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. 2026 Sep 30;23(10):e71771. doi: 10.1002/cbdv.71771

Seasonal and Regional Variation of the Chemical Composition and Antimicrobial Activity of Essential Oils From Piper marginatum Jacq.

Rebeca Gabriel de Camargo 1, Marta Cristina Teixeira Duarte 2, Diones Krinski 3, Ana Paula Palaro Klein Hendges 4, Aurea Portes Ferriani 1, Francisco de Assis Marques 1, Thalita Gilda Santos 5, Beatriz Helena Lameiro de Noronha Sales Maia 1,✉
PMCID: PMC13626260  PMID: 42814518

ABSTRACT

Essential oils (EO) from Piper marginatum Jacq. exhibit biological activities dependent on chemical compositions, which vary mainly due to environmental factors. This study investigated the seasonal and regional variation in the chemical composition and antimicrobial activity of EO from P. marginatum leaves collected in three Brazilian regions across four seasons. Sixty compounds were identified, primarily terpenoids and phenylpropanoids. Multivariate analyses revealed seasonal variations and distinguished the Northeast region EO, which contained the isomers pipermargine (3.38%–14.56%) and 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene (13.47%–14.87%)—reported for the first time in EO. The Central‐West winter EO exhibited the broadest antimicrobial spectrum, with strong activity against Candida albicans (MIC 0.5 mg mL−1) and Bacillus subtilis (MIC 0.5 mg mL−1), and a bacteriostatic effect against Staphylococcus aureus. All EO inhibited Escherichia coli, with the Northeast winter EO showing the strongest activity (MIC 0.25 mg mL−1). This work highlights the importance of studying the chemical and biological variability of P. marginatum EO for potential applications.

Keywords: antimicrobial activity, essential oil, gas chromatography, multivariate analysis, natural products

Evaluating Piper marginatum essential oils across three Brazilian biomes reveals significant chemical and antimicrobial variations driven by seasonal and regional factors. Sixty compounds are identified, including a novel (Z)‐phenylpropanoid reported for the first time. Multivariate analysis correlates environmental stressors with specialized metabolic profiles, establishing a strategic harvesting roadmap for potent, standardized natural antimicrobials for industrial applications.

graphic file with name CBDV-23-e71771-g005.webp

1. Introduction

Piperaceae Giseke is a family of pantropical distribution comprising more than 3500 species in five genera. The genus Piper L. is the largest, including approximately 2000 species, of which 289 occur in Brazil [1, 2].

Piper marginatum Jacq. is an aromatic shrub with occurrences reported in all Brazilian geographical regions. Studies have revealed several biological activities of its essential oils (EO), such as antiparasitic, anticholinesterase, ovicidal, larvicidal, insecticidal, and repellent [3, 4, 5, 6, 7, 8, 9].

Furthermore, EO from P. marginatum have shown antimicrobial potential, as evaluated previously [4, 10, 11, 12]. This is a highly desirable property important in fighting the ever‐growing issue of antimicrobial resistance not only in the pharmaceutical field, but also in the food industry, where EO are becoming a suitable alternative to synthetic preservatives [13, 14]. Other applications of EO with antimicrobial activities include formulation of animal dietary supplements, fabrication of textiles with functional finishes and agricultural biocontrol products [15, 16, 17].

Although seasonal and geographical influences on EO composition have been widely reported, these factors are often investigated separately. Seasonal variation is primarily associated with short‐term environmental fluctuations, including temperature, rainfall, photoperiod, and plant phenology, which directly affect secondary metabolite biosynthesis. In contrast, regional variation reflects broader ecological differences, such as altitude, soil characteristics, local temperature and rainfall patterns, and local climatic conditions, which may promote the establishment of distinct chemotypes [18]. While regional variability has been documented for P. marginatum across South and Central America [19, 20], there are no previous reports investigating its seasonal variation. However, relatively few studies have evaluated the combined influence of seasonal and regional factors within the same experimental design, making it difficult to distinguish whether observed variations are driven predominantly by temporal environmental fluctuations, geographical conditions, or their interaction.

Therefore, the novelty of the present study lies in being the first to simultaneously assess seasonal and regional variation in P. marginatum EO, allowing a more comprehensive understanding of the factors governing chemical composition and antimicrobial activity. Thus, the aim of this study was to evaluate the seasonal and regional variation of the chemical composition and antimicrobial activity of the EO from P. marginatum through multivariate analysis (principal component analysis [PCA] and hierarchical clustering analysis).

2. Results and Discussion

The EO yields from P. marginatum leaves collected across three Brazilian regions over four seasons varied from 0.15% to 1.74% (w/w) (Figure 1). Two‐way ANOVA confirmed that region, season, and their interaction had highly significant effects on extraction yields (p < 0.001), statistically demonstrating that geographical and temporal factors, along with their synergistic interaction, govern the oil accumulation.

FIGURE 1.

FIGURE 1

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Yields of the EO from P. marginatum.

The yields from the Northern region EO agreed with those from other studies of EO collected in the Brazilian Amazon Rainforest, which ranged from 0.3% to 2.0% [4, 21, 22]. On the other hand, the results for the Northeast region were substantially higher than other studies of Brazilian Atlantic Rainforest EO, which found yields of 0.39% and 0.11% [5, 23]. No previous investigations were found about EO collected in the Central Brazilian Savanna.

The variability of the yields observed in this study and reported in the literature may be attributed to several factors that may influence the accumulation of secondary metabolites such as physiological and geographic variations, environmental conditions, and genetic factors [18].

The chemical compositions of the EO were determined by GC–MS and GC‐FID (Table 1), which resulted in the identification of 60 compounds belonging mainly to the classes of phenylpropanoids, sesquiterpenes, and oxygenated sesquiterpenes (Figure 2).

TABLE 1.

Relative content (%) of the EO from P. marginatum collected in different Brazilian regions and seasons.

S. nos. Compounds a AI b Relative content (%) c
North region Central‐West region Northeast region
Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer
1 α‐Thujene 924 — — — — < 0.10 0.14 ± 0.02 0.23 ± 0.11 < 0.10 — — — —
2 α‐Pinene 931 0.61 ± 0.28 1.07 ± 0.54 1.98 ± 0.26 1.14 ± 0.58 0.69 ± 0.01 1.08 ± 0.12 1.20 ± 0.43 0.59 ± 0.24 3.44 ± 0.40 6.12 ± 2.26 5.71 ± 1.52 0.83 ± 0.37
3 Sabinene 970 — — — — < 0.10 0.17 ± 0.01 0.27 ± 0.06 < 0.10 — — — —
4 β‐Pinene 974 0.54 ± 0.21 1.03 ± 0.36 1.77 ± 0.36 1.09 ± 0.36 0.65 ± 0.11 0.83 ± 0.05 0.96 ± 0.16 0.45 ± 0.17 2.67 ± 0.26 5.31 ± 1.50 4.43 ± 1.14 0.67 ± 0.27
5 Myrcene 989 0.21 ± 0.07 0.44 ± 0.08 0.70 ± 0.15 0.43 ± 0.06 0.23 ± 0.04 0.65 ± 0.04 0.74 ± 0.16 0.38 ± 0.13 — — — —
6 α‐Phellandrene 1004 — — — — 0.19 ± 0.04 0.72 ± 0.07 2.79 ± 0.73 0.80 ± 0.22 3.50 ± 0.29 3.53 ± 0.82 3.97 ± 0.95 0.91 ± 0.24
7 δ‐3‐Carene 1009 0.37 ± 0.13 0.36 ± 0.08 1.14 ± 0.18 0.44 ± 0.09 — — — — < 0.10 < 0.10 0.80 ± 0.66 < 0.10
8 α‐Terpinene 1014 — — — — 0.22 ± 0.05 0.54 ± 0.07 1.46 ± 0.38 0.55 ± 0.15 — — — —
9 p‐cymene 1022 0.16 ± 0.02 0.27 ± 0.04 0.17 ± 0.05 0.53 ± 0.04 1.11 ± 0.11 3.86 ± 0.44 4.36 ± 0.33 1.42 ± 0.47 — — — —
10 Sylvestrene 1025 0.13 ± 0.02 0.24 ± 0.03 0.36 ± 0.08 0.28 ± 0.02 0.28 ± 0.05 0.73 ± 0.07 1.36 ± 0.23 0.45 ± 0.12 1.27 ± 0.09 1.61 ± 0.32 1.76 ± 0.37 0.32 ± 0.09
11 (Z)‐β‐Ocimene 1034 0.93 ± 0.21 2.62 ± 0.20 4.14 ± 0.87 2.39 ± 0.30 0.68 ± 0.14 2.88 ± 0.16 0.38 ± 0.06 1.26 ± 0.26 — — — —
12 (E)‐β‐Ocimene 1044 0.79 ± 0.18 3.17 ± 0.19 6.47 ± 1.34 3.13 ± 0.46 0.65 ± 0.13 3.87 ± 0.26 0.31 ± 0.04 1.60 ± 0.31 — — — —
13 γ‐Terpinene 1055 <0.10 0.14 ± 0.01 0.24 ± 0.05 0.24 ± 0.04 0.79 ± 0.18 1.62 ± 0.25 4.43 ± 0.73 1.66 ± 0.33 — — — —
14 Terpinolene 1086 — — — — 0.43 ± 0.09 1.09 ± 0.13 2.61 ± 0.40 0.98 ± 0.16 — — — —
15 Linalool 1099 0.31 ± 0.04 0.79 ± 0.02 0.88 ± 0.32 0.97 ± 0.33 0.39 ± 0.03 0.61 ± 0.08 0.73 ± 0.16 0.14 ± 0.04 — — — —
16 Safrole 1284 0.17 ± 0.01 0.11 ± 0.00 2.01 ± 0.20 0.72 ± 0.22 — — — — — — — —
17 δ‐Elemene 1333 0.41 ± 0.03 0.44 ± 0.04 0.57 ± 0.04 0.66 ± 0.07 1.10 ± 0.04 0.83 ± 0.06 0.60 ± 0.14 1.16 ± 0.18 1.53 ± 0.06 3.47 ± 0.19 2.92 ± 0.35 1.72 ± 0.34
18 Cyclosativene 1358 0.21 ± 0.01 0.19 ± 0.01 < 0.10 0.17 ± 0.02 0.48 ± 0.04 0.28 ± 0.03 0.40 ± 0.10 0.29 ± 0.04 — — — —
19 α‐Copaene 1370 2.83 ± 0.16 1.89 ± 0.08 0.60 ± 0.02 2.13 ± 0.30 6.77 ± 0.54 3.25 ± 0.30 4.32 ± 1.02 6.30 ± 0.92 2.24 ± 0.08 0.75 ± 0.06 1.93 ± 0.21 2.27 ± 0.21
20 β‐Bourbonene 1378 2.60 ± 0.20 4.98 ± 0.33 0.59 ± 0.04 2.56 ± 0.33 0.76 ± 0.06 1.12 ± 0.09 0.90 ± 0.37 0.83 ± 0.11 — — — —
21 β‐Cubebene 1385 — — — — 0.25 ± 0.01 0.15 ± 0.01 0.13 ± 0.02 0.22 ± 0.01 — — — —
22 β‐Elemene 1387 0.37 ± 0.02 0.46 ± 0.04 0.19 ± 0.00 0.45 ± 0.06 1.15 ± 0.07 0.45 ± 0.05 0.64 ± 0.16 0.59 ± 0.08 1.46 ± 0.11 0.50 ± 0.04 0.95 ± 0.09 1.34 ± 0.22
23 Methyl eugenol 1403 1.84 ± 0.22 1.76 ± 0.04 2.22 ± 0.16 1.39 ± 0.38 0.21 ± 0.01 0.43 ± 0.05 1.04 ± 0.12 0.61 ± 0.08 — — — —
24 β‐Caryophyllene 1412 9.82 ± 0.71 9.86 ± 0.71 4.78 ± 0.17 7.66 ± 1.08 10.39 ± 0.43 8.83 ± 0.66 8.65 ± 1.96 12.54 ± 1.17 9.95 ± 0.29 8.41 ± 0.52 13.74 ± 1.35 10.35 ± 1.73
25 β‐Copaene 1422 0.78 ± 0.04 0.92 ± 0.06 0.17 ± 0.01 0.73 ± 0.10 1.10 ± 0.07 0.55 ± 0.04 0.78 ± 0.19 0.70 ± 0.07 0.39 ± 0.01 0.61 ± 0.04 0.78 ± 0.08 0.40 ± 0.08
26 Aromadendrene 1431 0.22 ± 0.00 0.11 ± 0.00 < 0.10 0.20 ± 0.03 0.27 ± 0.02 0.24 ± 0.02 0.17 ± 0.05 0.24 ± 0.03 — — — —
27 α‐Humulene 1446 1.70 ± 0.07 1.46 ± 0.15 0.76 ± 0.09 1.26 ± 0.15 1.34 ± 0.01 1.15 ± 0.10 1.36 ± 0.32 1.53 ± 0.03 0.75 ± 0.02 0.54 ± 0.02 0.90 ± 0.11 0.80 ± 0.09
28 Croweacin 1453 — — — — — — — — 3.57 ± 0.15 < 0.10 1.42 ± 0.30 3.06 ± 0.76
29 allo‐Aromadendrene 1453 — — — — 0.67 ± 0.05 < 0.10 < 0.10 0.20 ± 0.00 — — — —
30 Ishwarane 1456 — — — — — — — — 7.01 ± 0.30 3.81 ± 0.35 4.13 ± 0.33 4.34 ± 0.91
31 cis‐Cadina‐1(6),4‐diene 1459 0.18 ± 0.02 0.33 ± 0.03 < 0.10 0.19 ± 0.03 — — — — — — — —
32 α‐Neocallitropsene 1466 0.10 ± 0.01 < 0.10 < 0.10 0.15 ± 0.01 0.49 ± 0.02 0.31 ± 0.03 0.26 ± 0.08 0.23 ± 0.02 0.73 ± 0.03 1.47 ± 0.07 2.39 ± 0.22 0.70 ± 0.17
33 γ‐Muurolene 1471 0.24 ± 0.02 0.12 ± 0.01 < 0.10 0.16 ± 0.03 0.84 ± 0.06 0.32 ± 0.03 0.53 ± 0.14 0.43 ± 0.04 — — — —
34 Germacrene D 1474 6.25 ± 0.98 7.86 ± 0.67 6.58 ± 0.31 10.79 ± 1.64 6.45 ± 0.30 2.41 ± 0.13 0.59 ± 0.09 3.43 ± 0.34 — — — —
35 β‐Selinene 1478 0.57 ± 0.04 0.33 ± 0.02 0.23 ± 0.01 0.45 ± 0.06 2.06 ± 0.11 0.54 ± 0.04 1.08 ± 0.29 0.94 ± 0.08 — — — —
36 Bicyclogermacrene 1489 2.98 ± 0.35 3.75 ± 0.33 2.84 ± 0.23 5.83 ± 0.90 6.02 ± 0.18 5.52 ± 0.35 1.49 ± 0.38 8.22 ± 0.87 9.51 ± 0.21 11.82 ± 0.53 13.90 ± 0.69 8.71 ± 1.72
37 α‐Muurolene 1493 0.80 ± 0.12 0.29 ± 0.01 0.28 ± 0.03 0.15 ± 0.08 0.87 ± 0.02 0.52 ± 0.04 0.38 ± 0.06 0.58 ± 0.15 0.17 ± 0.01 0.36 ± 0.02 0.42 ± 0.03 0.19 ± 0.05
38 α‐Bulnesene 1495 — — — — 1.07 ± 0.08 0.46 ± 0.03 0.54 ± 0.16 0.75 ± 0.09 — — — —
39 γ‐Cadinene 1508 0.25 ± 0.01 < 0.10 < 0.10 < 0.10 0.51 ± 0.01 0.12 ± 0.01 0.31 ± 0.04 0.46 ± 0.07 — — — —
40 Myristicin 1516 0.26 ± 0.00 0.21 ± 0.14 5.04 ± 1.58 1.66 ± 1.31 0.19 ± 0.02 0.37 ± 0.10 1.57 ± 0.08 0.28 ± 0.07 — — — —
41 δ‐Cadinene 1518 1.69 ± 0.02 0.55 ± 0.09 0.37 ± 0.05 0.57 ± 0.31 3.20 ± 0.06 1.31 ± 0.04 0.29 ± 0.11 3.55 ± 0.24 0.44 ± 0.03 0.29 ± 0.04 0.47 ± 0.03 0.58 ± 0.05
42 3,4‐Methylenedioxypropiophenone 1528 23.23 ± 1.71 24.86 ± 2.49 33.01 ± 0.89 24.81 ± 3.05 4.32 ± 1.04 22.31 ± 4.28 8.23 ± 2.38 11.69 ± 2.46 — — — —
43 Elemicin 1557 3.08 ± 0.25 2.77 ± 0.13 2.11 ± 0.19 3.44 ± 0.38 3.39 ± 0.27 2.31 ± 0.25 7.38 ± 1.65 2.80 ± 0.17 — — — —
44 (E)‐Nerolidol 1562 2.86 ± 0.04 1.40 ± 0.08 1.39 ± 0.07 1.77 ± 0.21 3.40 ± 0.05 2.96 ± 0.26 3.75 ± 0.05 4.81 ± 0.17 — — — —
45 Spathulenol 1569 8.55 ± 0.45 6.45 ± 0.34 2.42 ± 0.08 5.53 ± 0.72 8.85 ± 0.20 6.02 ± 0.60 8.36 ± 0.44 7.31 ± 0.05 1.57 ± 0.05 1.48 ± 0.24 2.46 ± 0.38 0.99 ± 0.05
46 Caryophyllene oxide 1574 6.49 ± 0.28 4.17 ± 0.15 1.09 ± 0.02 1.79 ± 0.23 6.73 ± 0.09 2.26 ± 0.21 7.92 ± 0.50 3.84 ± 0.04 1.51 ± 0.08 1.66 ± 0.14 1.64 ± 0.21 1.63 ± 0.15
47 Guaiol 1594 0.66 ± 0.01 0.60 ± 0.05 0.22 ± 0.01 0.45 ± 0.05 — — — — — — — —
48 Humulene epoxide II 1599 0.67 ± 0.09 0.34 ± 0.01 0.11 ± 0.02 0.16 ± 0.03 — — — — — — — —
49 β‐Asarone 1622 — — — — — — — — 0.77 ± 0.54 0.92 ± 0.54 0.63 ± 0.13 0.99 ± 0.33
50 Dillapiole 1622 1.73 ± 0.05 0.72 ± 0.06 1.05 ± 0.01 1.61 ± 0.18 3.56 ± 0.08 2.08 ± 0.13 3.10 ± 0.44 3.22 ± 0.18 — — — —
51 Isospathulenol 1626 2.73 ± 0.36 2.60 ± 0.55 3.63 ± 0.71 1.47 ± 0.68 0.61 ± 0.16 4.53 ± 0.77 1.16 ± 0.50 0.83 ± 0.11 — — — —
52 1,3,5‐Trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene d 1627 — — — — — — — — 13.47 ± 0.97 14.22 ± 2.54 13.91 ± 3.67 14.87 ± 0.42
53 epi‐α‐Cadinol 1634 0.63 ± 0.00 0.31 ± 0.03 0.23 ± 0.01 0.40 ± 0.05 1.07 ± 0.02 0.54 ± 0.02 0.19 ± 0.03 1.26 ± 0.11 — — — —
54 δ‐Cadinol 1640 1.56 ± 0.03 0.97 ± 0.08 0.73 ± 0.02 0.87 ± 0.09 1.23 ± 0.04 0.69 ± 0.04 0.64 ± 0.09 1.57 ± 0.14 — — — —
55 β‐Eudesmol 1643 0.34 ± 0.01 0.28 ± 0.02 0.13 ± 0.01 0.17 ± 0.02 0.36 ± 0.38 0.11 ± 0.10 0.29 ± 0.04 0.14 ± 0.05 1.01 ± 0.02 0.29 ± 0.05 0.43 ± 0.08 1.81 ± 0.03
56 α‐Cadinol 1647 1.26 ± 0.03 0.64 ± 0.08 0.77 ± 0.03 0.68 ± 0.16 1.07 ± 0.42 0.91 ± 0.13 < 0.10 1.85 ± 0.16 — — — —
57 Intermedeol 1650 — — — — 0.36 ± 0.05 0.12 ± 0.00 0.25 ± 0.05 0.15 ± 0.03 — — — —
58 Exalatacin d 1655 — — — — — — — — 11.90 ± 0.31 12.36 ± 1.80 9.30 ± 2.44 17.93 ± 1.54
59 α‐Asarone 1680 — — — — — — — — 2.40 ± 0.09 2.63 ± 0.21 0.92 ± 0.33 3.52 ± 0.17
60 1,3,5‐Trimethoxy‐2‐[(E)‐prop‐1‐enyl]benzene (pipermargine) d 1716 — — — — — — — — 10.42 ± 0.53 10.52 ± 1.57 3.38 ± 1.23 14.56 ± 1.38
Monoterpene hydrocarbons 3.79 ± 1.13 9.35 ± 1.53 16.98 ± 3.32 9.67 ± 1.83 6.04 ± 0.99 18.19 ± 1.00 21.10 ± 3.66 10.28 ± 2.58 10.93 ± 0.99 16.62 ± 4.86 16.67 ± 4.45 2.77 ± 0.98
Oxygenated monoterpenes 0.31 ± 0.04 0.79 ± 0.02 0.88 ± 0.32 0.97 ± 0.33 0.39 ± 0.03 0.61 ± 0.08 0.73 ± 0.16 0.14 ± 0.04 — — — —
Sesquiterpene hydrocarbons 31.99 ± 2.54 33.63 ± 2.44 18.16 ± 0.09 34.19 ± 4.99 45.79 ± 1.36 28.44 ± 1.82 23.46 ± 5.50 43.20 ± 4.07 34.18 ± 0.85 32.05 ± 0.96 42.53 ± 3.16 31.41 ± 3.83
Oxygenated sesquiterpenes 25.75 ± 1.21 17.76 ± 0.35 10.70 ± 0.67 13.29 ± 0.88 23.67 ± 0.80 18.15 ± 0.70 22.63 ± 0.86 21.75 ± 0.79 4.09 ± 0.14 3.44 ± 0.40 4.53 ± 0.68 4.43 ± 0.20
Phenylpropanoids 30.30 ± 2.25 30.44 ± 2.41 45.45 ± 2.24 33.63 ± 3.13 11.67 ± 1.31 27.50 ± 3.81 21.32 ± 4.19 18.59 ± 2.42 42.54 ± 1.22 40.69 ± 5.54 29.56 ± 7.26 54.93 ± 2.71
Total identified (%) 92.15 ± 0.18 91.96 ± 0.68 92.17 ± 0.39 91.74 ± 1.95 87.57 ± 0.21 92.89 ± 0.59 89.24 ± 0.70 93.97 ± 0.20 91.75 ± 0.07 92.79 ± 0.47 93.29 ± 0.34 93.54 ± 0.12
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a

Compounds are listed in order of their elution from a SH‐Rtx‐5MS capillary column.

b

AI: Linear retention indices relative to C9 to C25 n‐alkanes on a SH‐Rtx‐5MS capillary column.

c

Relative content values are presented as mean ± standard deviation of three determinations. Values lower than 0.10% are reported as < 0.10, and dashes (—) indicate that the compound was not detected in the sample.

d

Compounds isolated by TLC and identified by GC‐MS and 1H and 13C NMR.

FIGURE 2.

FIGURE 2

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Relative contents of the compound classes present in the EO from P. marginatum collected in (a) the Northern region, (b) the Central‐West region, and (c) the Northeast region.

Two major compounds from the Northeast region EO (combined concentrations of 17.30%–29.43%) could not be identified using standard GC–MS libraries and retention indices. Thus, they were isolated by TLC and identified by 1H and 13C NMR data comparison with literature as 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene (compound 2) [24] and pipermargine (1,3,5‐trimethoxy‐2‐[(E)‐prop‐1‐enyl]benzene (compound 3) [25]. The main constituent exalatacin (compound 1) was also isolated and identified [26]. For the novel (Z)‐isomer (compound 2), key 1H NMR signals were observed at δ 5.89 ppm (dq, J = 11.3 and 6.8 Hz, 1H), and δ 6.19 ppm (dd, J = 11.3 and 1.7 Hz, 1H), characteristic of the Z‐configuration of the propenyl group. The (E)‐isomer (pipermargine, 3) was distinguished by the larger coupling constant of the olefinic hydrogens at δ 6.46 ppm (dq, J = 16.0 and 6.0 Hz, 1H) and δ 6.55 ppm (dd, J = 16.0 and 1.5 Hz, 1H). Exalatacin (1) was identified as a phenylpropanoid featuring an aromatic ring substituted with a methylenedioxy bridge and two methoxyl groups, in addition to the allyl side chain. Its structure was confirmed by characteristic signals at δ 5.86 ppm (s, 2H), corresponding to the methylenedioxy group, and two singlets at δ 3.74 and 3.95 ppm, representing the two methoxyl groups. These data are in full agreement with literature values [24, 25, 26] and provide definitive evidence for the presence of these phenylpropanoids in P. marginatum EO. The 1D and 2D NMR spectra, along with the table of NMR chemical shifts for compounds 1–3, are provided in the (Figures S1–S10 and Table S1).

Although these compounds had previously been isolated from the roots and fruits of P. marginatum [24, 25], in terms of EO composition, compound 3 had only been reported in the EO of Peperomia lancifolia Hook. (sin. Piper patulum M. Martens and Galeotti) [27].

To the best of our knowledge, this is the first report of the presence of compound 2 in EO. In this work, compounds 2 and 3 showed AI values of 1627 and 1716, respectively.

To assess the differences in the chemical compositions of the EO samples and verify which constituents were responsible for them, PCA was performed. Figures 3, 4 and 5 show the scores and loadings for each region and Figure 6 shows the scores and loadings for all regions.

FIGURE 3.

FIGURE 3

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(a) Scores and (b) loadings obtained by PCA of EO from P. marginatum collected in the Northern region. PC1 and PC2 accounted for 65.90% and 22.56% of the total variance, respectively.

FIGURE 4.

FIGURE 4

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(a) Scores and (b) Loadings obtained by PCA of EO from P. marginatum collected in the Central‐West region. PC1 and PC3 accounted for 55.59% and 14.12% of the total variance, respectively.

FIGURE 5.

FIGURE 5

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(a) Scores and (b) loadings obtained by PCA of EO from P. marginatum collected in the Northeast region. PC1 and PC2 accounted for 59.39% and 24.97% of the total variance, respectively.

FIGURE 6.

FIGURE 6

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(a) Scores and (b) loadings obtained by PCA of EO from P. marginatum. PC1 and PC2 accounted for 45.35% and 22.79% of the total variance, respectively.

In the Northern region, the major compounds found were 3,4‐methylenedioxypropiophenone (23.23%–33.01%), germacrene D (6.25%–10.79%), β‐caryophyllene (4.78%–9.86%), spathulenol (2.42%–8.55%), caryophyllene oxide (1.09%–6.49%), (E)‐β‐ocimene (0.79%–6.47%), bicyclogermacrene (2.84%–5.83%), and myristicin (0.21%–5.04%).

Principal component 1 (PC1) and principal component 2 (PC2) of the Northern region samples explained 88.46% of the total variance and PC1 differentiated the spring and autumn EO (Figure 3). The spring sample presented the highest amounts of monoterpenes and phenylpropanoids and the lowest amounts of sesquiterpenoids amongst all Northern region EO. In contrast, the autumn sample possessed the highest levels of oxygenated sesquiterpenes and the lowest levels of monoterpenes (Figures 2a and 3b). By the proximity of the summer and winter samples, they are more closely related than the others, as both possess similar levels of sesquiterpenes and phenylpropanoids.

The monoterpenes (Z)‐β‐ocimene (4.14%) and (E)‐β‐ocimene (6.47%), as well as the phenylpropanoids safrole (2.01%) and myristicin (5.04%), were found at higher concentrations in the EO obtained during spring. In contrast, the sesquiterpenes bicyclogermacrene (5.83%) and β‐bourbonene (4.98%) were most abundant in summer and winter, respectively. The sesquiterpene β‐caryophyllene and the oxygenated sesquiterpenes spathulenol and caryophyllene oxide were found at higher concentrations in the EO obtained during autumn and winter. In contrast, 3,4‐methylenedioxypropiophenone was present at high concentrations in all EO collected from the Northern region.

In the Central‐West region, the main compounds present were 3,4‐methylenedioxypropiophenone (4.32%–22.31%), β‐caryophyllene (8.65%–12.54%), spathulenol (6.02%–8.85%), bicyclogermacrene (1.49%–8.22%), caryophyllene oxide (2.26%–7.92%), elemicin (2.31%–7.38%), α‐copaene (3.25%–6.77%) and germacrene D (0.59%–6.45%).

PC1 and principal component 3 (PC3) of the Central‐West region EO described 69.71% of the total variance (Figure 4). Although PC3 described less variability than PC2 (14.12% against 30.30%), the cumulative variance explained by the first three principal components (PC1, PC2, and PC3) was 100.0% (55.59%, 30.30%, and 14.12%, respectively), and PC3 better explained the data observed. PC1 and PC3 were able to differentiate the EO samples, as each one was in a different quadrant. The autumn and summer season EO both showed high amounts of sesquiterpenoids but differed in monoterpenes and phenylpropanoids levels (Figures 2b and 4b). The winter EO showed the highest concentration of phenylpropanoids amongst all Central‐West region samples, whilst the spring EO possessed the lowest concentration of sesquiterpenes.

The autumn EO was characterized by the highest concentration of germacrene D (6.45%). The winter EO exhibited the highest levels of (Z)‐β‐ocimene (2.88%), (E)‐β‐ocimene (3.87%), 3,4‐methylenedioxypropiophenone (22.31%), and isospathulenol (4.53%). In contrast, the spring EO showed the highest concentrations of α‐phellandrene (2.79%), γ‐terpinene (4.43%), terpinolene (2.61%), and elemicin (7.38%), whereas the summer EO was distinguished by elevated levels of β‐caryophyllene (12.54%) and bicyclogermacrene (8.22%).

In the Northeast region, the main constituents found were exalatacin (9.30%–17.93%), pipermargine (3.38%–14.56%), 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene (13.47%–14.87%), bicyclogermacrene (8.71%–13.90%), β‐caryophyllene (8.41%–13.74%), ishwarane (3.81%–7.01%), α‐pinene (0.83%–6.12%), and β‐pinene (0.67%–5.31%).

For the Northeast region EO, PC1 and PC2 elucidated 84.36% of the total variance (Figure 5). Each EO sample was in a different quadrant, thus presenting different profiles. While the spring EO presented a high concentration of monoterpenes and a low concentration of phenylpropanoids, the summer EO presented an opposite profile (Figures 2c and 5b). The winter and autumn EO both showed high amounts of sesquiterpenes and phenylpropanoids but differed in the monoterpenes levels.

The highest concentrations of α‐pinene, β‐pinene, δ‐elemene, and bicyclogermacrene were observed in the EO obtained during winter and spring. In contrast, croweacin and ishwarane were more abundant in the autumn EO, whereas exalatacin reached its highest concentration in the summer EO.

This is the first investigation of the seasonal variation of the chemical composition of EO from P. marginatum. For each studied region, the constituents present in the EO differed only quantitatively between seasons, which is consistent with results from studies with other Piper species [28, 29, 30].

In the PCA of all EO samples, the PC1 and PC2 accounted for 68.14% of the total variance (Figure 6). The analysis confirmed the distinction of the Northeast region EO, which were characterized by the presence of sesquiterpene ishwarane and phenylpropanoids α‐ and β‐asarone, 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene, exalatacin, and pipermargine, constituents only found in the EO from this region. Consequently, this clear separation establishes the Northeast population as the most distinctive and unique regional chemotype of P. marginatum in Brazil, reflecting a highly specialized metabolic adaptation to the Atlantic Rainforest biome.

The chemical composition of EO grown in different locations has been previously investigated and shows major quantitative and qualitative variability [12, 19].

Several studies have shown that the EO from P. marginatum from the Brazilian Amazon Rainforest (Northern region) is mainly composed of phenylpropanoids, which present variation of specific molecules and concentrations. Andrade et al. [21] analyzed twenty‐two samples from various locations of the Brazilian Amazon and observed the existence of seven different chemotypes based on the EO main constituents. The authors concluded that the plant metabolism was driven to produce either 3,4‐methylenedioxypropiophenone and analogues or anethole and isoosmorhizole. Other investigations also found 3,4‐methylenedioxypropiophenone as the main compound, in agreement with the results from this study for the Northern region EO [6, 7, 10, 31, 32].

As for the EO from the Brazilian Atlantic Rainforest (Northeast region), a study has shown they were mainly composed of phenylpropanoids and sesquiterpenoids, with α‐asarone, β‐asarone, bicyclogermacrene, and patchoulol as the main constituents [23]. In this investigation, the EO from the Northeast region also presented bicyclogermacrene as a major compound.

In another study, Dutra et al. [5] investigated the EO collected in the same region and found the phenylpropanoid exalatacin as one of the major compounds, agreeing with this research.

There are no previous reports of EO from P. marginatum collected in the Central Brazilian Savanna (Central‐West region).

The distinctions observed in the PCA for the three regions (Figure 6) suggest that the selective pressures of the Amazon Rainforest, Cerrado, and Atlantic Rainforest biomes play a crucial role in the chemical profile of the plants. This regional divergence reveals that while the EO share a common origin in the phenylpropanoid pathway, their specific biosynthetic routes are uniquely modulated by local environmental conditions.

The specialized metabolic profile of the Northeast population suggests differences in phenylpropanoid metabolism relative to the Northern and Central‐West populations. Whereas the latter predominantly accumulate 3,4‐methylenedioxypropiophenone and related methylenedioxy derivatives, the Northeast population is characterized by the accumulation of highly O‐methylated phenylpropanoids, including exalatacin, pipermargine, and its (Z)‐isomer. A plausible biosynthetic explanation involves differential metabolic channeling of phenylpropanoid intermediates, where O‐methylation acts as a crucial branch point in these pathways [33, 34]. In the Northeast population, this channeling may favor the activity of S‐adenosyl‐L‐methionine (SAM)‐dependent O‐methyltransferases (OMTs), enzymes that catalyze the methylation of phenolic hydroxyl groups to enhance the lipophilicity and antimicrobial potential of these specialized metabolites [34]. Conversely, in the Northern and Central‐West populations, phenylpropanoid intermediates may be preferentially directed toward the biosynthesis of methylenedioxy derivatives, resulting in the predominance of 3,4‐methylenedioxypropiophenone. Environmental conditions characteristic of the Atlantic Rainforest may also contribute to this metabolic differentiation by modulating specialized metabolism as an adaptive response to regional selective pressures, such as light and moisture fluctuations, which trigger stress‐responsive pathways [33]. Although this biosynthetic hypothesis remains to be experimentally validated, it provides a cohesive mechanistic framework consistent with the observed chemotypic divergence.

The antimicrobial activities of the EO from P. marginatum were assessed against seven microorganisms, of which only five of these were inhibited (Table 2) in the evaluated concentration range, up to 2.0 mg mL−1. The results show that the EO activities varied according to the season in which the plant was collected and the microorganism. In order to classify the activity of the different EO samples, the following classification was considered strong activity (MIC ≤ 0.5 mg mL−1), moderate activity (0.6 mg mL−1 ≤ MIC ≤ 1.5 mg mL−1), weak activity (MIC ≥ 1.6 mg mL−1) [35].

TABLE 2.

Antimicrobial activity (MIC/MBC mg mL−1) of the EO from P. marginatum leaves collected in different Brazilian regions and seasons.

Samples Yeasts Gram‐positive Gram‐negative
Regions Seasons Candida albicans Bacillus subtilis Staphylococcus aureus Escherichia coli Salmonella choleraesuis
MIC MFC MIC MBC MIC MBC MIC MBC MIC MBC
Northern Autumn * * 0.25 2.0 * 2.0 * * *
Winter 0.5 1.0 0.5 0.5 2.0 * 1.0 * * *
Spring 0.5 0.5 2.0 * * * 2.0 * 2.0 *
Summer 0.5 2.0 1.0 1.0 * * 1.0 * * *
Central‐West Autumn * * 0.25 1.0 1.0 * 2.0 * * *
Winter 0.5 2.0 0.5 * 2.0 2.0 1.0 * 2.0 *
Spring * * 0.5 * 2.0 * 2.0 * * *
Summer 0.5 2.0 0.25 0.25 1.0 * 2.0 * * *
Northeast Autumn * * * * * * 1.0 * * *
Winter 2.0 * * * * * 0.25 * * *
Spring 2.0 * * * * * 2.0 * * *
Summer 2.0 * * * * * 1.0 * * *
Antimicrobial Standard a 0.002 0.004 0.008 0.004 0.004
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a

Nystatin for yeast/chloramphenicol for bacteria, * MIC > 2.0 mg mL−1.

Among the EO samples tested and microorganisms evaluated, the EO with the broadest spectrum of action was the winter season from the Central‐West region, which inhibited five microorganisms, exhibiting strong activities against Candida albicans and Bacillus subtilis (MIC = 0.5 mg mL−1). All EO inhibited Escherichia coli, although only the winter season EO from the Northeast region presented a strong activity (MIC = 0.25 mg mL−1). No EO was able to inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa up to a 2.0 mg mL−1.

Regarding bactericidal/fungicidal activity, the EO samples obtained in the autumn and summer seasons from the Central‐West region were capable to cause the death of 99.9% of Gram‐positive bacteria cells, specifically, B. subtilis (MBC = 0.25 mg mL−1) and Staphylococcus aureus (MBC = 1.0 mg mL−1).

Finally, the Northeast region EO did not show a broad spectrum of antimicrobial inhibition. A previous study demonstrated that exalatacin, one of the main compounds present in P. marginatum EO, possessed antimicrobial activity against E. coli and B. subtilis [26]. Another study has shown that pipermargine showed fungicidal activity against C. albicans [36]. In this study, evidence strongly suggests that antimicrobial activity of EO cannot be associated with activities of the main compounds alone, and that complex interactions between constituents must be taken in consideration.

Antimicrobial EO are complex mixtures that offer a promising alternative to combat drug resistance. It is well accepted that their antimicrobial activity is mainly attributed to major compounds, although synergistic, additive, and antagonistic interactions among minor components also play an important role. The microorganisms evaluated in this study are relevant across several sectors, including human and animal health, food safety, cosmetics, and agriculture. Although the MIC and MBC values for P. marginatum EO were higher than those of pure commercial standards (Table 2), these complex mixtures act on multiple cellular targets simultaneously. This multi‐target mechanism makes it much harder for pathogens to develop resistance compared to single‐compound drugs. Therefore, MIC values between 0.25 and 1.0 mg mL− 1 are practical and useful for real‐world applications, such as topical formulations, skin antiseptics, and natural preservatives in food, cosmetic, and agricultural products.

To evaluate the similarities in the EO antimicrobial activities, Hierarchical Cluster Analysis (HCA) was performed, which separated them into three clusters (Figure 7). Cluster I grouped the autumn season EO from the Northern region and the autumn and spring season EO from the Central‐West region. These samples strongly inhibited B. subtilis and weakly inhibited E. coli, but were inactive against C. albicans and Salmonella choleraesuis. These EO contained the highest levels of oxygenated sesquiterpenes amongst all samples, especially spathulenol and caryophyllene oxide, which are shown in previous studies to be active against B. subtilis and E. coli [37, 38, 39].

FIGURE 7.

FIGURE 7

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Dendrogram obtained by hierarchical clustering analysis (Ward's Method) of the antimicrobial activities of EO from P. marginatum.

Cluster II grouped the winter, summer, and spring season EO from the Northern region and the summer and winter season EO from the Central‐West region. These samples were characterized by strong activity against C. albicans. The spring season EO from the Northern region showed strong fungicidal potential against C. albicans (MIC = MFC = 0.5 mg mL−1). This EO contained phenylpropanoids myristicin and 3,4‐methylenedioxypropiophenone, whose antifungal potentials have been shown in other studies [40, 41]. As opposed to our study, Duarte et al. [35] evaluated the antifungal activity of the EO against C. albicans and did not observe any activity up to 2.0 mg mL−1.

Cluster III grouped the Northeast region EO. These samples inhibited E. coli but were inactive against the Gram‐positive bacteria (B. subtilis and S. aureus) and the Gram‐negative S. choleraesuis. These EO were distinguished from the others, especially in phenylpropanoid composition. The winter EO showed the strongest activity against E. coli amongst all samples (MIC = 0.25 mg mL−1). Duarte et al. [42] observed in their study that the EO inhibited two strains of E. coli (MIC = 0.9 and 0.7 mg mL−1), agreeing with this study. Importantly, this biological grouping obtained by HCA is in excellent agreement with the regional chemotypic differentiation shown in the PCA (Figure 6), where the Northeast samples formed a highly distinct cluster due to their unique phenylpropanoid composition, while the Northern and Central‐West samples shared more overlapping chemical and antimicrobial profiles.

Previous studies have demonstrated potential applications of Piper spp. EO and extracts with antimicrobial activities. A study has shown that the combination of the EO from P. cernuum Vell. with gentamicin presented synergistic effects, increasing the EO activity against S. aureus [43]. Similarly, Carneiro et al. [44] have shown that the association of the EO from P. diospyrifolium Kunth. with gentamicin increased the EO antimicrobial effect against E. coli and S. aureus. Other studies have demonstrated the potential application of nano‐ and micro‐emulsions of EO from P. nigrum L. and P. betle L. against microorganisms in the food industry [45, 46]. In addition, de Pascoli et al. [47] reported the antibacterial activity of extracts from P. peltatum L. and P. marginatum in orange juice.

Furthermore, an integrated assessment of the EO yield, chemical composition, and antimicrobial activity reveals important trade‐offs for practical applications and optimal harvesting conditions. The Central‐West region produced the lowest EO yields (0.15%–0.35%); however, winter harvesting yielded the EO with the broadest antimicrobial spectrum, strongly inhibiting C. albicans and B. subtilis. This suggests that, for applications requiring broad‐spectrum antimicrobial agents, harvesting in the Brazilian Cerrado during winter is the most suitable strategy due to the specific accumulation of phenylpropanoids and sesquiterpenes. In contrast, for targeted activity against E. coli, winter harvesting in the Northeast region represents the optimal condition, providing a highly specific and potent EO (MIC = 0.25 mg mL− 1) together with a commercially attractive yield (∼0.9%). Notably, the EO from the Northeast collected during winter contains 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene as one of its major constituents, which is reported here for the first time in an EO. An integrated summary of the extraction yield, major chemical constituents, and antimicrobial activity for all 12 EO samples is provided in (Table S2).

This study provides a robust foundational mapping of the seasonal and regional chemical dynamics and antimicrobial potential of P. marginatum across three major Brazilian biomes. As the sampling was conducted at representative single locations for each region, the observed seasonal variations offer highly valuable baseline insights into the dynamic accumulation of these secondary metabolites and their corresponding biological activities. This work opens promising perspectives for more specific studies on the regionality of EO, establishing a strategic framework for the identification of elite chemotypes and the optimization of sustainable harvesting for the industrial and pharmaceutical sectors.

3. Conclusions

The chemical compositions and antimicrobial activities of P. marginatum EO across the Northern, Central‐West, and Northeast Brazilian regions exhibited significant seasonal and regional variations, driven by the unique selective pressures of the Amazon, Cerrado, and Atlantic Rainforest biomes. The Northeast EO provided the highest yields and revealed a distinct chemical profile characterized by specialized O‐methylation pathways, leading to the identification of exalatacin, pipermargine, and 1,3,5‐trimethoxy‐2‐[(Z)‐prop‐1‐enyl]benzene—the latter reported for the first time in EO. Biologically, the Central‐West winter EO displayed the broadest antimicrobial spectrum, while the Northeast winter EO showed the most potent targeted activity against E. coli. These varying responses underscore that EO bioactivities result from complex synergistic or antagonistic interactions rather than major constituents alone. Ultimately, these findings establish a strategic roadmap for the industrial exploitation of P. marginatum through targeted harvesting. Future research should prioritize: (i) integrating molecular tools and broader sampling to validate true chemotypes; (ii) conducting synergy bioassays to decode the antimicrobial interactions; and (iii) evaluating these chemically defined EO in real‐world commercial formulations.

4. Experimental Section

4.1. Plant Material

The leaves of P. marginatum Jacq. were collected in 2015 and 2016, in three Brazilian regions and in each season of the year. Prof. Dr. Diones Krinski identified the collected plants and voucher specimens were deposited in the Herbarium Tangará da Serra of Universidade do Estado do Mato Grosso (UNEMAT) (Table 3).

TABLE 3.

Collection sites and ecological characteristics.

Herbarium codes Regions City (state) Coordinates Collection months Biome
TANG 4643 Northern Novo Progresso (Pará) 7°07′43.6″ S, 55°23′22.1″ W Autumn: 04/2015 Amazon Rainforest
Winter: 07/2015
Spring: 10/2015
Summer: 01/2016
TANG 4644 Central‐West Tangará da Serra (Mato Grosso) 14°37′38.1″ S, 58°33′01.2″ W Autumn: 04/2015 Central Brazilian Savanna
Winter: 07/2015
Spring: 10/2015
Summer: 01/2016
TANG 4645 Northeast João Pessoa (Paraíba) 7°07′07.0″ S, 34°53′32.9″ W Autumn: 05/2015 Atlantic Rainforest
Winter: 08/2015
Spring: 11/2015
Summer: 02/2016
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4.2. Essential Oil Extraction and Analysis

The fresh collected leaves were air‐dried and heat‐dried at 40°C for 96 h. The EO were extracted by hydrodistillation of 100 g of dried leaves in 1.5 L of distilled water in a Clevenger‐type apparatus for 4 h in simultaneous triplicates. The oils were collected and dried over anhydrous sodium sulphate, filtered, transferred to vials, and stored at −20°C until further tests.

The EO chemical compositions were determined by gas chromatography coupled to mass spectrometry (GC–MS) using a Shimadzu GCMS TQ8040 system equipped with an electron ionization detector. The analysis was carried out using the same methodology described by Maduro et al. [29].

The samples were diluted in dichloromethane HPLC grade (J.T. Baker, USA) (0.1%, v/v) and were injected (1.0 µL) with a split ratio of 1:90. The injector operated at 250°C and the column temperature was programmed to the range from 60°C to 250°C at 3°C/min. The ionization energy of the mass spectrometer was 70 eV, the ion source and interface temperatures were 200°C and 300°C, respectively, and the scan ranged from 40 to 400 m/z.

Area normalization method was used to quantify the EO by gas chromatography with flame ionization detection (GC‐FID). The analysis was carried out using an Agilent 7890A system [29].

Components were identified through comparison of their mass spectra and arithmetical retention indices (AI) to the GC‐MS library (NIST 14), NIST WebBook [48] and literature [49]. The AI were calculated in reference to a standard mixture of n‐alkanes (C9–C25), injected under the same conditions as described above, using the Van den Dool and Kratz [50] equation.

4.3. Isolation and Identification of Essential Oil Compounds

The major constituents of the EO from P. marginatum collected in the Northeast region, which could not be identified as described previously, were isolated by thin‐layer chromatography (TLC).

The summer season EO sample (30 mg) was diluted in dichloromethane and applied on a glass plate (20 cm × 20 cm) coated with 1 mm layer of silica gel 60 F254 (Vetec, Brazil). The plate was developed twice with petroleum ether:ethyl acetate (98:2) and furnished three fractions (F1–F3), which were viewed under UV light at 254 and 366 nm.

Fraction F2 (11.0 mg) was applied on a pre‐coated silica gel 60 F254 aluminum plate (20 cm × 20 cm; layer thickness 200 µm; SiliCycle, Canada) impregnated with silver nitrate through immersion of the plate in a silver nitrate solution (10%, w/v) for 15 minutes, followed by heat‐drying at 40°C. The plate was developed with dichloromethane:ethyl acetate (85:15) and furnished fractions F2‐A and F2‐B, which were viewed under UV light at 254 and 366 nm.

The isolated compounds were identified by nuclear magnetic resonance (NMR), which were carried out at NMR spectrometers Bruker Avance 400 and Ascend 600. They observed the 1H nuclei at 400 and 600 MHz and the 13C nuclei at 100 and 150 MHz, respectively. Samples were diluted in deuterated chloroform with TMS as internal standard (Sigma–Aldrich, USA).

Fraction F3 was identified as compound 1 (6.2 mg) and fractions F2‐A and F2‐B were identified as compounds 2 (4.2 mg) and 3 (5.3 mg), respectively.

4.4. Antimicrobial Activity

The antimicrobial activities of P. marginatum EO were evaluated through the microdilution method, determining minimal inhibitory (MIC) and minimal bactericidal/fungicidal (MBC/MFC) concentrations against the bacteria B. subtilis (ATCC 6051), P. aeruginosa (ATCC 13388), S. aureus (ATCC 6538), E. coli (ATCC 11775), S. choleraesuis (ATCC 10708), S. epidermidis (ATCC 12228) and against the yeast C. albicans. The standardized bacteria and yeast C. albicans inocula were prepared following the protocol from CLSI (2012) [51]and CLSI (2017) [52], respectively, with adaptations validated for EO as described by Vanegas et al. [53]. Briefly, to ensure adequate solubilization and the emulsion stability of the EO, the broth medium of the sample dilutions was supplemented with 0.5% (v/v) Tween 80. The microdilution test to determine the minimum inhibitory concentration (MIC) was performed using a 96‐well microplate. Serial dilutions were prepared, and 100 µL of inoculum was added to each well. To prevent the evaporation of volatile metabolites during incubation, microplates were sealed with parafilm and incubated at 35 ± 2 °C for 24 h (bacteria) or 48 h (C. albicans ATCC 18804). The inhibition of microorganisms was confirmed by the addition of 50 µL of a triphenyl tetrazolium solution (0.1%) (Sigma) for the bacteria. After 2 h at 37°C, the last well without red coloration was visually identified as MIC. For yeast, a change in the color of the RPMI‐1640 (Cultlab) media indicated growth. The lowest concentration of the EO that visibly inhibited microbial growth was defined as the MIC. Minimum bactericidal concentration (MBC) was determined based on the MIC results by subculturing 10 µL from the contents of the wells corresponding to the MIC and three wells above them on Mueller–Hinton agar for bacteria and incubating the plates for 24 h. Likewise, for minimum fungicidal concentration (MFC), 10 µL from the contents of the wells corresponding to the MIC and three wells above them were plated on Sabouraud dextrose agar for Candida spp. and incubated for 48 h. The MBC/MFC of the EO was defined as bactericidal or fungicidal when no microbial growth was observed after incubation (99.9% cell death). Otherwise, the EO was considered bacteriostatic or fungistatic. Chloramphenicol and nystatin were included as positive controls for antibacterial and antifungal activity, respectively. Controls were also included to confirm the sterility of the culture medium and the viability of the inoculum. All assays were performed in triplicate.

4.5. Statistical Analysis

All EO extractions, chromatographic analyses, and antimicrobial bioassays were performed in independent triplicates (n = 3), serving as the basis for the multivariate analyses, which were performed to evaluate the associations among P. marginatum EO collected in different seasons and regions based on their chemical compositions and antimicrobial activities. PCA was carried out using PLS Toolbox 3.0 (Eigenvector Research, Inc.) in MATLAB 7.0.1 (MathWorks) based on the data presented in Table 1. HCA was performed using Origin 2019b (OriginLab) based on the data presented in Table 2. Prior to the multivariate analyses, the data were evaluated, and no outlier exclusion was necessary. Before PCA, the data were mean‐centered and scaled to unit variance (autoscaling), whereby each variable was centered by subtracting its mean and divided by its respective standard deviation. Before HCA, the data were normalized to the range of 0–1 (min–max normalization) to minimize the influence of differences in variable magnitude. HCA was performed using Euclidean distance as the similarity metric and Ward's linkage method. Additionally, a two‐way analysis of variance (ANOVA) was performed using Origin 2019b to evaluate the statistical significance of region, season, and their interaction on extraction yields and major chemical classes, with the significance level set at p < 0.05.

Author Contributions

Rebeca Gabriel de Camargo contributed to methodology, formal analysis, and writing – original draft. Marta Cristina Teixeira Duarte contributed to investigation, methodology, and formal analysis. Diones Krinski contributed to formal analysis and methodology. Ana Paula Palaro Klein Hendges contributed to writing – original draft, formal analysis, and methodology. Aurea Portes Ferriani contributed to writing – original draft and formal analysis Francisco de Assis Marques contributed to writing – review and editing and formal analysis. Thalita Gilda Santos contributed to supervision, writing – review and editing, and conceptualization. Beatriz Helena Lameiro de Noronha Sales Maia contributed to conceptualization, writing – review and editing, supervision, and project administration.

Funding

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through a scholarship granted to R.G.C (grant number 134535/2016‐2).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71771‐sup‐0001‐SuppMat.docx.

CBDV-23-e71771-s001.docx (2.2MB, docx)

Acknowledgments

The authors acknowledge the support from Universidade Federal do Paraná (UFPR) and open access funding enabled and organized by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supporting Information files.

References

  • 1. Calderón W. T. and Jaramillo M. A., “An Emended Description of Piper Cochleatum and a New Species of Piper (Piperaceae) from the Amazonian Slopes of the Colombian Andes,” Phytotaxa 408 (2019): 59–68. [Google Scholar]
  • 2. Flora e Funga do Brasil , “Jardim Botânico do Rio de Janeiro.”, Available at: http://floradobrasil.jbrj.gov.br/.
  • 3. de Souza M. T., de Souza M. T., Bernardi D., et al., “Chemical Composition of Essential Oils of Selected Species of Piper and Their Insecticidal Activity Against Drosophila Suzukii and Trichopria Anastrephae′ ,” Environmental Science and Pollution Research 27 (2020): 13056–13065. [DOI] [PubMed] [Google Scholar]
  • 4. Da Silva J. K. R., Silva N. N. S., Santana J. F. S., Andrade E. H. A., Maia J. G. S., and Setzer W. N., “Phenylpropanoid‐Rich Essential Oils of Piper Species From the Amazon and Their Antifungal and Anti‐Cholinesterase Activities,” Natural Product Communications 11 (2016): 1907–1911. [PubMed] [Google Scholar]
  • 5. Dutra K., Wanderley‐Teixeira V., Guedes C., et al., “Toxicity of Essential Oils of Leaves of Plants from the Genus Piper with Influence on the Nutritional Parameters of Spodoptera Frugiperda (J.E. Smith) (Lepidoptera: Noctuidae),” Journal of Essential Oil Bearing Plants 23 (2020): 213–229, 10.1080/0972060X.2020.1756423. [DOI] [Google Scholar]
  • 6. Gonçalves R., dos Santos Ayres V. F., Magalhães L. G., et al., “Chemical Composition and Schistosomicidal Activity of Essential Oils of Two Piper Species From the Amazon Region,” Journal of Essential Oil Bearing Plants 22 (2019): 811–820, 10.1080/0972060X.2019.1631720. [DOI] [Google Scholar]
  • 7. Macêdo C. G., Fonseca M. Y. N., Caldeira A. D., et al., “Leishmanicidal Activity of Piper marginatum Jacq. From Santarém‐PA Against Leishmania amazonensis ,” Experimental Parasitology 210 (2020): 107847, 10.1016/j.exppara.2020.107847. [DOI] [PubMed] [Google Scholar]
  • 8. Santana A. D. S., Baldin E. L. L., Lima A. P. S., et al., “New Challenges Demand New Solutions: Selected Essential Oils as an Alternative to Control Bemisia tabaci MED in Brazil,” Crop Protection 155 (2022): 105909, 10.1016/j.cropro.2022.105909. [DOI] [Google Scholar]
  • 9. De Lima C. H. M., Camara C. A. G., Monteiro V. B., et al., “Bioactivity of Piper Aduncum and Piper marginatum Essential Oils on Planococcus Citri (RISSO, 1813) (Hemiptera: Pseudococcidae),” International Journal of Tropical Insect Science 44 (2024): 2539–2548. [Google Scholar]
  • 10. Almeida C. A., Azevedo M. M. B., Chaves F. C. M., et al., “Piper Essential Oils Inhibit Rhizopus Oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity,” Canadian Journal of Infectious Diseases and Medical Microbiology 2018 (2018): 1–7, 10.1155/2018/5295619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Majolo C., Monteiro P. C., do Nascimento A. V. P., et al., “Essential Oils From Five Brazilian Piper Species as Antimicrobials Against Strains of Aeromonas Hydrophila,” Journal of Essential Oil Bearing Plants 22 (2019): 746–761, 10.1080/0972060X.2019.1645047. [DOI] [Google Scholar]
  • 12. Jaramillo‐Colorado B. E., Arroyo‐Salgado B., and Palacio‐Herrera F. M., “Antifungal Activity of Four Piper Genus Essential Oils Against Postharvest Fungal Fusarium spp. Isolated From Mangifera Indica L. and Persea americana Mill. Fruits,” Industrial Crops and Products 223 (2025): 120170, 10.1016/j.indcrop.2024.120170. [DOI] [Google Scholar]
  • 13. Pandey A. K., Kumar P., Singh P., Tripathi N. N., and Bajpai V. K., “Essential Oils: Sources of Antimicrobials and Food Preservatives,” Frontiers in Microbiology 7 (2017): 2161, 10.3389/fmicb.2016.02161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Nurjanah S., Suryadi E., Thoriq A., et al., “Essential Oils From Different Parts of Piper nigrum L.: Chemical Composition, Antibacterial, and Antioxidant Activities,” Food Science Nutrition 13 (2025): e71205, 10.1002/fsn3.71205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Ballou M. A., Davis E. M., and Kasl B. A., “Nutraceuticals: An Alternative Strategy for the Use of Antimicrobials,” Veterinary Clinics of North America—Food Animal Practice 35 (2019): 507–534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Chatha S. A. S., Asgher M., Asgher R., et al., “RETRACTED: Environmentally Responsive and Anti‐Bugs Textile Finishes—Recent Trends, Challenges, and Future Perspectives,” Science of the Total Environment 690 (2019): 667–682, 10.1016/j.scitotenv.2019.06.520. [DOI] [PubMed] [Google Scholar]
  • 17. Raveau R., Fontaine J., and Lounès‐Hadj Sahraoui A., “Essential Oils as Potential Alternative Biocontrol Products Against Plant Pathogens and Weeds: A Review,” Foods 9 (2020): 365, 10.3390/foods9030365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. da Silva J. K., da Trindade R., Alves N. S., Figueiredo P. L., Maia J. G. S., and Setzer W. N., “Essential Oils From Neotropical Piper Species and Their Biological Activities,” International Journal of Molecular Sciences 18 (2017): 2571, 10.3390/IJMS18122571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Figueiredo A. C., Barroso J. G., Pedro L. G., and Scheffer J. J. C., “Factors Affecting Secondary Metabolite Production in Plants: Volatile Components and Essential Oils,” Flavour and Fragrance Journal 23 (2008): 213–226, 10.1002/ffj.1875. [DOI] [Google Scholar]
  • 20. Raut J. S. and Karuppayil S. M., “A Status Review on the Medicinal Properties of Essential Oils,” Industrial Crops and Products 62 (2014): 250–264, 10.1016/j.indcrop.2014.05.055. [DOI] [Google Scholar]
  • 21. Andrade E. H. A., Carreira L. M. M., da Silva M. H. L., et al., “Variability in Essential‐Oil Composition of Piper marginatum Sensu Lato,” Chemistry & Biodiversity 5 (2008): 197–208, 10.1002/cbdv.200890011. [DOI] [PubMed] [Google Scholar]
  • 22. Souto R. N. P., Harada A. Y., Andrade E. H. A., and Maia J. G. S., “Insecticidal Activity of Piper Essential Oils from the Amazon Against the Fire Ant Solenopsis Saevissima (Smith) (Hymenoptera: Formicidae),” Neotropical Entomology 41 (2012): 510–517, 10.1007/s13744-012-0080-6. [DOI] [PubMed] [Google Scholar]
  • 23. Autran E. S., Neves I. A., da Silva C. S. B., Santos G. K. N., da Câmara C. A. G., and Navarro D. M. A. F., “Chemical Composition, Oviposition Deterrent and Larvicidal Activities Against Aedes aegypti of Essential Oils From Piper marginatum Jacq. (Piperaceae),” Bioresource Technology 100 (2009): 2284–2288, 10.1016/j.biortech.2008.10.055. [DOI] [PubMed] [Google Scholar]
  • 24. De Oliveira Chaves M. C., De B. V., and Santos O., “Constituents From Piper marginatum Fruits,” Fitoterapia 73 (2002): 547–549, 10.1016/S0367-326X(02)00167-3. [DOI] [PubMed] [Google Scholar]
  • 25. Santos B. V. D. O., Da‐Cunha E. V. L., Chaves M. C. O., and Gray A. I., “Phenylalkanoids From Piper marginatum ,” Phytochemistry 49 (1998): 1381–1384, 10.1016/S0031-9422(98)00100-9. [DOI] [Google Scholar]
  • 26. Masuda T., Inazumi A., Yamada Y., Padolina W. G., Kikuzaki H., and Nakatani N., “Antimicrobial Phenylpropanoids From Piper Sarmentosum,” Phytochemistry 30 (1991): 3227–3228, 10.1016/0031-9422(91)83180-S. [DOI] [Google Scholar]
  • 27. Cruz S. M., Cáceres A., Álvarez L. E., Apel M. A., and Henriques A. T., “Chemical Diversity of Essential Oils of 15 Piper Species From Guatemala,” Acta Horticulturae 964 (2012): 39–46, 10.17660/ActaHortic.2012.964.4. [DOI] [Google Scholar]
  • 28. Gasparetto A., Bella Cruz A., Wagner T. M., Bonomini T. J., Correa R., and Malheiros A., “Seasonal Variation in the Chemical Composition, Antimicrobial and Mutagenic Potential of Essential Oils From Piper Cernuum ,” Industrial Crops and Products 95 (2017): 256–263, 10.1016/j.indcrop.2016.10.030. [DOI] [Google Scholar]
  • 29. Maduro C. E. P., de Camargo R. G., Hendges A. P. P. K., et al., “Essential Oils From Piper Caldense C. DC. and Piper xylosteoides (Kunth) Steud.: Seasonal Variation of the Chemical Composition and Antimicrobial Activity,” Chemistry Biodiversity 18 (2021): e2100495, 10.1002/cbdv.202100495. [DOI] [PubMed] [Google Scholar]
  • 30. Marques A. M. and Coelho Kaplan M. A., “Seasonal Evaluation and Chemical Composition of Volatile Fractions From Piper Claussenianum by Hydrodistillation and SPME,” Journal of Essential Oil Research 23 (2011): 15–19, 10.1080/10412905.2011.9700475. [DOI] [Google Scholar]
  • 31. Alves C. M. G., Nogueira J. N., Luz J. G. R., Chaves F. C. M., and Tavares‐Dias M., “Essential Oil of Piper Callosum, Piper Hispidum and Piper marginatum (Piperaceae) Possesses In Vitro Efficacy Against Monogeneans of Colossoma Macropomum (tambaqui),” Aquaculture Research 52 (2021): 6107–6116, 10.1111/are.15473. [DOI] [Google Scholar]
  • 32. Ayres V. F. S., Oliveira M. R., Baldin E. L. L., Corrêa G. M., Guimarães A. C., and Takeara R., “Chemical Composition and Insecticidal Activity of the Essential Oils of Piper marginatum, Piper Callosum and Vitex Agnus‐castus ,” Anais da Academia Brasileira de Ciências 93 (2021): e20200616, 10.1590/0001-3765202120200616. [DOI] [PubMed] [Google Scholar]
  • 33. Ninkuu V., Aluko O. O., Yan J., et al., “Phenylpropanoids Metabolism: Recent Insight into Stress Tolerance and Plant Development Cues,” Frontiers in Plant Science 16 (2025): 1571825, 10.3389/fpls.2025.1571825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Liu Y., Fernie A. R., and Tohge T., “Diversification of Chemical Structures of Methoxylated Flavonoids and Genes Encoding Flavonoid‐O‐Methyltransferases,” Plants 11 (2022): 564, 10.3390/plants11040564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Duarte M. C. T., Figueira G. M., Sartoratto A., Rehder V. L. G., and Delarmelina C., “Anti‐Candida Activity of Brazilian Medicinal Plants,” Journal of Ethnopharmacology 97 (2005): 305–311, 10.1016/j.jep.2004.11.016. [DOI] [PubMed] [Google Scholar]
  • 36. Pauli A. and Kubeczka K.‐H., “Antimicrobial Properties of Volatile Phenylpropanes,” Natural Products Communications 5 (2010): 1387–1394. [PubMed] [Google Scholar]
  • 37. Ben Hsouna A., Ben Halima N., Abdelkafi S., and Hamdi N., “Essential Oil From Artemisia Phaeolepis: Chemical Composition and Antimicrobial Activities,” Journal of Oleo Science 62 (2013): 973–980. [DOI] [PubMed] [Google Scholar]
  • 38. Bougatsos C., Ngassapa O., Runyoro D. K. B., and Chinou I. B., “Chemical Composition and In Vitro Antimicrobial Activity of the Essential Oils of Two Helichrysum Species From Tanzania,” Zeitschrift für Naturforschung C 59 (2004): 368–372, 10.1515/znc-2004-5-614. [DOI] [PubMed] [Google Scholar]
  • 39. Rahman A., Sultana Shanta Z., Rashid M. A., et al., “In Vitro Antibacterial Properties of Essential Oil and Organic Extracts of Premna Integrifolia Linn,” Arabian Journal of Chemistry 9 (2016): S475–S479, 10.1016/j.arabjc.2011.06.003. [DOI] [Google Scholar]
  • 40. Reigada J. B., Tcacenco C. M., Andrade L. H., Kato M. J., Porto A. L. M., and Lago J. H. G., “Chemical Constituents From Piper marginatum Jacq. (Piperaceae)—Antifungal Activities and Kinetic Resolution of (RS)‐Marginatumol by Candida antarctica Lipase (Novozym 435),” Tetrahedron: Asymmetry 18 (2007): 1054–1058, 10.1016/j.tetasy.2007.05.006. [DOI] [Google Scholar]
  • 41. Valente V. M. M., Jham G. N., Jardim C. M., Dhingra O. D., and Ghiviriga I., “Major Antifungals in Nutmeg Essential Oil Against Aspergillus Flavus and A. ochraceus,” Journal of Food Research 4 (2014): 51, 10.5539/jfr.v4n1p51. [DOI] [Google Scholar]
  • 42. Duarte M. C. T., Leme E. E., Delarmelina C., Soares A. A., Figueira G. M., and Sartoratto A., “Activity of Essential Oils From Brazilian Medicinal Plants on Escherichia coli ,” Journal of Ethnopharmacology 111 (2007): 197–201, 10.1016/j.jep.2006.11.034. [DOI] [PubMed] [Google Scholar]
  • 43. Alves Borges Leal A. L., Fonseca Bezerra C., Esmeraldo Rocha J., et al., “ Piper cernuum Vell.: Chemical Profile and Antimicrobial Potential Evaluation,” Industrial Crops and Products 140 (2019): 111577, 10.1016/j.indcrop.2019.111577. [DOI] [Google Scholar]
  • 44. Pereira Carneiro J. N., da Cruz R. P., da Silva J. C. P., et al., “ Piper Diospyrifolium Kunth.: Chemical Analysis and Antimicrobial (Intrinsic and Combined) Activities,” Microbial Pathogenesis 136 (2019): 103700, 10.1016/j.micpath.2019.103700. [DOI] [PubMed] [Google Scholar]
  • 45. Amrutha B., Sundar K., and Shetty P. H., “Spice Oil Nanoemulsions: Potential Natural Inhibitors Against Pathogenic E. coli and Salmonella spp. From Fresh Fruits and Vegetables,” LWT—Food Science and Technology 79 (2017): 152–159, 10.1016/j.lwt.2017.01.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Basak S. and Guha P., “Use of Predictive Model to Describe Sporicidal and Cell Viability Efficacy of Betel Leaf (Piper betle L.) Essential Oil on Aspergillus Flavus and Penicillium Expansum and Its Antifungal Activity in Raw Apple Juice,” LWT 80 (2017): 510–516, 10.1016/j.lwt.2017.03.024. [DOI] [Google Scholar]
  • 47. de Pascoli I. C., dos Anjos M. M., da Silva A. A., et al., “Piperaceae Extracts for Controlling Alicyclobacillus Acidoterrestris Growth in Commercial Orange Juice,” Industrial Crops and Products 116 (2018): 224–230. [Google Scholar]
  • 48. National Institute of Standards and Technology OR National Bureau of Standards , U.S. Department of Commerce: Gaithersburg, MD. Available at: 10.18434/T4D303. [DOI]
  • 49. Adams R. P., Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, 4th edition (Allured Publishing: IL, 2007). [Google Scholar]
  • 50. van Den Dool H. and Kratz P. D., “A Generalization of the Retention Index System Including Linear Temperature Programmed Gas‐Liquid Partition Chromatography,” Journal of Chromatography A 11 (1963): 463–471. [DOI] [PubMed] [Google Scholar]
  • 51. Clinical and Laboratory Standards Institute (CLSI) , Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard–9th ed. (CLSI Document M07‐A9). (Wayne, PA: Clinical and Laboratory Standards Institute, 2012). [Google Scholar]
  • 52. Clinical and Laboratory Standards Institute (CLSI) , Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI Standard M27 (Wayne, PA: Clinical and Laboratory Standards Institute, 2017). [Google Scholar]
  • 53. Vanegas D., Abril‐Novillo A., Khachatryan A., et al., “Validation of a Method of Broth Microdilution for the Determination of Antibacterial Activity of Essential Oils,” BMC Research Notes 14 (2021): 439, 10.1186/s13104-021-05838-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supporting File 1: cbdv71771‐sup‐0001‐SuppMat.docx.

CBDV-23-e71771-s001.docx (2.2MB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supporting Information files.

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