new herbivores, pathogenic microbes, and competing plants as
antimicrobial therapies. With each passing year, the number well as serving as signaling molecules in ecological interactions.
of effective antibiotics against ESKAPE pathogens dwindles, Additionally, they help shield plants from abiotic stress factors,
highlighting the urgent need for new antibacterial agents.12 including ultraviolet (UV) radiation, freezing temperatures,
While Escherichia coli is not classified among the ESKAPE drought, and various other osmotic stresses.19
pathogens, it remains a major cause of infections, including Several theories have been proposed to explain the
sepsis, bloodstream infections, and urinary tract infections. As a emergence of plant secondary metabolites. One theory
common gut commensal in humans and animals, E. coli can suggests that plants developed secondary metabolism genes
acquire resistance genes from other bacteria, similar to the from their primary metabolism genes. Through gene
ESKAPE pathogens.13 duplication, the newly formed genes mutated to exhibit
Historically, the discovery of biologically active compounds novel metabolic functions, which were then preserved through
from nature has driven advancements in therapeutics, natural selection.20 Another theory posits that plants may have
benefiting both the health and pharmaceutical sectors.14 acquired secondary metabolism genes through horizontal gene
Natural plant-based antimicrobial compounds offer promising transfer from their bacterial symbionts. Alternatively, fungi
alternatives to traditional antibiotics, with different plant parts, living in symbiosis with plants could have directly supplied
including stems, roots, fruits, flowers, leaves, and seeds secondary metabolites to the host or transferred the
demonstrating strong inhibitory effects against bacteria and corresponding genes via horizontal gene transfer.21
fungi.15 Terpenes, a large group of phytochemicals, have Secondary metabolites can be classified in various ways,
shown significant antimicrobial activity and represent a including by their chemical structure (e.g., compounds
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containing rings or sugars), their composition (e.g., nitrogen-

containing or nitrogen-free), their solubility in different
solvents, and, most commonly, their biosynthetic pathways.22
The literature generally categorizes these natural products into
three major classes: phenolics, alkaloids, and terpenoids.23

3. BIOSYNTHESIS OF TERPENES
Plants have evolved and developed various mechanisms to
cope with biotic and abiotic stress conditions, leading to the
anabolism and accumulation of secondary metabolites. These
metabolites, numbering approximately 100,000, are synthe-
sized through diverse metabolic pathways and include
common products such as phenols, flavonoids, and terpenes.18
Among these, terpenes stand out as the most chemically
diverse group of plant secondary metabolites, that includes
more than 50,000 natural products.24 Terpenes play crucial
ecological and physiological roles, including allelopathy,
insecticidal activity, attracting insect pollinators, and function-
ing as plant hormones like abscisic acid and gibberellin.23
Structurally, terpenes are large hydrocarbons with cyclic or
acyclic structures, composed of 5-carbon isoprene (2-methyl- Figure 2. Biosynthetic pathways for terpenes in plants: the MVA
pathway (left) occurs in the cytoplasm and endoplasmic reticulum
1,3-butadiene: C5H8) units as their basic building blocks25
(ER), and the MPE pathway (right) occurs in plastids. Abbreviations
(Figure 1). (in order of appearance from left to right): AACT, acetoacetyl-CoA
Terpenes are categorized according to the number of thiolase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR,
isoprene units in their structure, leading to classifications such 3-hydroxy-3-methylglutaryl-CoA reductase; MVA, mevalonic acid;
as hemiterpenes (5 carbon atoms), monoterpenes (10 carbon MK, mevalonate kinase; MVAP, mevalonate 5-phosphate; PMK,
atoms), sesquiterpenes (15 carbon atoms), diterpenes (20 phosphomevalonate kinase; MVAPP, mevalonate diphosphate; MDD,
carbon atoms), sesterterpenes (25 carbon atoms), triterpenes mevalonate diphosphate decarboxylase; IDI, isopentenyl diphosphate
(30 carbon atoms), tetraterpenes (40 carbon atoms), and isomerase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl
polyterpenes (with the formula 5n carbon atoms). Terpenoids, diphosphate; FPPS, farnesyl diphosphate synthase; FPP, farnesyl
which are derivatives of terpenes, arise from modifications such diphosphate; GA-3P, D-glyceraldehyde 3-phosphate; DXS, 1-deoxy-D-
xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate
as the introduction or elimination of functional groups.26 In
reductoisomerase; MEP, 2-C-methylerythritol 4-phosphate; MCT, 2-
this work, both terpenes and terpenoids are referred to as C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-
terpenes. (cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-
Terpene metabolism in plants begins with the incorporation methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-
of cytochrome P450 and terpene synthase genes into plant hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, (E)-4-
genomes26,27 (Figure 2). hydroxy-3-methylbut-2-enyl diphosphate reductase; GGPPS, geranyl
In the MEP pathway, terpene synthesis starts with the Mapp diphosphate synthase; GPPS, geranyl diphosphate synthase;
condensation of pyruvate and D-glyceraldehyde-3-phosphate GPPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate.
(GA-3P) by 1-deoxy-D-xylulose 5-phosphate synthase (DXS),
resulting in the production of isopentenyl diphosphate (IPP)
and dimethylallyl diphosphate (DMAPP), utilizing three ing MVAPP into IPP, which is isomerized into DMAPP and
adenosine triphosphate (ATP) and three nicotinamide adenine then converted into C15 farnesyl diphosphate (FPP) by FPP
dinucleotide phosphate (NADPH). DXS controls the flow of synthase (FPPS), concluding the metabolic pathway.29,30 The
terpenes produced in this pathway, making it a key target for primary difference between these two biosynthetic pathways
biotechnological studies aiming to manipulate terpene lies in their occurrence: the MVA pathway supplies isopentenyl
production.28 and dimethylallyl diphosphates in most eukaryotes and
The MVA pathway involves the production of one archaea, while the MEP pathway is active in eubacteria, algal
isopentenyl molecule and uses three acetyl-CoA, three ATP, plastids, and higher plants. Additionally, the MEP pathway is
and two NADPH. Initially, the enzyme acetoacetyl-CoA responsible for the production of monoterpenes, diterpenes,
thiolase condenses two acetyl-CoA molecules to form and tetraterpenes, while the MVA pathway leads to the
acetoacetyl-CoA. This is followed by the condensation of synthesis of sesquiterpenes, triterpenes, saponin derivatives,
another acetyl-CoA molecule via 3-hydroxy-3-methylglutaryl- and sterols.31
CoA synthase (HMGS) to form HMG-CoA.29
The pathway continues with the reduction of HMG-CoA by 4. PREPARATION METHODS
HMG-CoA reductase (HMGR) to mevalonate in the Bioactive compounds, such as terpenes, can be obtained from
endoplasmic reticulum, consuming two NADPH, which is a plants through a systematic process that involves the separation
critical and regulatory step in the pathway. The enzyme and isolation of secondary metabolites using various methods
mevalonate kinase (MK) then phosphorylates mevalonate to followed by downstream analysis of their bioactivities (Figure
mevalonate-5-phosphate (MVAP), followed by another 3). The initial step in studying medicinal plants is selecting the
phosphorylation by phosphomevalonate kinase (PMK). The most appropriate biological material from different parts/
final step occurs through the consumption of ATP by organs of the plant, including flowers, leaves, bark, roots, and
mevalonate diphosphate decarboxylase (MDD), decarboxylat- fruits.32
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Figure 3. Isolation, identification, and downstream analyses of the bioactive compounds. In general, the process of obtaining terpenes from plants
for medicinal use involves the following steps: selection of the appropriate biological material from plants (leaves, bark, roots, fruits, etc.);
disruption of plant cells to liberate their chemical components via dry or wet methods; extraction of the bioactive compounds by employing an
appropriate solvent, or through methods like distillation or compound trapping, the target compound(s) can be isolated from the biological extract
using chromatographic techniques. Finally, the isolated compound(s) can be identified and used in downstream analyses.

Flowers often have a high concentration of these compounds or delicate plant material and is great in terms of yield,
as they serve to attract pollinators.33,34 Many aromatic plants, especially for plants with low terpene content.48
such as mint, rosemary, and eucalyptus, have high terpene Steam distillation is a heat-based technique in which steam is
concentrations in their leaves.34 Some plants, such as directed through plant material, leading to cell rupture and the
cinnamon, produce terpenes in their bark. Cinnamon bark, release of terpenes. The steam carries the volatile compounds
for example, contains compounds like cinnamaldehyde and (terpenes) to a condenser, where they are collected. This is a
other terpenoids.35 While less common, certain plants, such as simple and well-established process that produces high-quality
ginger and ginseng, have terpenes concentrated in their roots. pure terpene extracts, most commonly used for aromatic
Ginger, for instance, has terpenes like zingiberene that flowers and leaves.49 Hydrodistillation is a variant of steam
contribute to its flavor and medicinal properties.36 The peels distillation where water immersion followed by heating is used
of citrus fruits like oranges, lemons, and limes contain high to process the plant material. This process is very useful when
levels of terpenes such as limonene, which give the fruit its working with EOs.50
distinctive scent and flavor.37 Supercritical CO2 extraction involves using supercritical
The plant materials are first cleaned of foreign substances carbon dioxide (CO2) to extract terpenes from the plant
like soil, debris, and other contaminants.38 Subsequently, the material. The CO2 behaves as both a gas and a liquid, which
method of fragmentation is chosen, as smaller particle sizes allows it to penetrate plant cells and rupture the cell walls,
increase interaction with the solvent, enhancing the extraction extracting EOs and terpenes. This is a very efficient technique
efficiency and impacting the quantity of metabolites recov- and produces high-quality extracts without the use of heat or
ered.39−42 solvents. It is also suitable for extracting a wide variety of
Mechanical disruption techniques, such as cold pressing, terpenes without degradation.51
often applied to citrus fruits, involve physically rupturing the Modern extraction techniques, such as ultrasonic-assisted
plant surface to facilitate the release of volatile terpenes.43 extraction (UAE) and microwave-assisted extraction (MAE),
Grinding plant material into a fine powder or crushing it are are more efficient and environmentally friendly, requiring little
other methods that increase the surface area and allow for to no solvents.52 UAE uses ultrasonic acoustic vibrations to
easier extraction of terpenes through subsequent methods like break plant cells and release their contents into the solvent,
steam distillation or solvent extraction.44 Finally, hydraulic while MAE involves exposing the biological material, along
pressing is used to mechanically press fresh plant material, with a solvent, to electromagnetic radiation at 2.45 GHz.53 The
releasing essential oils (EOs). This method is sometimes used final step in recovering terpenoids involves selecting an
for delicate plant parts like flowers.45 appropriate separation method based on the compound
The methods used for terpene extraction can be categorized characteristics and the desired recovery yield, aiming to
into solvent-based, heat-based, supercritical CO2 extraction, minimize losses.54
and modern technologies like microwave-assisted and ultra- Once the terpene extract is obtained, it may undergo further
sound-assisted extraction.46 The choice of method is processing to purify and concentrate the terpenes or to remove
influenced by the nature of the plant material, the specific impurities such as solvents or residual plant matter. Some
terpenes being extracted, and the desired quality of the final common postextraction techniques include vacuum distillation,
product.47 fractional distillation, and chromatography.55 Adsorption
Solvents such as ethanol and hexane can be used to dissolve chromatography techniques, such as column chromatography
the EOs and terpenes from the plant material. The solvent and liquid chromatography, especially high-performance liquid
breaks down cell walls, releasing terpenes into the solvent. The chromatography (HPLC), and ultraperformance liquid chro-
resulting mixture is usually subjected to filtration, and the matography (UPLC), are particularly effective in purifying
solvent is evaporated to leave behind concentrated terpenes. natural products. These techniques can be integrated with a
This method is excellent for extracting terpenes from resinous range of detectors and analytical instruments to enable
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compound identification and quantification.56,57 Today, online new generations of ionic liquids have been formulated to
systems equipped with technologies like diode arrays, nuclear optimize the extraction of high-molecular-weight terpenes
magnetic resonance, and infrared spectroscopy have automated from challenging matrices. The use of green solvents like ionic
the analysis processes which can reduce human intervention liquids or biomass-derived solvents has gained traction as an
thus shortening and streamlining the time for analyses.58,59 ecofriendly alternative to conventional solvents such as
Once the terpenes are extracted and purified, they must be ethanol.74
properly stored to maintain their quality. Terpenes are highly When the maceration method is employed, the plant
volatile and sensitive to light, heat, and oxygen, which can material is immersed in the chosen solvent for 24−48 h in a
cause degradation over time.60 cool, dark environment, with periodic agitation of the mixture
In the past few years, significant advancements have been to enhance extraction efficiency. This technique allows the
made in terpene extraction, particularly focusing on sustain- solvent to penetrate the plant tissues, facilitating the
ability, efficiency, and automation. The shift toward green and dissolution of target compounds.75 Alternatively, reflux or
sustainable extraction technologies, such as the use of ionic Soxhlet extraction methods can be utilized.76
liquids, green solvents, and enzyme-assisted methods, has Postextraction, the solvent from the plant material should be
gained momentum in response to growing environmental filtered using a fine mesh or cheesecloth to remove solid
concerns.61 Additionally, the development of multitechnique particles. To concentrate the extract, a rotary evaporator can be
extraction approaches, including ultrasound-assisted super- used to remove the solvent under reduced pressure,
critical CO2 extraction and microwave-assisted solvent minimizing heat-induced degradation of the terpenes.77 If a
extraction, has enhanced both precision and yield, enabling rotary evaporator is unavailable, solvent removal can be
the selective targeting of specific terpenes.62 Furthermore, achieved through mild evaporation under heat in a fume
innovations in automation and the integration of extraction hood or vacuum chamber, though this may carry a higher risk
systems have streamlined production processes, improving of thermal degradation.78
scalability and minimizing human error.63 Supercritical CO2 extraction is another viable method for
For optimal volatile terpene extraction, first fresh leaves, nonvolatile terpenes, requiring optimization of temperature
flowers, and fruit peels are collected.55 It is best to harvest and pressure conditions, typically involving higher pressures
plant material early in the day when terpene levels are at their and lower temperatures than those used for volatile terpenes to
peak due to cooler temperatures.64 Ideally, the plant should be enhance extraction efficiency and selectivity.79 Microwave-
processed immediately after collection. If drying is necessary, assisted extraction has also advanced significantly in recent
plant material can be air-dried at low temperatures (below 40 years, offering rapid and efficient extraction of nonvolatile
°C/104 °F) in a well-ventilated area or using a dehydrator, but terpenes such as diterpenes and triterpenes. This method
freeze-drying is the preferred method for maximum terpene employs microwave energy to heat both the solvent and plant
preservation as it avoids heat and retains the aromatic profile.65 material, enhancing the efficiency and yield of the extraction
Using cryogenic grinding to freeze plant material before process.80
extraction has also emerged as a key technique in improving After solvent removal, vacuum drying of the concentrate is
the yield and purity of volatile terpenes.66 necessary to eliminate any remaining solvent traces. For further
Steam distillation, or its variant hydrodistillation, can be purification, column chromatography can be employed to
used afterward due to its efficiency and ability to preserve the separate specific terpenoids or nonvolatile terpenes based on
delicate nature of terpenes.67 For the best balance of efficiency, molecular weight or polarity, facilitating the isolation of desired
sustainability, and terpene preservation, supercritical CO2 compounds.81 For quality control or research purposes, the
extraction is an optimal option,51 while microwave-assisted final extract can be analyzed using gas chromatography−mass
extraction presents a faster, solvent-efficient alternative.68 The spectrometry (GC-MS) or high-performance liquid chroma-
last two methods minimize thermal degradation, preserve the tography (HPLC) to identify and quantify specific nonvolatile
volatile terpene profile, and can be scaled for industrial use.69 terpenes, ensuring that the extract’s composition meets
For isolating pure terpenes, techniques like fractional required standards.82
distillation or chromatography (e.g., GC-MS for terpene In recent years, Brazilian researchers have been using various
identification) can be employed.55 techniques to isolate terpenes. de Siqueira et al.83 employed
For the extraction of nonvolatile terpenes, such as stir bar sorptive extraction (SBSE) with polydimethylsiloxane
terpenoids, carotenoids, and larger molecular terpenes, it is (PDMS) and ethylene glycol (EG) silicone in both immersion
best to begin with dried plant material such as roots, stems, and headspace modes. This technique provided efficient
and seeds, as these parts generally contain higher concen- terpene isolation, offering higher selectivity and sensitivity for
trations of nonvolatile compounds.54 Drying should be done volatile compounds compared with conventional methods.
carefully to prevent degradation, typically under low heat and Supercritical fluid extraction (SFE) using CO2, Soxhlet
low humidity. Air drying or freeze-drying are effective extraction (SOX) with petroleum ether, and maceration
methods.54 Once dried, the plant material should be ground (MAC) with hexane were the techniques used by Santos et
into a coarse or medium-sized powder to increase the surface al..84 SFE was found to be the most selective for isolating
area, facilitating more efficient extraction.70 monounsaturated fatty acids (MUFAs), polyunsaturated fatty
Subsequently, solvent extraction can be employed using acids (PUFAs), and sesqui- and diterpenes, highlighting its
solvents such as ethanol, hexane, or methanol to selectively efficiency in terpene isolation compared to the other methods.
dissolve the target compounds.71 Ethanol is commonly chosen SFE was also used by Spyrou et al.,85 who investigated the use
due to its high polarity and efficiency in extracting a wide range of subcritical propane (scPropane), supercritical CO2 (scCO2),
of terpenoids and other nonvolatile compounds.72 Enzymatic and supercritical CO2 combined with ethanol (scCO2 +
formulations could be used for more efficient and selective EtOH) as cosolvent systems for extracting valuable com-
terpene extraction in combination with solvents.73 Recently, pounds, including terpenes, from ginger.
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Dalsasso et al.86 combined vacuum microwave drying containing a bacterial suspension at a concentration of 5 × 105
(VMD) with ultrasound-assisted extraction (UAE) at different CFU/mL. The first and second tubes are supplemented with
temperatures, along with microwave-assisted extraction (MAE) the test molecule or extract, typically at final concentrations of
for fresh ginger composition analysis. The use of VMD and 0.25 × MIC and 1 × MIC, while the third tube serves as the
UAE resulted in extracts richer in phenolics and antioxidant growth control. Incubation is carried out under suitable
activity while preserving the terpene profile. VMD facilitated conditions for varying time intervals (0, 4, 6, 8, 10, 12, and 24
bioactive extraction by breaking down cell structures, and UAE h).91,95 The proportion of dead cells is calculated in
selectively extracted compounds, minimizing losses due to
comparison to the growth control by quantifying the number
volatilization or thermal degradation. This combination offers a
of viable cells (CFU/mL) in each tube via the agar plate count
more efficient and tailored extraction process compared to
traditional methods. method. A bactericidal effect is generally observed when the
lethality reaches 90% after 6 h, which increases to 99.9%
5. METHODS FOR ANTIMICROBIAL EVALUATION OF lethality after 24 h.95 Additionally, this method can be used to
TERPENES AND TERPENOIDS assess synergism or antagonism between drugs when used in
The biological activity of terpenes and terpenoids has been combination.91,96
evaluated by using various methods to identify new
compounds with antibacterial properties. Some of these 6. TERPENES AND THEIR INHIBITORY PROPERTIES
methods are discussed below. AGAINST ESKAPE PATHOGENS
Agar diffusion techniques such as disk diffusion, well Terpenes represent a broad and varied class of plant-derived
diffusion, agar plug, and spot assays are commonly employed compounds with significant antimicrobial activity, making
in clinical microbiology laboratories for routine antimicrobial
them promising candidates in the fight against antibiotic
susceptibility testing due to their cost-effectiveness. The
Clinical and Laboratory Standards Institute (CLSI) has resistance. They exhibit both bacteriostatic and bactericidal
established numerous recognized guidelines for antimicrobial effects against various pathogens, with a more pronounced
susceptibility testing of bacteria and yeasts.87 These techniques impact on Gram-positive bacteria relative to Gram-negative
function by allowing antimicrobial agents to diffuse from paper bacteria.8 The multitarget effects of terpenes and their
discs, wells, or plugs into the agar medium, where they derivatives contribute to their potency as antimicrobial agents.
suppress the growth of the test microorganism spread across These effects include disrupting the cell envelope and
the surface.88 The effectiveness of the test compound against destabilizing the cytoplasm, which ultimately result in cellular
the specific microorganism is evaluated by measuring the damage. Terpenes interact with lipid membranes by engaging
inhibition zone, the clear area around the agent where with the lipophilic tails of intermembrane lipids and polar head
microbial growth has been inhibited or suppressed.89 However, groups, thereby affecting both the lipid intermembrane and
Griffin et al.90 noted that the disc diffusion method can be polar transmembrane pathways.97 Additionally, terpenes may
problematic, since its reliability largely hinges on the test interfere with essential microbial functions, including oxygen
agent’s water solubility and its capacity to diffuse effectively consumption and the process of oxidative phosphorylation,
through the agar medium. which are essential for cellular respiration.90 Low oxygen levels
Dilution methods are considered the most appropriate for
limit bacterial respiration rates, thereby inhibiting their
determining minimal inhibitory concentration (MIC) values,
growth.98 Moreover, terpene interaction with oxidative
as they enable the determination of antimicrobial concen-
trations in either agar (agar dilution) or liquid media (macro- phosphorylation can lead to alterations in cellular respiration
or microdilution). The MIC is the lowest concentration of an and the uncoupling of this energy-producing process in
antimicrobial agent required to prevent the visible growth of a microbes. Their antibacterial efficacy is shaped by factors
specific bacterium.91 The key distinction between agar dilution such as lipophilicity, hydrophobicity, and the presence of
and broth dilution methods lies in the medium used: agar hydroxyl groups.99
dilution utilizes agar plates, while broth dilution employs liquid The inhibitory effects of terpenes on bacterial efflux pumps
broth tubes. Both methods introduce different concentrations have been reported in several studies, either by downregulating
of the antimicrobial agent into the medium, followed by gene expression or by directly interacting with the binding sites
inoculation with a standardized number of microbial cells. The of membrane-associated efflux proteins.100 Due to their
MIC is determined by observing whether microbial growth multitarget effects, many terpenes and their derivatives exhibit
occurs.89 Additionally, the half-maximal inhibitory concen- strong antibacterial activity against multidrug-resistant organ-
tration (IC50) can be used to measure a drug’s efficacy. The isms, especially methicillin-resistant S. aureus (MRSA).16
IC50 represents the concentration of a compound required to While many terpenes are common across various plant
inhibit a biological process by 50%, providing a measure of an species worldwide, some are primarily found in Brazilian flora
antagonist drug’s potency and efficacy in pharmacological
due to the country’s unique biodiversity. Many of these
research.92
compounds are currently being studied for their potential in
The time-kill kinetics assay assesses antimicrobial efficacy by
exposing the test microorganism to different concentrations of medicine.101 Although terpenes are primarily found in EOs,
the antimicrobial agent over a defined period. Well-established they are also present in a variety of other natural sources, such
for bacteria, this test is outlined in the CLSI M26-A document. as oleoresins, propolis, and resins.49 The following sections will
It offers critical insights into the time- or concentration- first explore Brazilian terpenes isolated from EOs with
dependent effects of the agent and provides data on the antimicrobial properties against multidrug-resistant bacteria,
temporal progression of antimicrobial activity.93,94 The assay is including ESKAPE pathogens and E. coli, followed by terpenes
conducted in a broth culture medium with three tubes each derived from other sources.
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7. TERPENES ISOLATED FROM EOs OF BRAZILIAN 0.39−3.12 mg/mL for thyme, respectively, resulting in
PLANTS promising antibacterial alternatives against Staphylococcus
species. The results revealed that citral (40.9%), myrcene
EOs are highly concentrated plant extracts that contain volatile
terpenes responsible for their strong aroma and therapeutic (24.7%), and geraniol (1.9%) were detected in lemongrass EO
properties.48 Several Brazilian researchers have investigated the and thymol (44.2%), p-cymene (24.6%), and 1,8-cineole
antibacterial properties of these compounds against ESKAPE (9.9%) were detected in thyme EO.
pathogens. The main methods of obtaining these EOs and The EO of Mikania cordifolia and its major constituent
highlights found in these investigations are discussed below. limonene were evaluated against P. aeruginosa, E. coli, and S.
Hydrodistillation and steam distillation were the most used aureus by Justino de Araujo et al.106 In this study, the
methods used by Brazilian researchers to extract EOs from researchers did not find antibacterial activity of the natural
plants. Gas chromatography−mass spectrometry (GC-MS) products when tested alone. However, both compounds
served as the principal method for identifying the chemical enhanced the efficacy of antibiotics against resistant bacteria.
composition of EOs. GC-MS allows for the identification of The EO showed the most significant modulatory effect against
individual compounds, such as terpenes, which are often the P. aeruginosa, displaying synergistic interactions with gentami-
key active components in the oils.102 cin and norfloxacin. Additionally, the EO decreased the MIC
For antibacterial testing, the microdilution method was of norfloxacin against E. coli and of gentamicin against S.
widely used to determine the MIC. The minimum bactericidal aureus. Conversely, limonene demonstrated its strongest
concentration (MBC) was used in some studies to identify the modulatory effect against S. aureus.
concentration at which the EO completely kills the bacteria, Rezende et al.111 determined the antibacterial potential
confirming its bactericidal activity. E. coli and S. aureus were against E. coli and S. aureus of EOs from Satureja montana L.
the most common bacteria tested; some studies also included and Myristica fragrans H. Both EOs were effective against
other bacteria like P. aeruginosa and K. pneumoniae. bacteria tested with MIC = 6.25 μL/ml. As principal
Sesquiterpenes were a major component in several EOs, components, they found borneol, γ-terpineol, and carvacrol
especially those from Piper species.103 Other notable terpenes in the EO from S. montana, and sabinene and α-pinene were
include citral, thymol, carvacrol, limonene, borneol, and found in the EO from M. f ragrans.
caryophyllene, which were frequently identified in the oils. Porfirio et al.107 investigated antimicrobial and antibiofilm
EOs showed promising antibacterial activity with MIC values properties against S. aureus of the Lippia alba EO and its
ranging from 6.25 to 16 μL/mL. The oils were especially primary components (citral and carvone). Essential oils
effective against Gram-positive bacteria like S. aureus. The MIC derived from the aerial parts of three L. alba varieties showed
values varied significantly depending on the oil, with some EOs notable antibacterial effects, with the most effective concen-
being more potent than others (e.g., oils from lemongrass, tration (MIC) recorded at 0.5 mg/mL. At this same
thyme, and oregano).104,105 concentration, the oils also significantly suppressed biofilm
In some studies, EOs isolated from plants like Mikania development by S. aureus. Barbosa et al.112 produced EOs by
cordifolia demonstrated synergistic effects when combined with steam distillation from seven plants (Rosmarinus of ficinalis L.,
antibiotics, reducing the MIC of antibiotics against resistant Baccharis dracunculifolia DC, Vernonia polyanthes Less,
strains.106 Others EOs, particularly carvacrol and citral, showed Matricaria recutita L, Cinnamomum zeylanicum Blume,
activity against bacterial biofilm formation, which is a key Caryophyllus aromaticus L, Eugenia unif lora L.) and measured
factor in chronic infections. Oils from Lippia alba and oregano the antibacterial activities of the EOs alone and in
were specifically noted for their biofilm inhibition.107 combinations against ESKAPE pathogens such as S. aureus
EOs were found to exhibit both bactericidal (killing and P. aeruginosa. A chemical analysis of the EOs showed that
bacteria) and bacteriostatic (inhibiting growth) effects. For they were predominantly composed of terpenes and their
example, Piper corcovadense EO exhibited both bactericidal and derivatives. The MICs in this work varied from 0.25 to 92.40
bacteriostatic properties against E. coli.108 mg/mL.
Costa et al. 109 reported on the antimicrobial and The EO from P. corcovadense D. was extracted by steam
antivirulence activities of EO extracted from the leaves of distillation by Henrique Fontoura et al.108 The primary
Myrciaria pilosa. The EO, dominated by sesquiterpenes guaiol constituents identified included trans-sesquisabinene hydrate,
and (E)-β-caryophyllene, showed significant in vitro antimicro- trans-caryophyllene, β-pinene, trans-β-farnesene, 14-hydroxy-
bial activity against both standard strains and clinical isolates of caryophyllene, limonene, and p-cymene. At a concentration of
S. aureus, with the standard strain ATCC 6538 being more 2.61 μg/mL, the EO demonstrated both bactericidal and
susceptible than the clinical isolates. The compounds tested in bacteriostatic effects against E. coli.
this work showed a MIC of 5 μg/mL against the evaluated Amaral et al.105 evaluated Origanum vulgare (oregano) EO
strains and MBC ranging from 10 to 20 μg/mL. antibacterial properties against both standard laboratory strains
The antibacterial activity of the EOs of Piper species (P. and multidrug-resistant isolates of E. coli and K. pneumoniae.
arboretum, P. aduncum, P. gaudichaudianum) against standard Carvacrol emerged as the dominant compound, accompanied
and multidrug resistant E. coli and S. aureus was studied by da by β-caryophyllene, γ-terpinene, p-cymene, and thymol. The
Silva et al.110 Sesquiterpenes were considered as major EO was effective against all tested strains, with MIC values
components in all cases. In microdilution tests, the EOs ranging from 1.75 to 3.50 mg mL−1.
showed MICs between 16 and 1024 μg/mL. While some terpenes, such as limonene, carvacrol, and p-
Lopes et al.104 evaluated EOs from five plant species against cymene, are found in various global plant species, other
S. aureus. EOs from lemongrass and thyme demonstrated terpenes, such as trans-sesquisabinene hydrate, trans-β-
notable effectiveness, with MIC and MBC values of 0.39−3.12 farnesene and 14-hydroxycaryophylle, are either primarily
and 0.39−6.35 mg/mL for lemongrass and 0.39−1.56 and found or notably abundant in Brazilian plants such as P.
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Table 1. Antibacterial Activity of EOs from Brazilian Plants: Terpenes, Sources, and MIC Values
terpenes source bacteria tested antibacterial activity ref
sesquiterpenes (major components) Piper species (P. arboretum, P. E. coli (standard and MIC: 16 to 1024 μg/mL 110
aduncum, P. multidrug-resistant), S.
gaudichaudianum) aureus
citral (40.9%), myrcene (24.7%), geraniol (1.9%) lemongrass (Cymbopogon S. aureus MIC: 0.39 to 3.12 mg/mL, 104
citratus) MBC: 0.39 to 6.35 mg/
mL
limonene (major constituent) M. cordifolia P. aeruginosa, E. coli, S. not effective alone, but 106
aureus synergistic with antibiotics
borneol, γ-terpinol, carvacrol savory (S. montana) E. coli, S. aureus MIC: 6.25 μL/mL 111
sabinene, α-pinene nutmeg (M. fragrans) E. coli, S. aureus MIC: 6.25 μL/mL 111
citral, carvone L. alba S. aureus MIC: 0.5 mg/mL 107
camphor, limonene, myrcene rosemary (R. off icinalis) S. aureus, P. aeruginosa MIC: 0.25 to 92.40 mg/mL 112
nerolidol, spathulenol, germacrene B B. dracunculifolia DC E. coli MIC: 0.25 to 92.40 mg/mL 112
trans-sesquisabinene hydrate, trans-caryophyllene, β-pinene, P. corcovadense E. coli MIC: 2.61 μg/mL 108
trans-β-farnesene, 14-hydroxycaryophyllene, limonene, p-
cymene
carvacrol, β-caryophyllene, γ-terpinene, p-cymene, thymol oregano (O. vulgare) E. coli, K. pneumoniae MIC: 1.75 to 3.50 mg/mL 105

corcovadense.108 Table 1 summarizes the terpenes, sources, terpenes, such as phytol, camphor, and verbenone, identified
bacteria tested, and MIC values of the EOs studied. in Plectranthus barbatus extract, were active against MDR
Gram-negative bacteria.119 Additionally, oblongifolin B from
8. TERPENES ISOLATED FROM DIFFERENT SOURCES red propolis inhibited biofilm formation in S. aureus and
AS PROPOLIS AND OLEORESINS exhibited MBIC50 values ranging from 1.56 to 6.25 μg/mL,
When terpenes are isolated from sources other than EOs, while also reducing bacterial virulence through catalase
several extraction methods have been commonly employed. inhibition.120
Hydrodistillation was used to obtain volatile oils from Melipona These studies underscore the potential of plant-derived
quadrifasciata geopropolis.113 Chromatography techniques, terpenes, particularly kaurene diterpenes, triterpenes, and
including partitioning, thin-layer chromatography, and column volatile oils, as promising candidates for the development of
chromatography, were utilized across various studies to isolate natural antimicrobial agents.
specific compounds, such as diterpenes from Copaifera Geopropolis volatile oils from Melipona quadrifasciata,
species,114 Aspilia latissima,115 and Baccharis dracunculifolia.116 among other species, were obtained by hydrodistillation and
Maceration was employed to extract crude compounds from characterized by gas chromatography coupled with mass
brown propolis,117 while solvent extraction with dichloro- spectrometry (GC-MS) by.113 The lowest MIC obtained
methane was used for plants like Zizyphus joazeiro118 and against S. aureus was 219 μg/mL, and the main constituents
Baccharis dracunculifolia.116 Gas chromatography−mass spec- were α-pinene and β-pinene.
trometry (GC-MS) was applied to identify the chemical Leandro et al.114 evaluated the antibacterial, antibiofilm, and
composition of volatile oils.113 antivirulence properties of two key diterpenes isolated from the
For antibacterial analysis, the broth microdilution method oleoresins of Copaifera species using several chromatographic
was employed to determine minimum inhibitory concen- procedures against multidrug-resistant (MDR) bacteria,
trations (MICs), minimum bactericidal concentrations including S. aureus, E. faecium, P. aeruginosa, E. coli, and A.
(MBCs), and minimal biofilm inhibition concentration 50% baumannii. They focused on determining the MIC, MBC, and
(MBIC50) values.114,117,119 Some studies also included MBIC50; in addition, synergistic and antivirulence assays were
synergistic and virulence factor inhibition tests to assess the conducted for eight distinct diterpenes. Diterpenes ent-copalic
effect of compounds on biofilm formation and enzyme acid and ent-kaurenoic acid demonstrated the best results, with
inhibition (e.g., DNase and catalase).114 Additionally, IC50 MIC and MBC values ranging from 12.5 to 50 μg/mL against
determination was used to evaluate the potency of isolated the majority of MDR strains. These diterpenes demonstrated
compounds.116 The target microorganisms included both notable biofilm inhibitory activity, with MBIC50 values ranging
Gram-positive bacteria (e.g., S. aureus, including MRSA strains, from 3.12 to 25 μg/mL, showing potential against biofilm
and E. faecium) and Gram-negative bacteria (e.g., P. aeruginosa, formation, and inhibited DNase, a virulence factor produced
K. pneumoniae). by some S. aureus strains, at subinhibitory concentrations of
Regarding antibacterial activity, many studies demonstrated 6.25 μg/mL.
significant effects, particularly against S. aureus and P. Souza et al.115 assessed the antimicrobial properties of
aeruginosa. MIC values for compounds ranged from as low Aspilia latissima, a species native to the Pantanal wetlands of
as 0.5 μg/mL (e.g., Plectranthus barbatus extract) to as high as Brazil, against a range of microorganisms, including P.
500 μg/mL (e.g., Aspilia latissima leaf and root extracts). aeruginosa, E. coli, and S. aureus, where leaf and root crude
Several diterpenes, including ent-copalic acid, ent-kaurenoic extracts demonstrated antimicrobial effects against all exam-
acid, stigmasterol, and totarol, exhibited strong antibacterial ined strains. The chloroform fractions of both the leaves and
properties, especially against multidrug-resistant (MDR) roots exhibited the most notable activity, especially against S.
bacteria, and also showed significant activity against biofilm aureus, with a MIC of 500 μg/mL. The same active
formation in S. aureus.114,115,117 Triterpenes like methylceano- compounds (stigmasterol, kaurenoic acid, and kaura-
thate and alphitolic acid from Zizyphus joazeiro demonstrated 9(11),16-dien-18-oic acid) were found in both the leaves
notable antibacterial effects against S. aureus.118 Other and roots of the plant, explaining why both parts exhibited
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Table 2. Antibacterial Activity of Derivatives from Brazilian Plants: Terpenes, Sources, and MIC Values
terpenes source bacteria tested antibacterial activity ref
α-pinene, β-pinene M. quadrifasciata S. aureus MIC: 219 μg/mL 113
geopropolis
ent-copalic acid, ent-kaurenoic acid Copaifera spe- S. aureus, E. faecium, P. aeruginosa, E. coli, A. MIC: 12.5−50 μg/mL; 114
cies oleoresins baumannii MBIC50: 3.12−25 μg/mL
stigmasterol, kaurenoic acid, kaura-9(11),16-dien-18-oic acid A. latissima P. aeruginosa, E. coli, S. aureus MIC: 500 μg/mL 115
leaves and
roots
19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehy- Brazilian brown methicillin-resistant S. aureus (MRSA), E. coli, IC50: 10.9 to >200 mg/mL 117
droabietic acid, dehydroabietic acid, communic acid, iso- propolis P. aeruginosa, K. pneumoniae, E. faecium
pimaric acid (VRE)
diterpenes (ent-agathic acid, others) C. reticulata MRSA, E. faecium IC50: 1.6 to 10.7 μg/mL 121
oleoresin
methylceanothate, alphitolic acid Z. joazeiro Mart. 15 clinical strains of S. aureus (including MIC: 32 μg/mL and 16 μg/ 118
MRSA and MSSA) mL
ursolic acid, 2α-hydroxyursolic acid B. dracunculifolia methicillin-resistant S. aureus (MRSA) IC50: 5 μg/mL and 3 μg/mL 116
leaves
phytol, camphor, verbenone P. barbatus A. baumannii, K. pneumoniae, E. coli, P. MIC: 0.5−2.0 mg/mL 119
aeruginosa
2-oxo-populifolic acid A. cymbifera MRSA, MSSA, P. aeruginosa MIC: 500 μg/mL 101
oblongifolin B red propolis S. aureus (biofilm formation) MIC: 0.98 to 31.25 μg/mL; 120
MBIC50: 1.56 to 6.25 μg/
mL

similar antimicrobial activities. The antibacterial effects 2α-hydroxyursolic acid. These compounds exhibited anti-
observed in the plant are largely attributed to the presence bacterial activity against methicillin-resistant S. aureus, with
of kaurene-type diterpenes. IC50 values of 5 and 3 μg/mL, respectively.
Ribeiro et al.117 investigated the antibacterial activity of The antimicrobial activity of plant extracts from the
Brazilian brown propolis, which is primarily composed of Lamiaceae family which includes oregano, lavender, and
diterpenes such as 19-acetoxy-13-hydroxyabda-8(17),14-diene, mint, among others was studied by Assis et al.119 using the
totarol, 7-oxodehydroabietic acid, dehydroabietic acid, com- broth microdilution technique. The study focused on multi-
munic acid, and isopimaric acid. The study targeted drug-resistant Gram-negative bacteria, including A. baumannii,
methicillin-resistant S. aureus ATCC 1708 (MRSA), E. coli K. pneumoniae, E. coli, and P. aeruginosa. The Plectranthus
ATCC 2452, P. aeruginosa ATCC BAA-2018, K. pneumoniae barbatus extract, containing terpenes like phytol, camphor, and
ATCC 2146, and E. faecium (VRE) ATCC 700221, among verbenone, exhibited notable antibacterial activity with
other pathogens. The crude extract of brown propolis was minimum inhibitory concentrations (MICs) between 0.5 to
obtained through maceration with a hydroalcoholic solution, 2.0 mg/mL.
followed by partitioning and chromatographic techniques, and de Barros Machado et al.101 analyzed seven different species
the diterpene compounds demonstrated the most promising used in three commercial phytopharmaceuticals for their
antimicrobial activities. efficacy against multiresistant bacteria. The study isolated 2-
In another study, Pfeifer Barbosa et al.121 isolated diterpenes oxo-populifolic acid from Aristolochia cymbifera, which
from Copaifera reticulata. The oleoresin was fractionated by inhibited the growth of multiple strains, including MRSA,
treatment with potassium hydroxide followed by extraction MSSA, and multiresistant P. aeruginosa. Growth inhibition of
with diethyl ether, and the resulting diterpene acids were all methicillin-sensitive and -resistant S. aureus strains, as well
extracted and tested against MRSA DSM18827 and E. faecium as most P. aeruginosa strains, was observed at a concentration
DSM 20477. While most diterpenes exhibited significant of 500 μg/mL.
activity against both pathogens, ent-agathic acid showed no In 2021, de Souza Silva et al. 120 investigated the
antibacterial activity. antimicrobial properties of hydroalcoholic extracts of green
Leal et al.118 investigated the chemical composition and anti- and red propolis, as well as their isolated compounds, against
staphylococcal properties of the dichloromethane extract multidrug-resistant bacteria. The study revealed that red
derived from the Brazilian plant Zizyphus joazeiro Mart. The propolis extract (ERP) exhibited the strongest antimicrobial
study tested the extract against 15 clinical strains of S. aureus, activity. One of its key compounds, the terpene oblongifolin B,
including nine MRSA and six methicillin-sensitive S. aureus showed significant activity against biofilm formation in S.
(MSSA) strains. The bark extract was processed and subjected aureus, with MBIC50 values ranging from 1.56 to 6.25 μg/mL,
to chromatography, yielding five triterpenes, all of which and inhibited catalase. The MIC values for oblongifolin B
demonstrated antibacterial activity, with methylceanothate and against all tested Gram-positive bacteria ranged from 0.98 to
alphitolic acid being the most potent. The triterpenoid fraction 31.25 μg/mL. Table 2 summarizes the terpenes, sources,
exhibited bactericidal activity at 256 μg/mL, while the bacteria tested, and MIC values from sources other than EOs.
individual compounds inhibited all tested bacteria at Some of the terpenes mentioned in this review are
concentrations of 32 and 16 μg/mL, respectively. exclusively derived from Brazilian plants, such as kaurenoic
Filho et al.116 evaluated the antimicrobial activities of acid and kaura-9(11),16-dien-18-oic acid, which can be found
Baccharis dracunculifolia and its isolated compounds. The leaf- in Aspilia latissima (Souza et al., 2014);115 a plant from the
rinsed extract, obtained through immersion in dichloro- Brazilian Pantanal region. Other terpenes described in this
methane, was partitioned and chromatographed, leading to review are considered characteristic of Brazilian flora, such as
the isolation of eight compounds, including ursolic acid and 19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxode-
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hydroabietic acid, dehydroabietic acid, communic acid, and Authors

isopimaric acid found in Brazilian brown propolis (Ribeiro et Danae K. R. Bardaji − Thomas H. Gosnell School of Life
al., 2021);117 methylceanothate and alphitolic acid isolated Sciences, College of Science, Rochester Institute of Technology,
from Zizyphus joazeiro Mart118 and 2-oxo-populifolic acid Rochester, New York 14623, United States
isolated from Aristolochia cymbifera101 are from plant species Nagela B. S. Silva − Laboratory of Antimicrobial Testing,
native to Brazil. These compounds highlight the country’s rich Institute of Biomedical Sciences (ICBIM), Federal University
diversity in plant species with antimicrobial potential. of Uberlândia, Uberlândia, Minas Gerais 38405-314, Brazil
Renata R. Miranda − School of Chemistry and Materials
9. CONCLUSIONS, FUTURE PERSPECTIVES, AND Science, College of Science, Rochester Institute of Technology,
FINAL CONSIDERATIONS Rochester, New York 14623, United States
Carlos Henrique G. Martins − Laboratory of Antimicrobial
Microbial resistance poses an increasingly dire threat to global
Testing, Institute of Biomedical Sciences (ICBIM), Federal
health, making the discovery and development of novel
University of Uberlândia, Uberlândia, Minas Gerais 38405-
antimicrobial agents an urgent necessity. Secondary metabo-
314, Brazil
lites from Brazilian plants, particularly terpenes, present a
Michael A. Savka − Thomas H. Gosnell School of Life
promising avenue to address this crisis. The potential of these
Sciences, College of Science, Rochester Institute of Technology,
compounds is immense, yet there is a critical need to expedite
Rochester, New York 14623, United States
the analysis process for detecting antimicrobial activity. While
numerous studies have demonstrated the antibacterial proper- Complete contact information is available at:
ties of terpenes, few have advanced to the stage of clinical https://pubs.acs.org/10.1021/acsbiomedchemau.5c00069
application. By standardization of experimental methodologies,
the path to obtaining actionable results could be significantly Author Contributions
streamlined. This would enable researchers to focus on more DKRB, NBSS, RRM, CHGM, MAS, and AOH wrote and
sophisticated studies, such as structural modifications through edited the manuscript. CRediT: Danae K.R. Bardaji
chemical synthesis, using terpenes as templates to design conceptualization, writing - original draft, writing - review &
molecules with enhanced antimicrobial efficacy and reduced editing; Nagela B. S. Silva funding acquisition, writing -
risk of resistance development. original draft, writing - review & editing; Renata R Miranda
The rise in the consumption of herbal medicines globally, writing - original draft, writing - review & editing; Carlos
particularly in countries like Brazil with rich biodiversity, has Henrique Gomes Martins funding acquisition, writing -
fueled the pharmaceutical industry’s interest in plant-based original draft, writing - review & editing; Michael A Savka
secondary metabolites. Despite some clinical studies of natural writing - original draft, writing - review & editing; Andre O
products, challenges such as limited patient numbers or Hudson conceptualization, funding acquisition, supervision,
inconsistencies in preparation have hindered the full realization writing - original draft, writing - review & editing.
of their therapeutic potential. More robust studies are needed Funding
to establish the efficacy, safety, and side effect profiles of these
phytomedicines. Governments should actively support and A National Institutes of Health award (R15GM144862) to
incentivize research in this field, facilitating the removal of AOH in addition to the Fundação de Amparo a Pesquisa do
barriers that impede the development of new compounds, Estado de Minas Gerais (FAPEMIG - Scholarship grant
particularly those that are effective against antibiotic-resistant number 12138 and grant number APQ-02067-22) and by
pathogens. The wealth of existing research on the benefits of National Council for Scientific and Technological Develop-
phytomedicines, especially those rich in terpenes, offers a ment (CNPq) numbers 307974/2019-7 and 311568/2023-8
strong foundation for future advancements. funded this work.
This review delves into the characteristics of terpenes as Notes
plant secondary metabolites, summarizing key purification The authors declare no competing financial interest.
techniques, including fragmentation, extraction, and separation
methods like chromatography. It highlights studies by Brazilian
researchers that exemplify these methods and demonstrate the
antimicrobial activities of isolated terpenes against ESKAPE
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