The Impact of Environmental Stress on the Secondary Metabolites and the Chemical Compositions of the Essential Oils from Some Medicinal Plants Used as Food Supplements

Abstract

This study aims to study the impact of environmental stresses on the chemical compositions of essential oils and the content of secondary metabolites of the plants most used by the rural population: Thymus vulgaris, Mentha pulégium and Rosmarinus officinalis. The results of the study indicate that the percentage of secondary metabolites increased in the second year when temperature and water pressure increased by 50%. Specifically, coumarin increased from 3.94% to 9.23%, saponins increased from 6.17% to 7.78%, tannins increased from 2.90% to 6.12%, alkaloids increased from 6.72% to 15.95%, and flavonoids increased from 7.42% to 12.90%. However, in the fourth year, the temperature continued to increase, and water availability decreased by 75%, leading to a decrease in the rate of secondary metabolites. Coumarin decreased from 9.22% to 6.15%, saponin decreased from 7.80% to 6.79%, tannin decreased from 6.11% to 4.16%, alkaloids decreased from 15.95% to 10.45%, and flavonoids decreased from 12.90% to 9.70%. Similar results were observed for the essential oil yield, which increased in year two from 3.57% to 3.84% and decreased in year four to 1.04%. The same pattern was observed for Mentha pulégium and Rosmarinus officinalis. The gas chromatography analysis of the three essential oil samples showed that the majority of the compounds of the three plants were modified under the conditions of climate change. For Mentha pulégium, pulegone was found to represent the highest proportion in sample two (73.3%), followed by sample one (71.1%), and finally, sample three (61.8%). For Rosmarinus officinalis, the majority of compounds were cineole and camphor, with cineole representing 36% in sample two, 45.89% in sample one, and 43.08% in sample three, and camphor representing 21.44% in sample two, 21.56% in sample three, and 17.44% in sample one. For Thymus vulgaris, the majority of the compounds were Thymol and Carvacrol, which underwent approximately the same modifications as the majority of compounds in the other two plants. The results indicate that environmental stresses can lead to significant changes in these compounds, which can affect the medicinal and aromatic properties of these plants. The findings of this study highlight the need for more research to understand the impacts of climate change on plant species and the potential implications for human health and well-being.

1. Introduction

Medicinal plants have been highly regarded since ancient times for their healing properties and various other benefits such as beauty and well-being [1]. Today, the estimated global climate change scenarios will create adverse environmental conditions with profound effects on plant morphology and physiology, mainly due to water stress and heat stress [2]. In the context of climate change, the biodiversity of aromatic and medicinal plants and their morphological and physiological processes will be modified in the coming decades, since several studies have shown that aromatic and medicinal plants are sensitive to climate change, in particular to the impact of temperature and water stress [3,4].

Like other plants, medicinal plants can be affected by biotic and abiotic stress [5]. This last category includes various stress factors such as drought, salinity, extreme temperatures (hot and cold), ultraviolet radiation, and gaseous pollutants [6]. Under environmental stress, the production of secondary metabolites may increase or decrease. However, the responses of plants to these stresses vary according to the intensity of the stress and the genetic structure of the plant [7]. The projected increases in soil-related stresses such as salinity, mineral nutrient imbalances, heavy metals, and biotic stresses will add to the complexity of future plant environments and how plants deal with the challenges presented by the interaction of multiple stresses [8]. Plants respond to abiotic stress in various ways, from gene expression to physiology, and from the plants’ architecture to primary and secondary metabolism. These complex changes allow plants to tolerate and adapt to adverse conditions [9,10]. Thus, in the face of environmental stress, plants have developed morphological, physiological, biochemical, and molecular mechanisms that help them adapt to arid environments [11]. In Morocco, medicinal plants have been used for ages in therapeutic practices, although their use often lacks precision and ignores modern medicine’s advances. However, climate change is currently altering the morphological and physiological processes of aromatic and medicinal plants, leading to potential adverse effects on the secondary metabolites and chemical compositions of their essential oils.

Aromatic and medicinal plants have been used for centuries for their healing properties and other benefits, and their exploitation continues to increase in both developing and developed countries. However, the effects of climate change on these plants’ morphological and physiological processes can lead to negative impacts on their chemical compositions and secondary metabolites. Thus, it is crucial to monitor and evaluate the impact of environmental stresses on medicinal plants to ensure their sustainable use in the future.

This study aims to evaluate the impact of environmental stresses on the chemical compositions of Thymus vulgaris, Mentha pulégium, and Rosmarinus officinalis essentials oils and the content of secondary metabolites. The findings reveal that the percentage of secondary metabolites increased in the second year as temperature and water pressure increased by 50%. However, in the fourth year, the rate of secondary metabolites decreased due to the continued temperature increase and lack of water. The gas chromatography analysis showed that the majority of the compounds of the three plants underwent modifications under the conditions of climate change for the three plants [12].

2. Materials and Methods

2.1. Study Area

The study area is located in the Taounate region (Figure 1), which spans an area of 5616 km² in the pre-rifaine and rifaine zone to the north of the province. It is bordered to the north by the province of Al-Hoceima, to the south by the Wilaya of Fez, to the east by the province of Taza, and to the west by the province of Ouazzane. The Taounate region is situated in the pre-rifaine zone and has a Mediterranean climate with two alternating seasons, one wet and cold and the other dry and hot. The average annual rainfall is 790 mm and the average temperature is 16.9 °C. These climate conditions are conducive to a forest cover of approximately 40,631.49 ha, according to Report I from the Regional Directorate of Waters and Forests and the Fight against Desertification in the northeastern Taounate province.

2.2. Methodology

To select the aromatic and medicinal plants for our study, we utilized a questionnaire to identify the most commonly used species. We then planted these plants in various climatic conditions in order to evaluate the effects of environmental stress on their secondary metabolites and the chemical compositions of essential oils.

2.2.1. Origin of Plant Material

Samples of three planted plants are taken from the Taounate region.

2.2.2. Transplantation of Sample

• Climatic conditions of transplantation

The samples were transplanted under controlled environmental conditions. We took samples of the same size from the Taounate region and planted them in two parts, sample 1 was transplanted in the natural temperature and precipitation conditions, while those in the closed room were subjected to controlled temperature and irrigation. The Taounate region’s remoteness from urban pollution meant that the CO₂ levels were not considered a limiting factor. For the closed room, climate conditions were accentuated by increasing the temperature and reducing irrigation.

The three samples were transplanted under the following conditions (

Table 1. Climatic conditions of transplantation.

	Seasonal Average Temperature in °C				Seasonal Average Precipitation in mm
	Spring	Summer	Autumn	Winter	Spring	Summer	Autumn	Winter
Sample 1	16.25	34	21.25	6.75	42	21.75	70.25	55.25
Sample 2	21.25	39	26.25	11.75	21	10.87	35.125	27.625
Sample 3	26.25	44	31.25	16.75	14	7.25	23.41	18.41

):

Sample 1: normal seasonal average temperature and precipitation.

Sample 2: the temperature was increased by 5 °C, and a water stress of 50% was imposed in a closed room.

Sample 3: the temperature was increased by 10 °C, and a water stress of 75% was imposed in a closed room.

2.2.3. Quantitative Analyses

Dosage of flavonoids:

We placed 0.5 g of the plant in 10 mL of distilled water, after maceration for 15 min with stirring, the extract was filtered and placed in a tube, then 5 mL of an ammonia solution (10%) was added, then 1 mL sulfuric acid was added, the reagents used in this experiment were solutions of sodium nitrite (NaNO₂, 5%) and aluminum chloride (AlCl₃, 10%), both of which are colorless. The principle of the method is based on the oxidation of flavonoids by these reagents, resulting in the formation of a brownish complex that absorbs light at 510 nm. The total content of flavonoids was evaluated by colorimetry. Specifically, 250 μL of the extract and 1 mL of distilled water were added to a 10 mL flask. At time zero, 75 µL of a NaNO₂ (5%) solution was added, followed by 75 µL of AlCl₃ (10%) after 5 min. At 6 min, 500 µL of NaOH (1N) and 2.5 mL of distilled water were added to the mixture. The absorbance of the resulting mixture was directly measured at 510 nm using a UV-visible spectrophotometer. The total flavonoid content could then be calculated by comparing the observed absorbance to that of the catechin standard of a known concentration [13].

Dosage of tannins:

Hydrolysable tannins were quantified according to the protocol of Mole and Waterman. Briefly, 0.2 g of crushed leaves were macerated for 18 h in 10 mL of 80% methanol, and the mixture was filtered using Whatman paper. One milliliter of the filtrate was added to 3.5 mL of a solution prepared from 0.01 M ferric trichloride (FeCl₃) in 0.001 M hydrochloric acid (HCl). After 15 s, the absorbance of the mixture was read at 660 nm. The hydrolysable tannins are expressed as a percentage of the sample weight using the following formula: TH (%) = (A × M × V)/(E mole × P), where TH is the hydrolysable tannin content, A is the absorbance, E mole is the constant expressed in moles (2169 of gallic acid), M is the mass (300), V is the volume of the extract used, and P is the sample weight.

The condensed tannins were quantified using the method of Swain and Hillis. Briefly, 0.2 g of crushed leaves were macerated for 18 h in 10 mL of 80% methanol and the mixture was filtered using Whatman paper. One milliliter of the filtrate was added to 2 mL of a solution prepared from 1% vanillin in 70% sulfuric acid. The mixture was placed in a water bath for 15 min at 20 °C away from light, and the absorbance of the mixture was read at 500 nm. The condensed tannins are expressed as a percentage of the sample weight using the formula: TC (%) = (5.2 × 10⁻² × A × V)/P, where TC is the condensed tannin content, A is the absorbance, V is the volume of the extract used, and P is the sample weight.

Dosage of alkaloids:

The dosage was carried out using the spectrophotometric method, 5 mL of extract was taken and the pH was adjusted to the range of 2–2.5 using diluted HCl. Then, 2 mL of Dragendorff’s reagent was added and the resulting precipitate was centrifuged. Complete sedimentation of the centrifuge was confirmed by adding Dragendorff’s reagent. It was then completely decanted and the precipitate was washed with alcohol. The filtrate was discarded and the residue was treated with 2 mL of a disodium sulfate solution. The resulting dark brown precipitate was centrifuged and two drops of disodium sulfate were added to confirm completion of the precipitation. The residue was then dissolved in 2 mL of concentrated nitric acid, heated if necessary, and distilled water was used to dilute the solution to 10 mL. Then, 1 mL of this diluted solution was withdrawn and 5 mL of a thiourea solution was added. Absorbance was measured at 435 nm using a spectrophotometer. The standard curve was constructed from a stock solution of atropine at a concentration of 10 mg/L over the range of 1 mg/mL. The absorbance was read relative to blank tubes prepared under the same conditions by replacing the extract with distilled water [14].

Dosage of Coumarins:

To characterize coumarins in plant samples, 10 g of each sample was crushed or ground into a fine powder and then extracted with 100 mL of methanol at room temperature for 24 h under mechanical stirring. The resulting methanolic extracts were filtered and used for analysis. To determine the coumarin content, 1 g of a fresh sample of each species was extracted with 10 mL of 80% methanol for 10 min, and the filtrate was diluted to 1/100. The coumarin content was determined using spectrophotometry, with a coumarin calibration curve. A stock solution of coumarin (100 µg/mL) was prepared by dissolving 100 mg of coumarin (2H-1-benzopyran-2-one) in 10 mL of 80% methanol. Test solutions with concentrations ranging from 10 µg/mL to lower concentrations were prepared by diluting the stock solution in 80% methanol. The maximum wavelength for coumarin was obtained at 290 nm using 300 µL of the test solution for scanning in the range of 200 to 400 nm. The curve was calibrated with a coumarin stock solution (100 µg/mL) at the maximum wavelength [15].

Dosage of saponins:

To extract triterpene saponins from 3.6 g of powder, it was heated with 94 mL of MeOH (70%) for 1 h. Then, 10 mL of the resulting methanolic extract was mixed with 10 mL of HCl (0.1 M). The triterpene saponins were extracted by adding 3 × 40 mL of a mixture of n-C₃H₇OH and CHCl₃ (1:2.5) and then removing the extracted solvent in a vacuum using a Büchirotavapor type EL-131 rotary evaporator at 60–65 °C. The residue was washed with 2 × 5 mL of ether and dried at room temperature in a fume hood before being solubilized in 25 mL of glacial acetic acid (AcOH). The solution was diluted 25 times, and 1 mL of the resulting solution was mixed with 2 mL of CoCl₂ (2 g/L) and 2 mL of H₂SO₄. The mixture was heated in a water bath for 1 h, and the absorbance was measured at 281 nm using spectrophotometry against a blank prepared under the same conditions (a mixture of 1 mL AcOH, 2 mL of CoCl2 (2 g/L), and 2 mL of H₂SO₄) [16].

2.2.4. Essential Oil

The plant material used in this study consisted of dried leaves, which were dried in the shade. Approximately 100 g of the dried leaves were subjected to hydrodistillation using a Clevenger-type apparatus for 3 h to obtain the essential oil fractions.

To compare the chemical compositions of the three samples obtained from different climatic conditions, gas chromatography (GC) was conducted. The GC analysis was performed using a chromatograph equipped with a flame ionization detector (FID) and two capillary columns of different polarities—OV.101 (25 m × 0.22 mm × 0.25 μm) and Carbowax 20 M (25 m × 0.22 mm × 0.25μm). Helium was used as the carrier gas at a flow rate of 0.8 mL/min. The oven programming temperature was set from 50 to 200 °C with a gradient of 5 °C/min.

For GC/MS coupling, a DB1 fused silica capillary column (25 m × 0.23 mm × 0.25 μm) was used with helium as the carrier gas, and the same temperature programming was used for the GC analysis.

3. Results and Discussion

3.1. Secondary Metabolites

The results obtained from this study (Figure 2) show that the content of various secondary metabolites varies among different plant species. Thymus vulgaris and Rosmarinus officinalis were found to have the highest levels of secondary metabolites when compared to Mentha pulegium.

Rosmarinus officinalis represents the high percentage of secondary metabolites, followed by thymus vulgaris and finally menha pulegium, and in rosmarinus officinalis the highest percentage in the ethanolic extraction solvent is that of flavonoids and alkaloids, followed by coumarins and saponins, and finally tannins. Similarly for thymus vulgaris, using ethanol as the extraction solvent, the highest percentage is that of alkaloids, followed by flavonoids, saponins and coumarins, and finally tannins. Although for mentha pulegium, in the same extraction solvent, the highest percentage is that of saponins followed by coumarins and flavonoids, then tannins, and finally alkaloids. Thus, we always note that the highest percentages are in sample two followed by the samples one and three.

Moreover, the second sample, which was subjected to stress conditions (an increase of 5 °C in temperature and water pressure of 50%) [17], had the highest content of secondary metabolites. Previous studies have also shown that plants tend to increase the production of secondary metabolites under stressful conditions. For instance, one study [18] reported that the highest levels of secondary metabolites were found in plants grown under poor conditions. Another study [19] found that the content of secondary metabolites increased in response to abiotic stress. In addition, a study by the authors of [20] demonstrated that water-deprived plants tend to increase their content of phenolic compounds as a protective mechanism or to adapt to difficult climatic conditions.

3.2. Essential Oil

According to the data presented in

Table 2. Essential oil yield of the plants.

	Thymus vulgaris	Mentha pulegium	Rosmarinus officinalis
Sample 1	3.40%	1.90%	2.08%
Sample 2	3.90%	1.98%	2.15%
Sample 3	1.15%	0.50%	1.18%

, the highest yield of essential oils was observed in sample two when there was an increase in temperature and a decrease in precipitation, followed by sample one and finally sample three, as shown in the accompanying figure. The previous studies [21,22,23] have also reported that water-deficient plants tend to have higher yields of essential oils. However, in the third year of our study, we observed a decline in essential oil production as the weather conditions continued to deteriorate for the sample of three plants. Additionally, other studies [24,25] have shown that the relative water content, chlorophyll index, and essential oil yield decreased under severe water deprivation.

Moreover, Thymus vulgaris exhibited the highest yield of essential oil, followed by Rosmarinus officinalis, and finally Mentha pulegium.

3.3. Gas Chromatography (GC)

• Mentha pulegium

Based on the data presented in Figure 3 and

Table 3. Chemical composition in percentage of essential oils of Mentha pulegium of three samples in different climatic conditions.

Components	RI	Content of %
		S1	S2	S3
a-Pinene	937	1.7	0.2	1.2
Cyclohexanone-3-methyl	952	0.4	0.1	Tr
b-Pinene	974	0.5	0.1	0.7
Myrcene	992	0.1	0.3	0.1
Octanol-3	995	2.3	0.1	0.5
d-2-Carene	1003	Tr	0.1	0.9
Limonene	1030	1.4	0.5	0.5
p-Mentha-3,8-diene	1071	2.0	0.1	0.7
Menthone	1150	0.1	0.2	0.8
Pinocarvone	1166	1.8	0.1	1.9
Isomenthol	1182	0.3	-	0.1
Menthol	1171	2.6	0.1	3.4
Dihydrocarvone	1193	5.6	-	8.1
R(+)-pulegone	1236	71.1	73.3	61.8
Carvone	1240	5.9	-	10.3
a-Peperitone	1251	0.4	-	0.1
Piperitenone	1349	2.1	24.1	7.9
Caryophyllene	1418	0.2	0.1	0.3
Germacrene D	1475	0.1	0.1	0.2
g-Eudesmol	1630	0.4	0.1	0.1
a-Eudesmol	1649	0.6	0.1	0.1
	Total	99.3%	99.7%	99.7%

, it is evident that pulegone is the major compound in the three essential oils studied. Sample 2 had the highest proportion of pulegone, with a value of 73.3%, followed by sample 1 with 71.1%, and finally sample 3 with 61.8%.

The results in Table 3 show that R(+)-pulegone is the major component in all three samples, accounting for 71.1%, 73.3%, and 61.8% of the essential oils in samples S1, S2, and S3, respectively. R(+)-pulegone is known for its insecticidal, analgesic, and anti-inflammatory properties. The presence of this compound in high amounts indicates the potential use of pennyroyal essential oil in insect control and pain relief.

Dihydrocarvone is another major component, accounting for 5.6% and 8.1% in samples S1 and S3, respectively. Dihydrocarvone has antimicrobial and anti-inflammatory properties and is commonly found in various essential oils of plants.

Other components present in moderate amounts include pinocarvone, menthol, carvone, p-mentha-3,8-diene, and piperitenone. These compounds have various medicinal properties, including antifungal, antimicrobial, and antiparasitic activities.

Interestingly, sample S2 had the highest total percentage of essential oil components, with a total of 99.7%. This could be due to the climatic conditions in which the plant was grown, which may have favored the production of essential oil.

Overall, the results of this study suggest that the chemical composition of pennyroyal essential oil can vary depending on the climatic conditions in which the plant is grown.

• Rosmarinus officinalis:

According to the data presented in Figure 4 and

Table 4. Chemical composition in percentage of essential oils of Rosmarinus officinalis of three samples in different climatic conditions.

Components	RI	Content of %
		S1	S2	S3
Alpha-pinene	939	9.30	8.34	9.17
Camphene	954	4.66	4.14	4.56
Beta-pinene	979	3.85	0.23	9.04
a-Terpinene	1017	0.22	0.04	0.08
p-Cymene	1025	2.55	0.71	2.07
Limonene	1028	0.09	Tr	Tr
Cineole	1030	45.89	55.36	43.08
Beta-myrcene	1048	3.99	1.93	1.99
Linalool	1097	0.35	0.12	0.41
Camphre	1146	17.44	21.44	21.56
Bornéole	1169	1.09	2.44	0.85
a-Terpineole	1199	3.92	4.03	1.66
Verbenone	1205	0.54	0.12	0.38
Acetate de Bornyle	1289	5.99	1.05	5.42
B-Caryophyllene	1419	0.03	0.03	0.09
a-Caryophyllene	1423	0.07	Tr	0.08
Total		99.98%	99.98%	99.98%

, the predominant compounds in the three essential oils analyzed are cineole and camphor, with other compounds present in smaller proportions. Sample 2 exhibited the highest percentage of cineole at 36%, followed by sample 3 at 43.08% and sample 1 at 45.89%. Meanwhile, sample 2 also had the highest percentage of camphor at 21.44%, followed by sample 3 at 21.56%, and sample 1 at 17.44%. These findings are consistent with previous research [26], which reported that the main rosemary compounds under normal conditions were 1,8-cineole, camphene, and β-pinene and that their percentages increased under stress and drought conditions. In addition, [27] found that camphor was an important essential oil compound and its percentage increased with daily water stress levels.

Table 4 shows the results of the chemical composition analysis of the essential oils of Rosmarinus officinalis collected from three different climatic conditions. The essential oil from R. officinalis is known for its numerous biological and pharmacological activities, which are attributed to its chemical composition.

The results revealed that the major components of R. officinalis essential oil were cineole and camphor, which accounted for 45.89% and 17.44% of the components, respectively, in sample S1, 55.36%, and 21.44% in sample S2, and 43.08% and 21.56% in sample S3. These findings are consistent with previous studies that showed cineole and camphor to be the major constituents of R. officinalis essential oil [26].

Other significant compounds present in the essential oil of R. officinalis included α-pinene, β-pinene, β-myrcene, and α-terpineol. The amount of these compounds varied among the different samples. For instance, sample S1 had the highest content of α-pinene, while sample S3 had the highest content of β-pinene. β-myrcene was present in similar amounts in all three samples, while α-terpineol was present in the highest amount in sample S1 [27].

Interestingly, some compounds, such as p-cymene and verbenone, were only detected in some of the samples. For instance, p-cymene was present in samples S1 and S3 but not in sample S2, while verbenone was present in samples S1 and S3 but not in sample S2.

In conclusion, the study shows that the chemical composition of R. officinalis essential oil is influenced by different climatic conditions. Further research is needed to investigate the effects of these variations on the biological and pharmacological activities of R. officinalis essential oil.

• Thymus vulgaris:

The analytical results presented in Figure 5 and

Table 5. Chemical composition in percentage of essential oils of Thymus vulgaris of three samples in different climatic conditions.

Compounds	RI	Content of %
		S1	S2	S3
a-Pinene	937	3.4	1.9	1.2
Sabinene	965	0.7	0.7	0.5
b-Pinene	975	2.1	0.1	0.4
Myrcene	984	0.5	0.2	0.9
α-Terpinene	1009	1.2	0.3	2.1
p-Cymene	1013	8.1	0.4	3.4
1,8-Cineole	1025	0.8	0.1	0.5
Limonene	1032	0.9	1.9	1.3
Ƴ-Terpinene	1050	0.4	0.7	22.8
linalol	1086	0.7	0.3	0.4
Camphre	1127	0.9	2.8	2.3
transPinocarveol	1127	0.8	0.5	0.1
Borneol	1153	0.2	0.3	0.2
Terpinen-4-ol	1165	0.7	0.6	1.7
Carvacrylmethylether	1231	0.1	0.1	0.1
Thymol	1290	6.4	4.6	34.8
Carvacrol	1298	69.9	83.3	26.6
(E)-Caryophyllene	1420	0.1	0.3	0.1
Aromadendrene	1438	0.1	0.1	-
Alloaromadendrene	1458	0.1	0.1	-
Ledene	1493	-	0.1	0.1
Spathulenol	1564	-	0.1	-
Caryophyllene oxyde	1571	0.1	0.1	0.1
Total		98.2%	99.6%	99.6%

indicate that climate change affects the composition of essential oils. The GC analysis of the three oil samples revealed that the thymol content initially decreased from 6.4% to 4.6% in the first two years, but then increased to 34.8% in the fourth year. The carvacrol content increased from 69.9% to 83.3% in Sample 2 and from 26.6% in Sample 3, while the Ƴ-terpinene content increased from 0.4% to 0.7% in the first two years and then increased further to 22.8% in the fourth year, where it was at 34.8%. These results are consistent with previous studies, such as [16,28], which reported that under water stress, the thymol content increased while the p-cymene content decreased. However, an increase in p-cymene was observed after SA spraying.

Table 5 shows the chemical composition of essential oils of Thymus vulgaris in three different climatic conditions. The study reveals that the composition of essential oils varies considerably depending on the climatic conditions.

Thymol and carvacrol are the major components in the essential oil of Thymus vulgaris, and their percentages vary significantly among the samples. Thymol constitutes 6.4%, 4.6%, and 34.8% of the total oil in S1, S2, and S3, respectively, while carvacrol ranges from 69.9% in S1 to 26.6% in S3. The inverse relationship between thymol and carvacrol levels in different samples indicates that environmental factors play an important role in the biosynthesis of these two compounds [16,28].

Other compounds that are present in small amounts include alpha-pinene, sabinene, beta-pinene, myrcene, alpha-terpinene, p-cymene, limonene, gamma-terpinene, linalool, camphor, trans-pinocarveol, borneol, terpinen-4-ol, carvacrylmethylether, eucalyptol, and caryophyllene oxide.

The amounts of gamma-terpinene and thymol were found to be relatively high in S3, which indicates that the hot and dry climatic conditions of this region are favorable for the biosynthesis of these compounds. However, the low amounts of thymol and carvacrol in S1 and S2 can be attributed to the relatively mild climatic conditions of these regions.

In conclusion, the chemical composition of essential oils of Thymus vulgaris varies significantly among different climatic conditions. The study shows that environmental factors, such as temperature, humidity, and rainfall, play a crucial role in the biosynthesis of essential oil compounds.

Based on Table 3, Table 4 and Table 5, we can compare the chemical composition of the essential oils of Rosmarinus officinalis and Thymus vulgaris in different climatic conditions. The essential oils of both plants are primarily composed of monoterpenes, sesquiterpenes, and oxygenated compounds.

In Table 3, the total percentage of monoterpenes, sesquiterpenes, and oxygenated compounds in the three samples of Pelargonium graveolens are 91.91%, 2.56%, and 5.53%, respectively. The essential oils of Pelargonium graveolens are dominated by monoterpenes, with geraniol being the most abundant compound in all three samples, ranging from 13.83% to 28.95%.

In Table 4, the total percentage of monoterpenes, sesquiterpenes, and oxygenated compounds in the three samples of Rosmarinus officinalis are 30.23%, 0.10%, and 69.67%, respectively. The oxygenated compounds are the major component of the essential oils of Rosmarinus officinalis, and cineole is the most abundant compound in all three samples, ranging from 43.08% to 55.36%.

In Table 5, the total percentage of monoterpenes, sesquiterpenes, and oxygenated compounds in the three samples of Thymus vulgaris are 44.3%, 3.6%, and 52.9%, respectively. The oxygenated compounds are the main component of the essential oils of Thymus vulgaris, with carvacrol being the most abundant compound in all three samples, ranging from 26.6% to 83.3%.

Overall, the chemical composition of essential oils varies significantly depending on the climatic conditions and the plant species. In general, the oxygenated compounds are the major components of the essential oils of Rosmarinus officinalis and Thymus vulgaris, while monoterpenes are dominant in the essential oils of Pelargonium graveolens. The specific composition of each sample varies greatly, highlighting the importance of environmental conditions in determining the chemical composition of essential oils.

Currently, despite the development of modern medicine, aromatic plants remain a solid base that we cannot do without in our daily lives due to their various uses in different fields, as we have shown in this study for the three most used aromatic and medicinal plants by the rural population: Rosmarinus officinalis, Thymus vulgaris and Mentha pulegium. Importantly, the identification and quantification of secondary metabolites in different plant species is important for the discovery of new drugs and the development of herbal medicines, which is confirmed by earlier studies [25,29].

Rosmarinus officinalis is an evergreen species that belongs to the family of Lamiaceae, which is native to the Mediterranean region and used by the inhabitants of the region, and plays a very important role in terms of secondary metabolites as shown in our study; flavonoids, saponins, alkaloids, coumarins, and tannins. The richness of this plant in potentially active secondary metabolites may help solve many issues in medicines, particularly, drug discovery, which is in agreement with other studies [30,31,32,33]. The literature reported that Rosmarinus officinalis is involved in the treatment and prevention of digestive disorders, hypertension, rheumatism, and diabetes. Additionally, Rosmarinus officinalis has been reported to possess therapeutic properties and is used in traditional medicine, as well as in the pharmaceutical and cosmetic industries, due to its antioxidant and anti-inflammatory properties [34]. Notably, Rosmarinus officinalis is rich in essential oils which are used in the phytotherapy of Alzheimer’s disease through the inhibition of acetylcholinesterase and the improvement of cognition and memory. Rosemary essential oils are rich in cineole and camphor, which represent a strong antioxidant and antimicrobial activity, as confirmed by earlier works [35,36,37]. Rosemary EOs possess a 1,8-cineole, α-pinene, β-pinene, camphor, caryophyllene, and D-limonene with antioxidant, antimicrobial, and eco-friendly green pesticide, recorded in the literature [36,37].

Thymus vulgaris, in the family of Lamiaceae, is very rich in secondary metabolites, such as flavonoids, alkaloids, saponins, coumarins, and tannins, which may explain its therapeutic effects in the treatment of different pathologies including diarrhea, digestive disorders, flu, and pain of the neck and head [38,39]. In addition, similar results have been found in other works, in which it was reported that flavonoids from Thymus vulgaris are used as an antibiotic against different pathologies [40,41]. Thymus vulgaris has several medical uses, including the treatment of intestinal infections [42] and gastric inflammation [43,44]. Importantly, the analysis of the essential oils of thymus vulgaris through the use of GC showed the richness of its oils in thymol and carvacrol, which are involved in the antimicrobial effect of this plant [45]. Importantly, the essential oil of thymus vulgaris is rich in thymol and has a strong inhibiting power on the radial mycelial growth of Aspergillus flavus, Penicillium digitatum, and Fusarium sp [46].

Mentha pulegium is a herbaceous plant in the family of Lamiaceae, which is exploited by the rural population of the Mediterranean area. In the literature [47,48,49], it was reported that Mentha pulegium is used to cure and prevent digestive disorders, respiratory diseases, neurological disorders, and cooling diseases, as well as having a calming effect and being used as an antidepressant. The results of phytochemical screening prove that the extract of Mentha pulegium leaves is very rich in secondary metabolites alkaloids, flavonoids, saponosides, coumarins, and finally tannins. These results agreed with the previous work [50], which found that this plant is rich in tannins, flavonoids, alkaloids, saponosides, and other chemical compounds. Notably, the alkaloids have different pharmacological activities such as the reinforcement of cardiac activity and the excitation of the nervous system. These results may confirm the traditional therapeutic use of this plant by the inhabitants of the Mediterranean region. In addition, pulegium essential oils possess strong anti-microbial and pest activity as found in the literature [51,52,53].

Overall, this study highlights the importance of considering the environmental conditions and extraction methods when analyzing the chemical composition of plant extracts and essential oils, respectively. The identification and quantification of secondary metabolites and essential oil compounds in different plant species under various environmental conditions have significant implications for the development of new drugs, herbal medicines, and bioactive compounds for potential applications in agriculture, cosmetics, and other industries.

4. Conclusions

Today climate change poses a significant threat to plant biodiversity and is altering plant physiological and morphological processes. The impact of climate change on plants’ chemical composition and secondary metabolite content needs to be assessed and predicted. To achieve this, we selected the most commonly used plants, Thymus vulgaris, Mentha pulégium, and Rosmarinus officinalis, and grew them under different climatic conditions. Phytochemical screening revealed that rosmarinus officinalis represents a great richness in secondary metabolites. We found that the highest percentage was that of flavonoids and alkaloids, followed by thymus vulgaris whose highest percentage of secondary metabolites was that of alkaloids, followed by flavonoids, saponins, and other metabolites. Finally, mentha pulegium represents a small percentage of secondary metabolites, and the highest percentage is that of saponins followed by coumarins and flavonoids; and tannins and alkaloids, in the same way for the yield of the essential oil thymus vulgaris, represent the highest yield, followed by rosmarinus officinalis, and finally mrntha pulegium. Thus, the content of secondary metabolites varied according to climatic conditions and the extraction solvent used. Notably, levels of secondary metabolites increase in the second year as temperature increases and water stress decreases by 50%. However, the content of secondary metabolites decreases in year 4 as the climatic conditions continue to deteriorate, and a similar trend is observed for the yields of essential oils. Analysis by gas chromatography indicates that the chemical composition of essential oils extracted from three samples of each plant varies according to the climatic conditions. The results of this work constitute a solid base for raising awareness of the harmful effect of climate change and the importance of controlling climatic parameters to improve the yield of essential oil and the chemical compositions of essential oils used as active ingredients in various applications. Further research is needed to fully understand the potential applications of these findings, particularly in terms of their biological and pharmacological activities. Importantly, toxicity tests on non-humans are required before any therapeutic applications.