Exploring the impact of seasonal variations on the chemical composition, antinociceptive, and anti-inflammatory properties of Pogostemon heyneanus Benth. essential oil

Background: Pogostemon heyneanus leaves infusions are relevant in ethnopharmacology for treating colds, coughs, headaches, and asthma. Purpose: The essential oil chemical composition of a Pogostemon heyneanus specimen was monthly monitored from October 2021 to July 2022 to evaluate the climatic influences on its yield and chemical composition and antinociceptive, andanti-inflammatory properties. Methods: The leaves, collected monthly over a 10-month period, were submitted to hydrodistillation. The oils obtained were analyzed by gas chromatography coupled to a mass spectrometer and gas chromatography coupled to flame ionization detector. The P. heyneanus essential oil (PhEO) was tested in vivo to evaluate its peripheral analgesic actions through the abdominal writhing test induced by acetic acid, and peripheral analgesia by tail immersion. Neurogenic and inflammatory pain were evaluated by formalin test, and acute oral toxicity of the oil was also verified. Results: PhEO presented 27 chemical constituents with the highest predominance of patchoulol (43.6%–76.9%), α-bulnesene (0.2%–12.7%), α-guaiene (0.4%–8.9%), seychellene (3.8%–5.1%) and pogostol (0.0%–8.2%). The climatic parameters insolation, humidity, rainfall, and temperature did not influence the essential oil yield or the main chemical constituents, except for pogostol, which presented a strong (r = 0.73) and statistically significant (p < 0.05) correlation with temperature. PhEO did not display toxicity at the maximum 300 mg/kg dosage. The oil showed low peripheral and central analgesic action at 100 mg/kg, while in the neurogenic and inflammatory pain inhibition tests, no actions related to PhEO were observed. In the carrageenan-induced peritonitis test, PhEO did not reduce the migration of leukocytes to the peritoneal cavity compared to the control group. Conclusion: Pogostemon heyneanus is a resistant plant to seasonal influences and a source of patchoulol. Despite ethnopharmacological indications, no in-vivo biological activities such as neurogenic or inflammatory pain were identified in the present work. So, the low influence of the climatic parameters on chemical composition can infer that the low pharmacological activity is also not subject to climatic variations, that is, it does not change due to the climate.


Introduction
Among the species in the Amazon, Pogostemon heyneanus Benth.(Lamiaceae), commonly known as oriza, patchouli, or pathuli, is an herbaceous species native to Southeast Asia, widely distributed in India, Malaysia, and Indonesia (Lorence et al., 2020).
There are only a few studies on the chemical constituents of P. heyneanus essential oils.Maia and Andrade (2009) demonstrated that P. heyneanus essential oil from the Amazon had patchoulol (patchouli alcohol), α-guaiene, aromadendrene, and α-bulnesene as its main components.Another phytochemical study carried out with the volatile oil of this species, obtained in Italy, described the occurrence of α-bulnesene, seychelene, α-guaiene, and patchoulol (Souza Filho et al., 2009).
Moreover, many studies reported that the chemical composition and essential oils yield depend on age, circadian rhythm, soil, temperature conditions, plant organ, and extraction method, among others (Benomari et al., 2023).Therefore, studies are necessary to analyze the influence of these factors on the chemical variability of essential oils, knowledge of which is essential for applying essential oils for phytotherapeutic purposes.
The research for essential oils anti-inflammatory activities has increased during the last years (Miguel, 2010).Moreover, many essential oils contain active anti-inflammatory compounds, which mediate anti-inflammatory effects via different mechanisms.Thus, the treatment with essential oil can decrease the expression of proinflammatory cytokines and mediators, neutralize excess reactive oxygen species, alleviate tissue edema, and accelerate wound healing due to infection (Zhao et al., 2023).
Compared to conventional anti-inflammatory drugs and small molecules (which target a specific pathway and their known side effects), complex essential oil compositions can safely target multiple signaling pathways associated with inflammation.However, only a few clinical trials have been conducted to evaluate the antiinflammatory effect of PEOs (Zhao et al., 2023).
Although P. heyneanus essential oil has in-vitro biological potential, few in-vivo studies are available in the literature on the chemical composition and analgesic and anti-inflammatory actions, such as the toxic activity of its essential oils.Therefore, this work aimed to evaluate the influence of seasonality on the chemical composition of P. heyneanus essential oil, its acute oral toxicity, and its antinociceptive and anti-inflammatory.The information gained from this study could have an impact as to when this medicinal plant is collected and the implications for treatment protocols and expectations.

Plant material and essential oil extraction
The P. heyneanus Benth.Leaves were collected in Mosqueiro Island, city of Belém, Pará State (Latitude 1 °5′58.095″S;longitude 48 °24′1.530″W), the single specimen samples were obtained following Brazilian laws regarding the protection of biodiversity (SISGEN AED714B).For the seasonal study, fresh leaves (30 g) were collected on the second day of each month, at 10 a.m. from October 2021 to July 2022.After identification, a voucher (MG246329) was deposited in the Herbarium "João Murça Pires", from the Museu Paraense Emílio Goeldi.
Leaves of P. heyneanus were allowed to dry for 7 days at room temperature.Hydrodistillation of the dry leaves was carried out using a glass Clevenger apparatus for 3 hours in triplicate; the condenser was maintained between 10 °C and 15 °C with a recirculating chiller.The essential oil was dehydrated by centrifugation (5 min, 3,000 rpm) with anhydrous Na 2 SO 4 and repeated centrifugation.The percent yield of essential oil expressed in percent (v/w) and calculated from humidity-free biomass through the relationship between oil volume, mass plant sample, and humidity calculated as follows 100% × volume essential oil/mass dry leaves (Barros et al., 2022).

Climatic data
The climatic parameters of insolation, precipitation, relative humidity, and average temperature were obtained monthly in parallel to the collection periods on the website of the National Institute of Meteorology (http://www.inmet.gov.br/portal/,accessed on 24 April 2023) from the Brazilian Government (INMET, 2023).Meteorological data were recorded using the automatic station A-201, located in Belém, Pará state, Brazil, equipped with a Vaisala system, model MAWS 301 (Vaisala Corporation, Helsinki, Finland).

GC and GC-MS analyses
Gas chromatography coupled to mass spectrometer (GC-MS) and gas chromatography with flame ionization detector (GC-FID) were used to analyze the composition of P. heyneanus essential oils.A Shimadzu Model QP 2010 ultra instrument (Shimadzu, Tokyo, Japan) equipped with Rtx-5MS fused silica capillary column (30 m, 0.25 mm; 0.25 μm film thickness) as stationary phase was used (Restek, Bellefonte, PA, United States).Helium gas was adjusted to 1.0 mL/min at 57.5 kPa as carrier gas.The injection of diluted essential oils in hexane (1µL, 5 μL of each oil: 500 μL of n-hexane) was injected in the split mode (1:20).The injector and interface temperatures were 250 °C; The programmed oven temperature was 60 °C-240 °C (3 °C/min), followed by a 10 min isotherm.Electron ionization mass spectrometry (EIMS) at 70 eV, the ion source temperature was 200 °C.
The mass spectra were obtained using automatic scanning, with fragment mass in the 35-400 m/z range.The comparison of mass spectra and retention indices presented by the samples was made using the similarities between the mass spectra and retention indices presented in the commercial libraries FFNSC-2 (Mondello, 2011) and Adams (2007).The retention indices of volatile constituents were calculated using the linear equation of Van Den Dool and Kratz (1963), using a homologous series of hydrocarbons (C 8 -C 40 , Sigma-Aldrich, St. Louis, MO, United States) under the same chromatographic conditions.GC-FID analysis was performed for each essential oil sample on a Shimadzu QP-2010 instrument (Shimadzu, Tokyo, Japan) equipped with an FID detector under the same conditions described above, except hydrogen was used as the carrier gas.The percentage composition of the oil sample was calculated from the GC-FID peak areas.Analyses were carried out in triplicate (i.e., three different essential oil samples injected separately).

Animals and ethical statement
Adult male mice (Mus musculus, 30-40 g, Swiss) were obtained from the Evandro Chagas Institute and retained in the "Luiz Carlos de Lima Silveira" animal facility of the Universidade do Estado do Pará (UEPA).The mice were maintained under standard conditions of 12-h light/dark cycle, temperature 22 °C-25 °C, humidity 65%, food and water available ad libitum.The research was approved by the UEPA Animal Use Ethics Committee (CEUA) under protocol 04/2023.

Acute oral toxicity
Acute oral toxicity was assessed according to Organization for Economic Co-operation and Development (OECD) guidelines 423 (OECD, 2002).The animals were randomly distributed into two groups.The first group was treated with distilled water, and the second group was treated with P. heyneanus essential oil (PhEO) at 300 mg/kg.Treatments were carried out by gavage (10 mL/kg).After the treatments, the animals were observed for 2 h and then every 24 h for 14 consecutive days.During this period, behavioral and physiological changes were observed (alertness, spontaneous motor activity, locomotion, apathy, response to touch, stereotypy, aggression, ataxia, sweating, urination, diarrhea, convulsions, and death).On the 15th day, the animals were subjected to induced euthanasia (ketamine 300 mg/kg and xylazine 30 mg/kg) (Souzados et al., 2021).

Analysis of analgesic activity 2.6.1 Peripheral analgesic effect in the abdominal contortion test
The acetic acid-induced abdominal contortion assay was used to assess the peripheral analgesic potential (Koster, 1959;Conegundes et al., 2021).The mice were randomly divided into five groups of five mice: Control group (treated with 0.9% saline solution, p.o.), three treatment groups (PhEO doses of 50, 100, and 300 mg/kg, p. o.), and a positive control group (5 mg/kg indomethacin, p.o.).Sixty minutes after treatment, the mice were subjected to 10 mL/kg of 0.6% acetic acid i.p.After administration of the acetic acid, the animals were confined for 30 min and the abdominal contortions and hind paw extensions were recorded.The analgesic effect was assessed based on the reduction in the number of abdominal contortions.

Tail immersion test
The tail immersion test evaluates the central analgesic activity by submerging the animals' tail in high temperatures.This test evaluates the latency time (s), defined as the period for the animal to remove its tail.Before the start of the test, the animals were subjected to a pre-test, and those with latency values greater than 5 s were excluded.The selected animals were separated into groups (n = 6) that were treated with vehicle (1% Tween 80 diluted in sterile saline solution, p. o.), morphine administered at 5 mg/kg (i.p.) or PhEO at doses of 100 and 300 mg/kg (p.o.).Subsequently, ⅓ (approximately 2 cm) of the animals' tails were submerged in the liquid with a fixed temperature of 55oC ± 0.5 °C, and the complete tail withdrawal response was evaluated at times 0, 30, 60, 90, and 120 min (Hamura et al., 2000;Gammoh et al., 2023).

Formalin test
The mice were separated into four groups: Control group (1% Tween 80 in distilled water, 10 mL/kg, p.o.), treatment groups (PhEO at doses of 100, and 300 mg/kg, p.o.), and positive control (morphine, 4 mg/kg, i.p.).After 1 h, the animals received 20 µL of formalin (1.0%) in the right hind paw (subplantar area) and were then placed individually in plastic containers.The time (seconds) during which the animal licked the injected paw was recorded during the first phase (0-5 min), attributed to the neurogenic phase, and the second phase (15-30 min) was characterized as inflammatory pain (Hunskaar and Hole, 1987;Gomes et al., 2007;Liu et al., 2021).

Analysis of anti-inflammatory activity 2.7.1 Carrageenan-induced peritonitis test
The carrageenan-induced peritonitis assay was used to assess cell migration during the inflammatory process (Souza and Ferreira, 1985).The mice were divided into five groups of six mice: Control group (no treatment), saline group (0.9% saline solution, i.p.), treatment groups (PhEO at doses of 100 and 3,000 mg/kg, i.p.), and positive control group (dexamethasone 1 mg/kg, i.p.).After 1 h, the control group received saline and the other groups received carrageenan (300 μg/mL in sterile saline), i.p.Four hours after injection, the mice were euthanized and their peritoneal cavities were rinsed with 3 mL of ice-cold phosphate-buffered saline (PBS) containing ethylenediaminetetraacetic acid (EDTA, 3 μM).Total leukocytes in the peritoneal fluid were determined by pipetting 20 µL of peritoneal fluid into 400 µL of Turk's solution.An aliquot was read in a Neubauer chamber (Kasvi, Brazil) using a Nikon Eclipse E100 binocular microscope (Nikon Europe BV).

Statistical analysis of chemical composition and in vivo test
Statistical analysis was evaluated using the Tukey test (p < 0.05), and Pearson correlation coefficients (r) were calculated to determine the relationship between the analyzed parameters such as insolation, precipitation, relative air humidity, and average temperature), using the GraphPad Prism software method, version 8.0.Principal component analysis (PCA) was applied to check the interrelationship in essential oil components (>1.0%) (OriginPro trial version, OriginLab Corporation, Northampton, MA, United States).Hierarchical cluster analysis (HCA), considering Euclidean distance and Ward linkage, was used to check the similarity of oil samples based on the distribution of constituents selected in the previous PCA analysis (da Cruz et al., 2022).
Statistical evaluations of the in-vivo experimental data were carried out using GraphPad Prism version 5.0.The data are expressed as means ± standard errors.Statistically relevant differences were determined using the ANOVA test, followed by Tukey's post hoc test.Differences were deemed to be statistically significant between treatment groups when p < 0.05 (de Jesus et al., 2023).

Seasonal effects on oil yield
Pogostemon heyneanus essential oil yields varied from 3.8% (April) to 10.5% (December), with an average of 6.9% ± 2.4% during the studied period (Figure 1).Essential oil production did not show a significant difference (p < 0.05) during the period of lowest rainfall intensity (7.1% ± 2.2%) and highest intensity (6.6% ± 3.2%) in the Tukey test.Regarding climatic parameters with essential oil content, no significant correlation was observed with climatic parameters (temperature, humidity, insolation, and precipitation) (p > 0.05).

FIGURE 1
Relationship between climatic parameters and oil yield of Pogostemon heyneanus during the seasonal study.
The constituents patchoulol, α-bulnesene, α-guaiene and seychellene and their respective classes did not significantly correlate with seasonal parameters.However, the pogostol constituent showed a negative and statistically significant correlation (p < 0.05) with temperature (r = −0.73),and the monoterpene hydrocarbons class showed a negative very strong correlation with temperature (r = −0.92),strong correlation with insolation (r = −0.62)and weak correlation with precipitation (r = −0.47),as shown in Table 2 below.Frontiers in Pharmacology frontiersin.org06

Multivariate analysis of Pogostemon heyneanus
The chemical variability of oil samples of P. heyneanus was evaluated by multivariate statistical analyses (HCA, hierarchical cluster analysis; PCA, principal component analysis) using constituents with levels greater than 1.0%.
Applying hierarchical cluster analysis (HCA) provided the dendrogram presented in Figure 3; the analyzed volatiles' compositions were included in three groups.Group I is comprised of oils from leaves collected in February and June.Group II includes oil samples from October, November, and December; Group III includes samples from January, March, April, May, and July.
Applying additional multivariate analysis, in the heat map analysis combined with the hierarchical cluster analysis (Figure 5), with the classes of compounds, the color pattern that varied with the intensity of the color increased, indicating from the lowest to the highest degree.The clustered heatmap confirmed the clustering results obtained in PCA and HCA.However, correlations were observed between climatic parameters and oil constituents and their compound classes, as mentioned previously (see Table 2).

Acute oral toxicity
After 14 days of analysis, no signs of mortality were observed, and there was little change in the behavioral patterns of the animals treated with the 300 mg/kg of PhEO.Furthermore, there were no observed differences in the relative organ masses between the PhEO treatment groups and the control group (data not shown).
There were no differences in biochemical parameters related to renal function (creatinine and urea) between the group treated with PhEO and the control.The biochemical parameters for evaluating liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP)), only AST and ALP, were statistically significant compared to control (Table 3).However, these differences are within the reference values for the species used in the study (Silva-Santana et al., 2020).

Peripheral analgesic effect of PhEO in the acetic acid-induced abdominal writhing test
In the current report, it was possible to verify, in Figure 6, a 41% contortions reduction in the group that received the dose of 100 mg/kg of PhEO compared to the control group (p < 0.01).No reduction in writhing was observed in the other groups treated with PhEO (50 and 300 mg/kg).Therefore, in the other antinociception tests, only 100, and 300 mg/kg doses were adopted.In animals treated with the standard drug (indomethacin 5 mg/kg), it was observed that the percentage of writhing inhibition was 77% (p < 0.01).

Effects of PhEO on the formalin test
The results of the formalin test, both the first phase (neurogenic pain) and the second phase (inflammatory pain), are shown in Figure 8.Neither the PhEO dose of 100 mg/kg nor the dose of 300 mg/kg Principal components Analysis of main components in Pogostemon heyneanus essential oil.
Frontiers in Pharmacology frontiersin.org08 significantly decreased the licking time compared to the control group (p > 0.05).However, morphine, at 4 mg/kg, did significantly decrease licking time in both the first phase (81.67%, p < 0.01) and the second phase (92.46%, p < 0.01) compared to the control group.

Discussion
The Brazilian Amazon presents a precipitation regime modulated by dynamic atmospheric systems on micro, meso, and  large scales.The Amazon region is characterized by only two seasons: dry and rainy.With the humid and hot climate, the Amazon has the highest rainfall from December to April, the rainy season, with the lowest rainfall from June to November in the dry season, and the other months are considered periods of transition between the seasons (Loureiro et al., 2014;da Costa et al., 2020).In a study that evaluated the effect of seasonality and essential oil, the climatic parameters obtained in 2022 showed an atypical Analgesic effect of Pogostemon heyneanus essential oil (PhEO, at 50, 100 and 300 mg/kg) in the abdominal writhing test induced by acetic acid (0.6% in saline, v/v) in mice.Each column represents the mean ± SEM (standard error of the mean) of 6 animals.**p < 0.01 was considered statistically significant compared to the control (ANOVA, followed by Tukey's post-test).

FIGURE 7
Central analgesic effect of Pogostemon heyneanus essential oil (PhEO, at 100 and 300 mg/kg) in the tail dip test in mice.Each column represents the mean ± SEM (standard error of the mean) of 6 animals.**p < 0.01 was considered statistically significant compared to the control (ANOVA, followed by Tukey's post-test).climate in the year studied, with a period of greater rainfall intensity of just 3 months (March to May) (Santos et al., 2022).
During the study, patchoulol (PA) was the constituent with the highest oil content from P. heyneanus leaves.PA is a tricyclic oxygenated sesquiterpene and has several important pharmacological properties, such as immunomodulatory, antiinflammatory, antioxidant, antitumor, antimicrobial, insecticidal, antiatherogenic, antiemetic, whitening, and sedative activities (Li et al., 2011;Hu et al., 2017).
PCA and HCA analyses did not differentiate P. heyneanus oil samples during the dry and rainy seasons.A previous study on the seasonality of essential oils from Psidium friedrichsthalianum leaves from Brazil showed no separation of samples from the dry and rainy seasons (Santos et al., 2023).Some species present variations in the content of constituents but cannot be separated in chemometric analyses due to their metabolism not correlating with biotic, abiotic, or climatic parameters, which can interfere with metabolic pathways (Santos et al., 2022;Guimarães et al., 2023).The results demonstrate that the essential oil was not toxic to animals.Based on the OECD guidelines (OECD, 2002), PhEO, with no acute deaths at 300 mg/kg, can be considered to have low acute oral toxicity.The intraperitoneal injection of acetic acid causes the release of inflammatory mediators such as prostaglandins, histamine, and several pro-inflammatory cytokines, which results in pain sensation (abdominal writhing) in the test animals (Dantas et al., 2020;Lee and Neumeister, 2020).The tail immersion test is an important evaluator of central analgesic activity in mice when studying the potential antinociceptive activity of a drug (Li et al., 2021).When the drug is effective, its activity in the nociceptive response determines a longer latency time in the animal compared to the control group, indicating a decrease in the activation of central nociceptors.
The formalin test is one of the most used models for selecting antinociceptive compounds.It consists of injecting formalin into the mouse's hind paw, which produces nociception, which occurs in two phases (Gomes et al., 2007).The first phase, which assesses neurogenic pain, occurs in the 0-5 min range, and sees the activation of nociceptors, and primary afferent fibers, which release bradykinin and substance P. In the second phase, which analyzes inflammatory pain, occurs between 15 and 30 min, and causes the release of histamine, prostaglandins, serotonin, and bradykinin due to peripheral and spinal sensitization.This test is used because it is the one that most resembles clinical pain.Therefore, it was noticed that P. heyneanus essential oil does not reduce neurogenic pain and/or pain caused by inflammation (Dantas et al., 2020).
Carrageenan has been used for decades to induce inflammation in different animal models as a screening method in research to assess potential anti-inflammatory substances.Injection of carrageenan into the peritoneal cavity induces several proinflammatory mediators such as prostaglandins, cytokines (IL-1β, TNF-α, IL-6), LTB 4 and C5a components, leukotrienes (B4 and PGE2) and chemokines (CXCL8) and CCL2).These inflammatory mediators stimulate increased vascular permeability and the migration of leukocytes and neutrophils to the abdominal cavity (Hall et al., 1998).Leukocyte migration has the role of recognizing and neutralizing foreign agents/aggressors, thus being an important step in the development of the inflammatory process (Kumar et al., 2014).The decrease in cell migration indicates the antiinflammatory activity of new substances (de Jesus et al., 2023;de Lima et al., 2023).The results demonstrate that P. heyneanus essential oil did not have an acute anti-inflammatory effect.
So, although PhEO displayed low anti-inflammatory activity, Patchoulol, its major component, is known to inhibit the production of iNOS and IL-6, activate NF-κB via suppressing IκB-α degradation and p65 nuclear translocation, inhibit ERK1/2 activation by suppressing their phosphorylation, and to have anti-inflammatory activity mediated by inhibiting ERK-derived NF-κB activation (Jeong et al., 2013).This fact can be due to the essential oil constituents acting synergistically because their main components when used as references have less or more activity than the essential oil.Therefore, synergism and antagonism studies must be developed (Miguel, 2010).

Conclusion
Pogostemon heyneanus essential oil composition was minimally influenced by climatic parameters over a seasonal period.Furthermore, the species is a source of patchoulol, the major component of its essential oil.
Pogostemon heyneanus essential oil does not present acute toxicity, nor were significant effects of central and peripheral analgesia observed.Tests about neurogenic and/or inflammatory pain proved inefficient, indicating that P. heyneanus essential oils do not act on certain biological activities despite the plant having broad ethnopharmacological indications.Moreover, the low influence of the climatic parameters on chemical composition can infer that this pharmacological activity is also not subject to climatic variations, that is, it does not change due to the climate.

FIGURE 2
FIGURE 2Chemical constituents identified in the essential oils of Pogostemon heyneanus.

FIGURE 3
FIGURE 3Hierarchical cluster analysis of the monthly Pogostemon heyneanus essential oils collection.

FIGURE 5
FIGURE 5Heat map of class clustering in essential oils from Pogostemon heyneanus samples.

FIGURE 8
FIGURE 8Central and peripheral analgesic effect of Pogostemon heyneanus essential oil (PhEO, at 100 and 300 mg/kg) in the formalin test in mice.(A) first phase, assesses neurogenic pain; (B) second phase, assesses inflammatory pain.Each column represents the mean ± SEM (standard error of the mean) of 6 animals.**p < 0.01 was considered statistically significant when compared to the control (ANOVA, followed by Tukey's post-test).

FIGURE 9
FIGURE 9Effect of Pogostemon heyneanus essential oil (PhEO, at 100 and 300 mg/kg) on carrageenan-induced peritonitis test.Each column represents the mean ± SEM (standard error of the mean) of 6 animals.*p < 0.05 compared to the control group.##p < 0.01 compared to the saline group.(ANOVA, followed by Tukey's post-test).

TABLE 1
Constituents identified in Pogostemon heyneanus essential oils during the seasonal study.

TABLE 2
Correlation between yield, main components, and classes of compounds of Pogostemon heyneanus essential oil and climatic parameters.

TABLE 3
Blood biochemical parameters of mice treated with Pogostemon heyneanus essential oil and the control group.Alkaline phosphatase (ALP).Statistical differences were observed using ANOVA, followed by Tukey's post-test.Each column represents the mean ± SEM, of 5 animals per group.*p < 0.05 and **p < 0.001 when compared to the control group.ns, No statistical difference.