H. crenata was collected in Vila de Chiquita (rural area, Figure 1) in the city of Salvaterra, Marajó, Pará state, Brazil (Lat. 0°51′43.71″S, Long. 48°37′23.33″W), in accordance with biodiversity protection laws, and the registration of access to genetic heritage under number AEC4B1F (SISGEN).

For the seasonal study, 12 monthly collections were carried out, with approximately 150 g of botanical material, on the first day of each month, starting in September 2021 and ending in August 2022, at 3:00 p.m.

The botanical identification was made by morphological comparison with authentic samples, and a voucher was incorporated into the collection of the Herbario of Museu Paraense Emílio Goeldi in Belém, Pará, under registration number MG-243648.

Climatic data such as atmospheric humidity, rainfall, average temperature, and solar radiation were collected on the website of the National Institute of Meteorology (INMET) of the Brazilian Government. The seasonal parameters were recorded through the automatic station located in Belém, State of Pará, Brazil, which is approximately 78 km as the straight line from the collection site.

The extraction of leaves essential oil from a single specimen of H. crenata was carried out using the hydrodistillation technique with a Clevenger-type device over a 3-h period, in triplicate. In this process, 50 g of dried and crushed leaves were added to a 2000-mL glass flask with 100 mL of distilled water. This system was coupled to the condenser, which was cooled at 10°C.

The extracted oils were centrifugated at 3,000 rpm for 5 min to allow total water separation and further dehydrated with anhydrous sodium sulfate (Na₂SO₄) under the same conditions. After this dehydration, the masses of the oils were determined using an analytical balance with an accuracy of 0.0001 g, stored in amber glass vials, and kept refrigerated at 5°C (Jerônimo et al., 2024). The essential oil yields were calculated from the moisture-free biomass, relating the plant mass, oil, and residual moisture, according to the Equation below. % EO = obtained   oil   volume   g plant   material   mass   g ‐ plat   material   mass   g   x   humidity   % 100   x   100 %

The residual moisture content of the leaves was calculated by water loss in an oven at 110°C until the material reached a constant weight.

The obtained essential oils were diluted in n-hexane in a ratio of 2 μL of oil to 500 μL of solvent and analyzed simultaneously in these two systems: gas chromatography with a flame ionization detector (GC-FID, Shimadzu Corporation, Tokyo, Japan) and gas chromatography with a mass spectrometer (GC-MS, Shimadzu Corporation, Tokyo, Japan) as stabilized protocol (Jerônimo et al., 2024). The system was equipped with an auto-injector: AOC-20i, and an Rtx-5MS silica capillary column (30 m; 0.25 mm; 0.25 μm film thickness) under the following operating conditions: temperature program: 60°C–240°C (3°C/min); injector temperature: 250°C; carrier gas: helium (1 mL/min); injection: split type 1:20 (solution of 5 μL of essential oil: 500 μL of hexane); mass spectra: were obtained by electronic ionization at 70 eV; ion source temperature: 200°C.

To determine the chemical composition, the retention times of each peak (constituents) were converted in retention indices using a homologous series of C₈–C₄₀ n-alkanes (Sigma-Aldrich, Milwaukee, WI, United States) according to the linear method of van Den Dool and Kratz (Van Den Dool and Kratz, 1963). Each mass spectrum and retention index were compared with Adams and FFNSC-2 libraries (Adams, 2007; Mondello, 2011). The Relative amounts of individual components were calculated by peak area normalization using the flame ionization detector (GC-FID).

Principal component analysis (PCA) was applied to the essential oil components of H. crenata leaves (>1.5%) (OriginPro Learning Edition, OriginLab Corporation, Northampton, MA, United States). Hierarchical cluster analysis (HCA) was performed considering the unique distance and Ward linkage. Statistical significance was assessed using the Tukey test (p < 0.05) and Pearson correlation coefficients (r) were calculated to determine the relationship between the analyzed climatic parameters (sunlight, relative humidity, temperature, and precipitation), using the GraphPad Prism software, version 8.0.

The climatic parameters: temperature, solar radiation, precipitation, and relative humidity were monitored over the 12 months (September/2021 to August/2022) to evaluate the influence of seasonality on the yield and composition of H. crenata leaves essential oil. During the study periods, insolation values ranged from 106.4 h (March) to 253.4 h (August), monthly precipitation from 103.9 mm (August) to 527.4 mm (March), temperature from 25.9°C (January) to 27.6°C (October) and relative humidity from 79.7% (August) to 93.0% (April).

According to precipitation data, the dry period in the region comprised the months of September to February and June to August, with an average precipitation of 215.58 ± 76.26 mm, and the rainy period from March to May, with an average precipitation of 472.53 mm ± 60.22 mm (Figure 2).

In the seasonal study, H. crenata leaves essential oil yields varied from 1.6% (September) to 2.8% (January), averaging 2.05% ± 0.39% during the studied year. Statistical Tukey test showed no significant difference in essential oil production during the dry (1.97% ± 0.2%) and rainy (2.07% ± 0.44%) seasons (Figure 3). Furthermore, no significant correlations (p > 0.05) were observed between essential oil yield and precipitation (r = −0.23), temperature (r = −0.12), humidity (r = −0.20), and insolation (r = −0.36) indicating that H. crenata specimen presents the same essential oil production regardless of the climatic conditions. Variations in oil yield may be linked to other abiotic factors, edaphic and/or genetic.

Essential oils (extracted by hydrodistillation) from the dry and fresh aerial part of H. crenata collected in Marajó Island (Brazilian Amazon) presented yields between 0.6% and 0.9%, respectively, in samples from Melgaço and São Sebastião da Boa Vista (Zoghbi et al., 2002); in Salvaterra a yield of 1.4% was described (Rebelo et al., 2009). Other reported samples showed yields of 0.2%, 0.6%, and 1.4% in Tocantins (Zoghbi et al., 2002), Cuiabá (Violante et al., 2012), and Mato Grosso do Sul respectively. None of these specimens showed a higher yield than the present study. However, little information has been found regarding phytochemical studies of this species. Moreover, Hyptis marrubioides showed high variability in the concentration of the essential oil components due to seasonality variability probably related mainly to the rainfall regime (Botrel et al., 2010a).

The chromatographic analysis identified 74 constituents in H. crenata leaf essential oils, representing an average of 97.96% (92.95%–99.16%) of the total chemical composition of the oils analyzed during the 12 months. The constituents are listed below in ascending order of their respective retention indexes (RI) in Table 1.

Oxygenated monoterpenoids (40.82%–53.82%, 46.21% ± 4.49) and monoterpene hydrocarbons (37.22%–47.29%, 43.08% ± 3.5) were the predominant in the essential oil, followed by sesquiterpene hydrocarbons (3.64%–9.26%, 5.44% ± 1.78) and oxygenated sesquiterpenoids (0.73%–3.56%, 1.72% ± 0.73).

The oxygenated monoterpenoid 1,8-cineole (eucalyptol) was the main constituent throughout the study, ranging from 22.48% (July/2022) to 36.90% (October/2021), presenting an annual average of 28.48 ± 4. 32%; followed by α-pinene, whose levels varied from 13.55% (July/2022) to 22.17% (May/2022), with an annual average of 19.58% ± 2.29%; the camphor content varied from 8.86% (November/2021) to 15.90% (July/2022), with an annual average of 11.98% ± 2.54%; β-pinene ranged from 6.35% (August/2022) to 11.20% (April/November/2021), with an annual average of 9.19% ± 1.47%; limonene, despite not occurring in the months of September, October, December (2021), and January (2022), presented an average annual content of 6.12% ± 3.15% with variations of 5.32% (April/2022) at 7.14% (August/2022); α-terpineol presented amounts ranging from 1.89% (May/2022) to 2.77% (November/2021) with an annual average of 2.42% ± 0.25% and borneol concentrations ranging from 1.74% (November/2021, February/2022) to 3.38% (May/2022) with an average concentration of 2.34% ± 0.48%. The chemical structures of these compounds are shown in Figure 4.

The chemical composition of H. crenata essential oil varies depending on the time of year and the location where the leaves are collected (Santos et al., 2023). Several studies report different chemical characteristics among essential oils of the Hyptis genus. A study on H. marrubioides showed that the composition of the essential oil varied significantly due to seasonal factors, with no qualitative variation in the composition of the oil throughout the seasons (Botrel et al., 2010b). Furthermore, it was found that the chemical composition of H. crenata essential oils presents notable variability, with different main constituents identified in the oils obtained from different samples (Lima et al., 2023).

Intraspecific variability in chemical composition was also noted in other Hyptis species, indicating the presence of several chemical compounds. Studies report the existence of different chemical characteristics among the essential oils of H. crenata, which generally has the constituents 1,8-cineole, borneol, camphor, limonene, α- and β-pinene, E-caryophyllene, p-cymene, all of which vary according to the time of year and place of collection (Scramin et al., 2000).

Based on Pearson’s correlation data between the climatic parameters and the H. crenata chemical composition, it was possible to identify only a statistically significant correlation (p < 0.05) between the amounts of β-pinene and sesquiterpene hydrocarbons with the data of precipitation and humidity, the other constituents such as α-pinene, camphene, limonene, 1,8-cineole, and camphor, as well as the classes of hydrocarbon/oxygenated monoterpenes and oxygenated sesquiterpenes showed statistically insignificant correlations.

β-pinene showed a moderate negative correlation between insolation (r = −0.47) and temperature (r = −0.49) but without statistical significance (p > 0.05); however, this constituent showed a moderate correlation positive correlation with precipitation (r = 0.58) and humidity (r = 0.58) with statistical significance (p < 0.05), in the same way the class of sesquiterpene hydrocarbons showed a moderate and negative correlation (r = −0.58) with humidity as displayed in Figure 5.

These data suggest that, despite the four climatic parameters analyzed, the production of the main constituents and classes of terpenoids remains with few qualitative and considerable quantitative variations. However, correlations with β-pinene suggest increasing precipitation and humidity, producing higher monoterpene concentrations in this H. crenata specimen. Likewise, humidity influences the class of sesquiterpene hydrocarbons; that is, the increase in this climatic parameter leads to a decrease in the concentration of this terpene class.

α-Pinene and β-pinene are constituents of several essential oils. They have a broad pharmacological action spectrum, described in several studies, with effects such as anti-inflammatory, antitumor and antimicrobial, antibacterial (Leite et al., 2007; Silva et al., 2012), anxiolytic (Santos et al., 2022), antidepressant, anticonvulsant, hypotensive, myorelaxant, antispasmodic (Nikitina et al., 2009; Silva et al., 2012; Felipe et al., 2019; Salehi et al., 2019). β-Pinene, with a woody aroma, occurs in several plants of the Lamiaceae and naturally plays an indirect role in the defense of the plant, attracting natural enemies (predators and/or parasitoids) that help control the population of herbivores (Kutty and Mishra, 2023).

Furthermore, this H. crenata specimen displayed low acute toxicity and significant anti-inflammatory activity, with peripheral and no central antinociceptive action (de Lima et al., 2023). However, the chemical composition variation due to seasonality may change the pharmacological activity of H. crenata essential oil.

In this way, it is understood that some abiotic or biotic factors that the plant is exposed to favor the significant biosynthesis of α-pinene and, consequently, the decline of camphor since these constituents come from the same formation pathway. In the present study, it was possible to identify that there was no significant correlation between the four climatic parameters analyzed and the α-pinene and camphor amounts.

Furthermore, α-pinene is found in the essential oils of many plants, such as conifers, has a distinct pine aroma, and is known for its various biological properties. In medicinal and/or aromatic plants, α-pinene is involved in various actions, including plant defense mechanisms and repelling insects with its distinct aroma (Kutty and Mishra, 2023).

Furthermore, the chemical composition of essential oils can vary depending on the time and place of harvest. Furthermore, the chemical composition of these oils can undergo biotransformation processes to produce other compounds, such as verbenone, which complements the antibacterial activity of α-pinene (Dewick, 2009).

Using hierarchical cluster analysis (HCA), a dendrogram was obtained showing two groups formed with the essential oils of H. crenata (see Figure 6). Group I comprised all months of study except July (2022), which formed group II.

Principal Component Analysis (PCA, Figure 7) confirmed the formation of two distinct groups and elucidated 68.01% of the data variability. PC1 explained 27.62% of the data and showed negative correlations with α-pinene (r = −0.96), camphene (r = −1.12), myrcene (r = −0.10), p-cymene (r = −1.25), 1,8-cineole (r = −1.49), borneol (r = −0.03), α-terpineol (r = −1.33), and α-longipinene (r = −0.89). The second component (PC2) explained 23.39% of the data and showed negative correlations α-pinene (r = −2.01), β-pinene (r = −0.92), limonene (r = −1.00), 1,8-cineole (r = −0.36), and γ-terpinene (r = −0.33). The third component (PC3) explained 17.00% of the data, presenting positive correlations with α-pinene (r = 0.39), camphene (r = 0.73), β-pinene (r = 1.27), p-cymene (r = 0.88), 1,8-cineole (r = 1.94), γ-terpinene (r = 0.43), terpinolene (r = 0.32), camphor (r = 0.69), borneol (r = 1.48), and caryophyllene oxide (r = 1.03).

Group I is represented by oils from all months except July, this group was characterized by the chemical type of 1,8-cineole (24.15%–36.90%), α-pinene (17.14%–22.17%), α-terpineol (1.89%–2.77%), α-longipinene (0.70%–1.99%), limonene (5.32%–7.14%), E-caryophyllene (1.25%–3.04%), myrcene (1.43%–1.79%), camphene (2.65%–3.79%), caryophyllene oxide (0.46%–0.91%), β-pinene (6.35%–11.20%), and borneol (1.74%–3.38%). However, group II was characterized by the highest camphor levels (15.90%); that is, July was the only month that formed a distinct group, marked by the high occurrence of camphor compared to the other months. Moreover, in July H. crenata presented the lowest content of α-pinene (13.55%).