Impact of climatic disturbances on the chemical compositions and metabolites of Salvia o ﬃ cinalis

: Aromatic and medicinal plants in ecosystems are subject to various climatic disturbances that impact their morphological and physiological processes. Although plants have mechanisms to adapt to their climatic conditions, such as periods of drought and lack of precipitation, their metabolism is still a ﬀ ected. This study aimed to predict and evaluate the behavior of Salvia o ﬃ cinalis under climatic disturbances. Over a period of 4 years in a controlled environment, three treatments were applied to the plant: Treatment 1 with normal monthly average temperature and precipitation in the ﬁ rst year; Treatment 2 with a temperature increase of 5°C and a 50% reduction in water supply in the second year; and Treatment 3 with a temperature increase of 10°C and a 75% reduction in water supply in the fourth year. The results show that the percentage of primary metabolites, including nutritional values, changed with increasing temperature and decreasing precipitation. Treatment 1 had 7.13% protein, 6.21% carbohydrate, 1.35% lipid, and 4% dietary ﬁ ber, while Treatment 2 had 7.05% protein, 5.12% carbohydrate, 1.01% lipid, and 3.01% dietary ﬁ ber, and Treatment 3 had 6.86% protein, 3.02% carbohydrate, 0.52% lipid, and 2.34% dietary ﬁ ber. The mineral composition of the plant also changed with each treatment, with Mg decreasing from 10.02 to 8.55 to 0.05%, Fe decreasing from 8.18 to 8 to 7.62%, K decreasing from 5.55 to 5.05 to 4.02%, Mn decreasing from 5.54 to 5.11 to 3.48%, Ca decreasing from 4.65 to 2.75 to 1.23%, and P decreasing from 3.37 to 3.05 to 2.25%. Regarding secondary metabolites, the percentage of alkaloids, ﬂ avonoids, saponins, coumarins, tannins, and essential oil yield changed as well. Treatment 2 showed an increase in secondary metabolites, while Treatment 3 showed a decrease. Alkaloids increased from 9.56 to 13.68 to 11.3%, ﬂ avonoids increased from 7.53 to 13.48 to 10.49%, saponins increased from 5.23 to 7.44 to 6%, coumarins increased from 3.35 to 4.85 to 3.99%, tannins increased from 2.26 to 3.22 to 2.62%, and essential oil yield increased from 0.53 to 0.80 to 0.62%. Gas chromatography analysis revealed that the major compounds of the essential oils of Salvia o ﬃ cinalis , such as α -thujone, manool, β -caryo- phyllene, α -humulene, viridi ﬂ orol, 1,8-cineol, and camphor, were also modi ﬁ ed by temperature and water stress


Introduction
Healing, happiness, and magnificence are just a few of the virtues that have been bestowed upon medicinal plants since the dawn of time [1].The thirst for acclimatization and a return to nature has fueled the popularity of herbal teas, capsules, essential oils, and homemade elixirs [2].As a result, traditional medicine based on fragrant and restorative plants has experienced a remarkable revival of safe and proven value, even though modern medicines often ignore them [3].This article explores the significance of traditional medicine and the effect of climatic parameters on the metabolites of aromatic and medicinal plants [4].The study focuses on Salvia officinalis, a commonly used plant in local populations, to offer suggestions for adjusting to the impacts of climate change.
Traditional medicine is a set of practices, methods, knowledge, and ideals inherited from one generation to another to diagnose, treat, and maintain human health [5].Traditional medicine has been used for thousands of years and is still widely used today.In Africa, up to 80% of people use aromatic and medicinal plants for preventive or curative purposes.Developed countries have also seen a surge in the exploitation of medicinal plants, with Australia, Canada, France, and China all reporting significant usage rates [6][7][8].
In ethnobotanical practices, sage has been used for various purposes, including as an antiseptic, anti-inflammatory, and anti-spasmodic agent.It has also been used to treat digestive disorders, respiratory infections, and menstrual problems.Additionally, it has been used topically to treat skin conditions and wounds [8].
Studies have shown that sage essential oil possesses several biological activities, including antimicrobial, antioxidant, and anti-inflammatory properties.The oil has been shown to be effective against various bacterial and fungal strains, as well as having potential antiviral activity.Additionally, sage oil has been found to exhibit significant antioxidant activity, which may help protect against oxidative stress and related diseases.
Furthermore, the sage essential oil has been investigated for its potential use in cancer therapy due to its ability to induce cell death and inhibit cancer cell proliferation.It has also been shown to have anxiolytic and antidepressant effects, making it a potential treatment option for anxiety and depression [7].Morocco is a prime example of a country with a rich history of medicinal plant use.Its geographical location, with its varied bioclimatic floors and double marine facade, has led to a unique panoramic diversity and essential plant diversity.Of the 4,200 species and subspecies found in Morocco, 800 are used for medicinal purposes.However, despite the long history of use, the exploitation of medicinal plants in Morocco often lacks precision and ignores advances in modern medicine [9].
Climate change poses a significant threat to the chemical composition of aromatic and medicinal plants.Meteorological factors such as temperature and precipitation have a direct impact on biomass production and an indirect influence on the chemical makeup of plants.
For instance, water availability can affect the growth and development of plants, which can subsequently influence their essential oil composition.High humidity can also impact the yield and quality of essential oils.Soil pH can affect nutrient availability and plant metabolism, ultimately affecting the chemical makeup of essential oils.Additionally, air quality, including pollution levels and atmospheric pressure, can also play a role in the chemical composition of essential oils.
Therefore, it is essential to consider multiple climatic parameters when investigating the effects of climate on essential oil composition.
Consequently, alterations in meteorological conditions can result in shifts in the chemical composition of plants.
This is particularly concerning for plants used for medicinal purposes, as changes in chemical composition can affect their effectiveness [10] To deepen the knowledge of the responses of commonly used aromatic and medicinal plants to climate change, a study was conducted on Salvia officinalis.The plant was grown under different climatic factors for 4 years, and the evolution of its chemical composition was analyzed.The study found that an increase in temperature and a decrease in precipitation led to changes in the chemical composition of Salvia officinalis.This highlights the need for further research into the effect of climatic parameters on the chemical composition of medicinal plants and the need for adaptation strategies to minimize these effects [11].
Traditional medicine based on fragrant and restorative plants has played a significant role in human health for thousands of years.The exploitation of medicinal plants continues to grow, and their significance cannot be ignored.However, the effect of climatic parameters on the chemical composition of medicinal plants poses a significant threat to their effectiveness.The study of Salvia officinalis highlights the need for further research into the impact of climate change on medicinal plants and the need for adaptation strategies to minimize these effects.By preserving the chemical composition of medicinal plants, we can ensure their continued effectiveness in treating and maintaining human health.

Establishment of a cultural center
Adequate soil preparation was conducted in both fields a few weeks prior to transplanting, aimed at preventing grass growth and ensuring a diverse blend of soil factors.This approach helped to avoid root suffocation during watering.Furthermore, a weekly weed elimination routine was implemented post-transplantation to mitigate nutrient competition with the intended plants.
Transplanting of the samples was executed under specific circumstances.In the open section, cuttings were transplanted while being exposed to the prevailing temperature and precipitation conditions.Conversely, the closed section underwent controlled adjustments, involving regulated temperature and irrigation.This manipulation simulated climatic conditions by intensifying temperatures and reducing irrigation.
The three samples were transplanted under the subsequent conditions: Treatment 1: involved subjecting the samples to typical monthly average temperature and precipitation conditions.
Treatment 2: the temperature was elevated by 5°C, and water stress was intensified by 50% within an enclosed chamber.
Treatment 3: saw a temperature rise of 10°C, coupled with a 75% escalation in water stress within a closed chamber.

Phytochemical screening 2.2.1 Detection of flavonoids
Extraction of flavonoids was performed using the "Cyanidin" reaction.To prepare the extract, 2 mL of each sample was mixed with a few drops of concentrated HCl and a small number of magnesium shavings.Flavonoids were indicated by an orange-to-red coloration [12].For the Cyanidin reaction, the mixture was heated for 10 min in a water bath without the addition of magnesium chips.Leucoanthocyanins produced a cherry-red or purplish color, while catechols produced a brown-red hue.To detect flavonoids in the plant, 0.5 g of the sample was combined with 10 mL of distilled water.
The sample was macerated for 15 min with stirring and then filtered.Next, 5 mL of 10% ammonia solution and 1 mL of sulfuric acid were added to the filtrate.The presence of flavonoids was indicated by the appearance of a yellow color [13].

Tannin detection
To detect the presence of tannins, a few drops of 10% aqueous FeCl 3 solution (mass/volume) is introduced into 3 mL of each extract.A blue-black or blue-green color indicates a positive test [14].
To test for tannins in the plant, 1.5 g of dry plant powder is mixed with 10 mL of 90% methanol and stirred for 15 min.The resulting solution is filtered and a few drops of 1% ferric chloride (FeCl 3 ) is added.The color changes to blue-black in the presence of gallic tannins and greenishbrown in the presence of catechin tannins [15].

Coumarin detection
Extract: The presence of coumarins can be determined by adding 0.5 mL of NH 4 OH (25%) to 2 mL of each extract, followed by observation under a 366 nm UV lamp.The appearance of intense fluorescence is indicative of the presence of coumarins, as reported by Farhan et al. [16].
Plant: To check for the presence of coumarins in the plant, 1 mg of plant powder is combined with 2 mL of distilled water and 1 mL of NH 4 OH (25%) in a test tube.After filtration, the solution is observed under a UV lamp emitting light at a wavelength of 366 nm.The emergence of strong fluorescence signifies the presence of coumarins [16].

Detection of alkaloids
Alkaloids can be identified through precipitation reactions utilizing Bouchardat's, Mayer's, and Dragendorff's reagents.
In the process of testing the extracts, 1 mL of each of the reagents (Mayer, Dragendorff, Bouchardat) is introduced to 3 mL of each extract.Subsequently, the solution is allowed to settle for a duration of 10 min.A positive test outcome is indicated by an orange precipitate when using Dragendorff's reagent, a yellowish-white precipitate with Mayer's reagent, and a brown precipitate with Bouchardat's reagent [17].
For plant testing, 10 g of the plant is macerated with 50 mL of 10% H 2 SO 4 for 24 h.The solution is filtered and diluted with distilled water up to 50 mL.One milliliter of the filtrate is taken and placed into two tubes to which five drops of Mayer's or Dragendorff's reagent are added.The confirmation of alkaloid presence is established by the emergence of an orange-red or reddish-brown precipitate when treated with Dragendorff's reagent, and a yellowishwhite precipitate when treated with Mayer's reagent [15].

Detection of saponins
Extract: The presence of saponins in the extracts was confirmed using the foaming test, where 5 mg of each extract was diluted in 5 mL of distilled water and vigorously shaken for 15 s in a test tube.The development of a persistent and stable foam, reaching a height exceeding 1 cm and lasting for a duration of 15 min, serves as an indication of the significant existence of saponins [12].
Plant: The presence of saponins in the plant was determined by preparing a decoction of 2 g of the plant in 100 mL of water, which was heated in a water bath at 95°C for 30 min.After filtration, the test tubes were agitated horizontally for 10 s.The presence of saponins was confirmed by the appearance of a foam larger than 1 cm, which persisted for 15 min [13].

Quantitative analyses 2.3.1 Dosage of flavonoids
The following reagents were utilized for the analysis: a colorless solution of sodium nitrite (NaNO 2 , 5%) and aluminum chloride (AlCl 3 , 10%).The procedure relies on the oxidation process of flavonoids facilitated by these reagents, resulting in the creation of brown complexes that exhibit a peak absorbance at 510 nm.The quantification of total flavonoid content is achievable by comparing the measured absorbance with values obtained from established concentrations of catechin standards.
The total flavonoid content of the extract was analyzed by adding 250 μL of the extract to a 10 mL flask, followed by 1 mL of distilled water.Subsequently, 75 µL of NaNO 2 (5%) solution was added at time zero, followed by the addition of 75 µL of AlCl 3 (10%) 5 min later.Following a span of 6 min, 500 μL of NaOH (1 N) was introduced and succeeded by incremental additions of 2.5 mL of distilled water.Subsequently, the absorbance of the resulting solution was directly assessed using a UV-Vis spectrophotometer set at a wavelength of 510 nm [18].

Dosage of tannins, alkaloids, Coumarins, and saponins
Upon the completion of qualitative identification for major metabolite families, the focus shifted toward a quantitative analysis of secondary metabolites.Well-established techniques as referenced in previous studies [18][19][20][21]42,43] were used to quantify these metabolites.These methods offer a robust foundation for precise measurement and determination of the quantity or concentration of identified metabolites within plant samples.

Essential oil
The plant material utilized in this research consists of leaves that were carefully dried in a shaded environment.
For the extraction of essential oils, a segment of the dried leaves was subjected to hydrodistillation using a Clevengertype apparatus.

Gas chromatography (GC)
After the extraction of essential oil from the three samples, GC was conducted to compare their chemical compositions under different climatic conditions.The analysis was carried out using a chromatograph equipped with a flame ionization detector and two OV-type capillary columns with different polarities, namely 101 (25 m × 0.22 mm × 0.25 µm) and Carbowax 20M (25 m × 0.22 mm × 0.25 µm).
Helium was used as a carrier gas at a flow rate of 0.8 mL/min and the oven programming temperature was set at 50-200°C with a 5°C/min ramp.In addition, GC/MS coupling was performed using a DB1 fused silica capillary column (25 m × 0.23 mm × 0.25 μm) with helium as the carrier gas and the same temperature programming as the GC analysis.
3 Results and discussion  [22][23][24] have found that water stress negatively affects the primary metabolites of the vine.This is consistent with previous research [25,26], which found that water stress affects the amino acids in Salvia officinalis.

Mineral compositions
Figure 3 shows that the mineral compositions present in the three treatments differed.As seen in Figure 3, the percentage of mineral compounds varied between the treatments.The most abundant compounds, in decreasing order, were Mg, Fe, K, Mn, Ca, and P. It was observed that the percentage of these compounds decreased as the climate change parameters intensified.Specifically, the percentage of Mg decreased from Treatment 1 (10.02%) to Treatment 3 (8.55%),Fe decreased from Treatment 1 (8.18%) to Treatment 3 (7.62%),K decreased from Treatment 1 (5.55%) to Treatment 3 (4.02%),Mn decreased from Treatment 1 (5.54%) to Treatment 3 (4.8%),Ca decreased from Treatment 1 (4.65%) to Treatment 3 (1.23%), and P decreased from Treatment 1 (3.37%) to Treatment 3 (2.25%).Other compounds were present in low percentages, as shown by [27].They found that water stress causes phosphorus deficiency in plants, leading to a reduction in leaf area and leaf water content.Other researchers have found that water stress limits mineral uptake by slowing the rate of diffusion of nutrients from the soil to root absorptive surfaces and reducing movement within the plant [28,29].D'Oria et al. also found that under stressful environmental conditions, uptake of the majority of mineral compounds is reduced [30].

Secondary metabolites
The findings depicted in Figure 4 illustrate the fluctuation in the proportion of secondary metabolites, which is contingent upon the prevailing climatic circumstances and the specific extraction solvent used.Among the extraction methods, ethanol yielded the highest percentage of alkaloids, succeeded by flavonoids, saponins, coumarins, and tannins.Moreover, the distribution of secondary metabolites varied across the treatments, with an upsurge detected in the second treatment and a decline in the third treatment, as demonstrated in the subsequent sequence: (Treatment 1, Treatment 2, Treatment 3): alkaloids (9.56, 13.68, 11.3%), flavonoids (7.53, 13.48, 10.49%), saponins (5.23, 7.44, 6%), coumarins (3.35, 4.85, 3.99%), and tannins (2.26, 3.22, 2.62%).Past research has documented that water stress has the potential to trigger an augmentation in both secondary metabolites and essential oils, corroborated by findings in previous studies [31,32].Additionally, Shil and Dewanjee [33] reported that drought and water stress can cause both an increase and decrease in the content of secondary metabolites.

Essential oil yield
Figure 5 displays data indicating that the yield of essential oil of Salvia officinalis exhibits an increase in Treatment 2 and a decrease in Treatment 3 as the climate conditions continue to worsen.This finding is consistent with previous research, such as Mirniyam et al. [34], who found that severe water deficit results in a decrease in the yield of essential oil, and Mohammadi et al. [35], who found that the content of essential oil increases in the case of water stress.

GC
Based on the GC analysis results (Figures 6-8 and Table 1), it can be observed that environmental factors have a significant impact on the essential oil compounds of Salvia officinalis, and the composition of these compounds varies depending on the treatment applied.Specifically, the percentage of α-thujone decreases from Treatment 1 to Treatment 3 (0.78, 10.44, 0.09%), while manool and β-caryophyllene increase in Treatment 2 and then slightly decrease in Treatment 3 (manool: 11.87, 13.67, 13.09%; β-caryophyllene: 12.08, 6.89, 14.01%).In addition, the percentage of α-humulene decreases significantly in Treatment 2 (10.80-0.01%),while the percentage of viridiflorol and 1,8-cineol fluctuates slightly between the three treatments (viridiflorol: 6.54, 7.55, 6.01%; 1,8-cineol: 8.71, 7.08, 9.07%).Camphor percentage decreases slightly from Treatment 1 to Treatment 3 (6.70,4.78, 5.75%).These findings are consistent with previous studies [36,37] which found that water stress can lead to changes in the composition of essential oils and their major compounds.Also, Russo et al. [38] found that the GC/ MS analysis of the essential oils of 18 samples under the various climatic conditions shows that these 18 samples have approximately the same compositions, which are αthujone, camphor, borneol, γ-muurolene, and sclareol, but their percentage varies according to environmental conditions.Rioba et al. [39] also discovered that the treatment does not affect the essential oil content.Fifty-four compounds have been identified in the essential oil.Nitrogen and irrigation frequency affected β-pinene production, and interactive effects of nitrogen and Phosphorus (NxP) on αand β-thuyone, and of nitrogen irrigation frequency on αthuyone were noted.There was a negative correlation between 1,8-cineole and chlorophyllol.The camphor percentage is above the threshold recommended by the ISO trade standard for sage essential oil.Similarly, Tundis et al. [40] confirmed this result and found that environmental conditions such as climatic and edaphic factors affect the chemical compositions of sage.Karalija et al. [41] also found that environmental stresses, such as increasing soil salinity and dryness and changes in average annual temperatures, pose serious risks to the commercial production of sage essential oil, which is produced commercially in many European countries and also carry the chemical compositions of salvia.Climate change is a well-established phenomenon that has been shown to have significant impacts on plant growth, biomass, and physiological and morphological processes.However, our study, which was conducted in a closed chamber over 4 years from 2019 to 2022, under controlled temperature and irrigation conditions, reveals that increasing temperature and decreasing irrigation limit mineral intake, while having almost the same results for the percentage of amino acids and nutritional values.Among the notable consequences of climate change on plants, a crucial area of impact is observed in their secondary metabolites and essential oils.Our research outcomes underscore that the synergistic impact of escalating temperatures and intensified water stress culminates in a reduction of the proportional representation of these compounds within the leaves of Salvia officinalis.
The outcomes of this investigation establish a robust groundwork for forthcoming studies focused on comprehending and evaluating the ramifications of climate change on aspects such as plant biodiversity, morphology, and chemical composition.Safeguarding this invaluable natural resource holds paramount significance, and the insights from this research will play a pivotal role in pinpointing critical factors that necessitate attention for the purpose of alleviating the adverse impacts of climate change on plant species.
It is important to note that our study was conducted under controlled conditions, and the findings may differ under natural conditions.Nonetheless, it furnishes invaluable insights into the conceivable repercussions of climate change on plant species, illuminating the imperative requirement for sustained research endeavors aimed at comprehensively grasping and tackling the forthcoming challenges.In summation, our study underscores the pivotal significance of devising strategies to counteract the repercussions of climate change on plant biodiversity, thereby fostering the conservation of this indispensable asset for the well-being of generations to come.
The toxicological effects of climatic changes on the essential oil composition of Salvia officinalis will depend on the specific compounds present in the oil and their concentrations.Changes in climatic conditions can affect the concentration and composition of essential oil components, potentially leading to an altered toxicity profile.Some essential oil components may exhibit toxicity at high concentrations or with prolonged exposure, while others may be safe for use in certain concentrations.Therefore, it is essential to characterize the essential oil composition under different climatic conditions and conduct toxicity studies to evaluate the safety of its use.The method of administration (e.g., topical, oral, inhalation) can also influence the toxicity of essential oils, further emphasizing the need for additional studies to assess the toxicological effects of changes in essential oil composition due to climatic changes.

Figure 3 :
Figure 3: Mineral compositions of the three samples of Salvia officinalis at three different treatments.

Figure 6 :
Figure 6: Chromatogram of essential oil of Salvia officinalis in Treatment 1.

Figure 7 :
Figure 7: Chromatogram of essential oil of Salvia officinalis in Treatment 2.

Figure 8 :
Figure 8: Chromatogram of essential oil of Salvia officinalis in Treatment 3.

Table 1 :
Main compounds of Salvia officinalis essential oils in the three treatments