Phytochemical and Antioxidant Profile of the Medicinal Plant Melia azedarach Subjected to Water Deficit Conditions

Environmental stress triggered by climate change can alter the plant’s metabolite profile, which affects its physiology and performance. This is particularly important in medicinal species because their economic value depends on the richness of their phytocompounds. We aimed to characterize how water deficit modulated the medicinal species Melia azedarach’s lipophilic profile and antioxidant status. Young plants were exposed to water deficit for 20 days, and lipophilic metabolite profile and the antioxidant capacity were evaluated. Leaves of M. azedarach are rich in important fatty acids and oleamide. Water deficit increased the radical scavenging capacity, total phenol, flavonoids, and catechol pools, and the accumulation of β-sitosterol, myo-inositol, succinic acid, sucrose, d-glucose and derivatives, d-psicofuranose, d-(+)-fructofuranose, and the fatty acids stearic, α-linolenic, linoleic and palmitic acids. These responses are relevant to protecting the plant against climate change-related stress and also increase the nutritional and antioxidant quality of M. azedarach leaves.


Introduction
According to the Intergovernmental Panel on Climate Change [1], the increasing frequency of extreme weather events related to climate change, such as droughts and heatwaves, represents a severe threat to agriculture and forestry ecosystems. Moreover, the climatic projections forecast indicate that the climate change impacts on the functioning of the ecosystem will increase in intensity and frequency, largely due to the occurrence of more severe drought events [2]. Drought severely impacts plant development and productivity. The photosynthetic apparatus is one of the main targets of drought stress, decreasing net CO 2 assimilation rate and PSII efficiency [3]. Plants respond to drought stress by triggering several defense mechanisms. The activation of the antioxidant system, which comprises enzymatic and non-enzymatic defense mechanisms, plays an important role in controlling reactive oxygen species (ROS) excessive production. The enzymatic system includes several enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione peroxidase, and the non-enzymatic system comprises the ascorbate, glutathione, carotenoids, tocopherols, and phenolic compounds [4]. Within the phenolic compounds, the flavonoids are involved in plant physiological functions, demonstrating protective effects against biotic and abiotic stresses, including drought and UV-B radiation [5]. These The global recognition of the health benefits of plant phytocompounds promotes an increased use in the pharmaceutical and food industries and a search for novel natural antioxidant compounds [16]. This tendency highlights the need to tackle how climate change-related stressors modulate medicinal species' phytochemical composition and redox state [7,8,12,16]. Thus, we aim to study how M. azedarach plants adjust their metabolite and antioxidant status to deal with a water deficit condition and decipher how drought modulates leaves' bioactive compounds, promoting their nutritional and economic value. We hypothesized that M. azedarach metabolite plasticity to drought conditions involves an adjustment of the lipophilic metabolites and antioxidant status.

Results and Discussion
Climate change stressors can reduce plants' growth and phytocompound levels [14]. We reported previously that water deficit (WD) conditions reduce M. azedarach water potential, photosynthesis, and cell membrane stability but activate the antioxidant enzyme system (e.g., superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase) and increase the ascorbate pool [22].
A commonly used marker to evaluate the level of plant dehydration or water status is the leaf relative water content (RWC). In the present work, M. azedarach plants under well-watered (WW) conditions presented a leaf RWC of 91.1 ± 1.77%, while the ones under WD conditions showed a leaf RWC of 74.1 ± 4.19%. These data indicated that WD treatment induced severe stress since the RWC levels decreased to values below 80% [27]. Severe drought stress can cause several physiological impairments in plants, such as a decrease in photosynthesis, and when this stress condition is extended for a long period, plant growth and productivity can be compromised [28].
Long-chain alkanes are presented in high amounts in M. azedarach leaves, and WD conditions stimulated the accumulation of these compounds (Table 1). Leaf cuticular wax is mainly composed of long-chain alkanes, triterpenes, alcohols, aldehydes, and fatty acids [34]. The increase in long-chain alkanes in WD M. azedarach plants may represent an investment to strengthen cuticle structure and, therefore, a protective barrier against water loss.
Within the organic acids, M. azedarach leaves are particularly rich in the polyunsaturated fatty acids α-linolenic and linoleic acids, which are compounds that decrease the risk of human cardiovascular disease [35], and also in the saturated fatty acids, palmitic and stearic acids. Moreover, WD promoted the increase in the levels of Values are means ± standard deviation (n = 6). Asterisks indicate statistical differences between treatments (p < 0.05). exert many valuable positive effects, acting as an antidiabetic, anti-inflammatory, antioxidant, and anticancer agent [50]. Our work demonstrates that M. azedarach leaves can be enriched by myo-inositol through exposure to WD, potentially increasing the health benefits. Long-chain alkanes are presented in high amounts in M. azedarach leaves, and WD conditions stimulated the accumulation of these compounds (Table 1). Leaf cuticular wax is mainly composed of long-chain alkanes, triterpenes, alcohols, aldehydes, and fatty acids [34]. The increase in long-chain alkanes in WD M. azedarach plants may represent an investment to strengthen cuticle structure and, therefore, a protective barrier against water loss.
Within the organic acids, M. azedarach leaves are particularly rich in the polyunsaturated fatty acids α-linolenic and linoleic acids, which are compounds that decrease the risk of human cardiovascular disease [35], and also in the saturated fatty acids, palmitic and stearic acids. Moreover, WD promoted the increase in the levels of these fatty acids (except linoleic acid) ( Table 1). Studies in Populus simonii plants suggested that drought stress promotes the biosynthesis of long-chain fatty acids (LCFAs) through the upregulation of the enzyme acetyl-CoA carboxylase 1 involved in the elongation of LCFAs [36]. However, the mechanisms behind the fatty acid adjustments in response to drought are not well understood.
The TCA-cycle intermediate, succinic acid, was also stimulated by the WD treatment and is positively correlated with the antioxidant battery, ABTS, total phenols, catechols, and flavonoids (Table 1, Figure 2; r ≥ 0.85 and p ≤ 0.04). In fact, this acid (and its derivatives) has antioxidant properties and plays an important role as an antidiabetic agent for type 2 diabetes, lowering blood glucose and glycosylated hemoglobin [37]. In addition, several studies also demonstrated that the treatment with this acid improves motor behavior and ameliorates the cognitive deficits in animal models with neurodegenerative diseases [38].

Plant Material and Stress Treatment
Seeds of Melia azedarach Linn. were germinated in plastic pots (500 mL) with tur vermiculite (2:1). After germination, seedlings were grown in a climatic chamber at °C, a 16/8 h (day/night) photoperiod, and with a photosynthetic active radiation of 20 μmol m −2 s −1 (250 ± 20 μmol m −2 s −1 at the top of the plants) provided by Osram la To our knowledge, this is the first report of oleamide presence in M. azedarach leaves. The levels of oleamide were not affected by the WD treatments, despite some works referred this compound putative stress protector [8]. So, the role of oleamide in plant physiology remains unknown, possibly acting in growth and development regulation and pathogen interactions [39]. Moreover, oleamide is an emerging drug with several pharmacological properties (e.g., neuro-signaling and antitumoral) [40], and the high content of this amide (compared to other species, such as Coxi lachrymal-jobi [41], Lemna minor [42], Nigella sativa [43] and O. europaea [8]) make M. azedarach a valuable source of this compound.
The leaves of M. azedarach are also rich in sterols, and we identified sitosterol, stigmasterol, and campesterol (Table 1). Along with sphingolipids and glycerolipids, sterols are structural components of cell membranes that maintain membrane permeability and fluidity [15]. Under abiotic stress conditions, changes in sterols' total content, as well as variations in their profile (particularly the composition ratio of sitosterol and stigmasterol) can occur affecting the state of the cell membrane [29,44,45]. In the present work, only sitosterol increased in response to WD treatment, indicating that this treatment induced changes in the ratio of campesterol/sitosterol and sitosterol/stigmasterol, a process of stress compensation [44]. Nevertheless, considering that plant sterols reduce human total and LDL cholesterol by inhibiting cholesterol absorption [46], the increase in sitosterol levels by the WD treatment represents an improvement of the nutritional quality of M. azedarach leaves.
In M. azedarach, the levels of carbohydrates changed in response to WD, leading to a significant increase in sucrose, D-glucose and derivatives, D-psicofuranose, and D-(-)-fructofuranose ( Table 1). The increase in the sugars pools is a typical response of plants to drought stress [3,13]. For instance, sucrose and glucose increase under water deficit conditions can act as osmolytes to maintain cell turgor, while the increase in other sugars, such as fructose, can be linked with the upregulation of some secondary metabolite synthesis [47]. Moreover, the increase in these soluble sugars in M. azedarach is positively correlated with total phenols, catechols, flavonoids, and total antioxidant activity (TAA) (evaluated by the ABTS assay) (except for the case of D-psicofuranose) (Figure 2; r ≥ 0.83 and p ≤ 0.04), suggesting their involvement in antioxidant responses, possibly helping in ROS balance and oxidative stress control, membrane protection, and enzyme/protein stabilization as reported by You and Chan [48]. Despite this role in stress tolerance, the increase in carbohydrate contents also represents an augmenting of energy availability, enriching the nutritional level of M. azedarach plants.
The polyalcohol myo-inositol is a plant signaling molecule and under drought conditions can act in osmoregulation processes and/or in oxidative control (e.g., ROS scavenger) [49]. This protective role seems to be important in M. azedarach plants, as their levels increase under WD conditions and a positive correlation with the antioxidant battery, TAA, total phenols, flavonoids, and catechols was observed (Table 1, Figure 2; r ≥ 0.85 and p ≤ 0.03). Additionally, in humans, myo-inositol and its phosphate derivatives exert many valuable positive effects, acting as an antidiabetic, anti-inflammatory, antioxidant, and anticancer agent [50]. Our work demonstrates that M. azedarach leaves can be enriched by myo-inositol through exposure to WD, potentially increasing the health benefits.

Plant Material and Stress Treatment
Seeds of Melia azedarach Linn. were germinated in plastic pots (500 mL) with turf and vermiculite (2:1). After germination, seedlings were grown in a climatic chamber at 20 ± 2 • C, a 16/8 h (day/night) photoperiod, and with a photosynthetic active radiation of 500 ± 20 µmol m −2 s −1 (250 ± 20 µmol m −2 s −1 at the top of the plants) provided by Osram lamps. Light intensity in the climate chamber was measured with the external PAR sensor of the LI-6400XT (Portable photosynthesis system, LI-COR Bioscience, Lincon, NE, USA). Before the beginning of the water deficit treatment (WD), all plants were well-watered (100% field capacity). Two-month-old plants (60 days-old) with a height of 13.5 ± 3.2 cm were randomly assigned as well-watered (WW) (plants watered at 100% of field capacity (n = 12)), and WD plants were watered at 20% of field capacity for 20 days (n = 12). The pots were watered at 100% or 20% field capacity by restoring the quantity of water lost every second day (water lost was measured by weighing the pots with a scale). After these treatments' leaves from the third top node (the youngest fully expanded) were collected for determination of the relative water content. Additionally, leaf samples (collected above the second node from the bottom) were immediately frozen in liquid nitrogen and stored at −80 • C.

Plant Water Status
Plant water status was determined by measuring the leaf relative water content (RWC). The leaves' fresh weight was determined, and then the leaves were immersed in distilled water for 24 h at 4 • C. After this period, the turgid weight was determined, and the leaves were dried to calculate the dry weight. The percentage of RWC was determined using the formula: (FW-DW)/(TW-DW) × 100, where the FW was the leaf fresh weight, the DW the leaf dry weight, and the TW the leaf turgid weight.

Total Antioxidant Activity, Total Polyphenols, Catechols, and Flavonoids
Approximately 100 mg of frozen leaf samples were ground with 1 mL of methanol [8] for the determination of the total antioxidant activity, total polyphenols, catechols, and flavonoids. After incubation for 30 min at 40 • C the homogenate was centrifuged (5000× g for 5 min at 4 • C) and the supernatants were used for analysis.
For the total antioxidant activity (TAA), determined by the ABTS +• free cation radical scavenging activity method, one aliquot of the leaf extract (10 µL) was incubated for 10 min at 30 • C with 200 µL of ABTS (2,20-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) as described by Re et al. [51]. Then, the absorbance of the supernatant was recorded at 734 nm, and the total antioxidant activity was calculated using a calibration curve for the gallic acid. Total antioxidant activity is expressed as µM Gallic Acid Equivalents per mg of dry extract.
For the determination of total polyphenols, the Folin-Ciocalteu method was used. One aliquot of the leaf extract (20 µL) was homogenated with 405 µL of a Folin-Ciocalteu reagent, and 75 µL Na 2 CO 3 (20%). After an incubation period of 30 min (at 37 • C) the supernatant was read at 765 nm. Total polyphenols content was determined based on a gallic acid calibration curve. Total polyphenols are expressed as µM Gallic Acid Equivalent per mg of dry extract.
Catechols were determined using the molybdate assay. A sodium molybdate solution (5%) was prepared in 50% methanol, and 40 µL were mixed with 160 µL of leaf extract [52]. After incubation at 20 • C for 15 min, the absorbance of the supernatant was recorded at 370 nm. Catechol contents were determined based on a gallic acid calibration curve. Catechols are expressed as µM Gallic Acid Equivalents per mg of dry extract.
For flavonoid determination, one aliquot of the extract (37.5 µL) was homogenized with methanol, 75 µL of NaNO 2 at 5% and 75 µL of AlCl 3 at 10%. After 6 min in dark, 125 µL of 1 M NaOH was added, and the absorbance was recorded at 510 nm. Flavonoids content was determined based on a rutin calibration curve. Total flavonoids are expressed as µM Rutin Equivalents per mg of dry extract.

Extraction of Metabolites and Chromatography Analysis
For each treatment (WW and WD) leaves of M. azedarach were ground in a mill. Leaf powder (10 g) was mixed with 100 mL of hexane. The leaf metabolites were extracted at room temperature in three cycles of magnetic stirring for 72 h. The hexane was evaporated using a rotatory evaporator and the extract solutions were left to dry. Before analysis samples were silylated. In a glass tube, 250 µL of pyridine, 250 µL of N,Obis(trimethylsilyl)trifluoroacetamide, 50 µL of trimethylsilyl chloride, 450 µL of the leaf extract (~10 mg mL −1 ), and 450 µL of tetracosane (1.4 mmol L −1 ) were mixed and placed at 70 • C (in a water bath) for 35 min. After that, the silylated extracts were injected into the gas chromatography mass spectrometer (QP2010 Ultra Shimadzu, DB-5-J&W capillary column of 30 m × 0.25 mm id and a film thickness of 0.25 µm) as described by Dias et al. [7,8].
Briefly, helium was used as carrier gas (35 cm s −1 ) and the initial temperature was fixed to 80 • C for 5 min with a temperature rate of 4 • C min −1 up to 285 • C for 10 min. The injector worked at a temperature of 250 • C and transfer-line temperature was 290 • C with a split ratio of 1:50. The mass spectrometer functioned in the electron impact mode with energy of 70 eV, and data were stored at a rate of 1 scan s −1 (m/z 33-750). The temperature of the ion source was 250 • C. The peak obtained in the chromatograms were identified by comparison with the library entries of mass spectra database (WILEY Registry TM of Mass Spectra Data and NIST14 Mass spectral). For metabolite quantification, calibration curves of pure compounds representative of the chemical families identified in these extracts (palmitic acid, cholesterol, maltose, octadecanol, and tetradecane) were prepared and injected in the GC-MS as described above for the leaf extracts.

Statistical Analysis
Data were analyzed by t-test at a significance level set to 0.05 using the statistic program SigmaStat for Windows version 3.1 (Systat Software, San Jose, CA, USA). Pearson correlations were performed in the SigmaStat for Windows version 3.1 (Systat Software, San Jose, CA, USA). Fold change was determined in Microsoft ® Excel for windows (version 10).

Conclusions
We characterized the antioxidant status (total antioxidant activity, catechols, total phenols, and flavonoids) and the leaf lipophilic profile of the multipurpose species M. azedarach and demonstrated that leaves are a source of important fatty acids, polyalcohol, sterols, TCA cycle-related metabolites, amides, and carbohydrates. Moreover, we demonstrated for the first time in this species that metabolite adjustments and the increase in the total antioxidant pool (total antioxidant activity, polyphenols, flavonoids, and catechols) represent a protective response to drought, helping M. azedarach plants to survive and tolerate this stress. Drought conditions can stimulate the accumulation of important compounds, contributing to increase the nutritional and bioactive richness of this species and thus its commercial value.