Influence of Soil Nutrient Toxicity and Deficiency from Three Ecuadorian Climatic Regions on the Variation of Biological, Metabolic, and Nutritional Properties of Moringa oleifera Lam.

Moringa oleifera Lam. contains numerous essential constituents found in all plant parts (leaves, pods, and seeds). From all its edible parts, the leaf represents an effective remedy with high potential for medicinal applications. Ecuador is part of the new promising cultivation areas for Moringa, and therefore our study is emphasized to determine the influence of soil nutrition, toxicity (excess), and deficiency, from three main areas of this country, correlated with its climatic characteristics, on the mineral components, bioactive compounds’ synthesis, and antioxidant capacity of Moringa. Different analyses were performed in soil and especially leaf samples for phytochemical content, antioxidant activity, calcium, protein, and vitamin C determination to identify the relationship between soil nutrients, abiotic conditions, and the therapeutic potential of this species cultivated in Ecuador. The obtained values using methods such as DPPH, FRAP, and ABTS showed a high antioxidant capacity of the leaves from the Coastal Ecuadorian region, related with total phenolic compounds’ content (through the Folin–Ciocalteu method) and flavonoids in samples, with results obtained under the positive influence of high soil nutrients such as Ca, Mg, Mn, and Fe. We can conclude that M. oleifera from the coastal area of Ecuador presents the right environmental and soil conditions to positively influence its mineral and phytochemical content, making it suitable for incorporation into foods and medicines to solve the nutritional and medical problems in Ecuador and worldwide.


Introduction
M. oleifera represents one of the most known species of the Moringa genus, native to India and cultivated across the world, a fast-growing, drought-resistant plant species, that tolerates a wide range of soil conditions. It was acclimatized to the tropics and subtropics, being known as horse reddish or ben tree [1]. Recently, research concerning the growing/acclimatization of this species in Europe was also reported [2].
M. oleifera is a very important plant species used in traditional phytomedicine due to its nutrients. Various phytochemicals can be found among all parts of the plant, which makes them all edible [3]. It is considered a highly nutritional food, providing vitamins (vitamin C, vitamin A), minerals (calcium, potassium, iron, zinc), protein, phytosterols, amino acids, and PUFAs [4]. Scientific research outlines the appreciation of M. oleifera's uses for different Considering the medicinal properties of M. oleifera and its widespread cultivation in different areas of Ecuador, it is important to evaluate, as in this study, the influence of the abiotic components such as soil nutrients (toxicity (excess) and deficiency) and ambient conditions characteristic for three areas, Andean, Amazonian, and Coastal regions, on the synthesis of constituents responsible for the biological effect and on its components with nutritional properties. In this view, the objective of the present study was to test the variability of the antioxidant potential and biochemical and nutritional constituents of Moringa under the influence of soil macro-and micro-nutrient toxicity (excess levels) and deficiency, under local climatic conditions characteristic of three different Ecuadorian regions.

Sample Collection for Soil Analysis
For the soil analysis, a zig-zag sampling was carried out, trying to cover the entire lot to be studied belonging to elected Provinces from the three Ecuadorian areas. For this purpose, 20 subsamples, representing 10 cores collected from a 30 cm depth, were taken for each lot with a shovel, and then all the samples were mixed and transported in a cooler, in plastic bags, to the laboratory for further soil analysis using the standard procedures of the Agricultural Research and Education Center (AREC), Belle Glade. The pH of the soil was carried out in a 1:2.5 dilution (1 part soil in 2.5 parts deionized water).
Plant Material was represented by the fresh young leaves collected from middle M. oleifera branches. The samples were selected from three Ecuadorian growing regions: Andean, Coastal, and Amazonian, chosen based on different altitudes and representative climatic characteristics, respectively ( Figure 1).

Sample Collection for Foliar Analysis
For the foliar analysis, Moringa leaves were collected from three different regions of Ecuador: the Coastal region, in the province of Esmeraldas at 10 MASL (meters above sea level), with a mollisol-type soil, the Andean region, at 200 MASL (Cotopaxi province, with a selected area with the lowest altitude in this region), with an inceptisol-type soil, and from the Amazonian region, represented by Napo province at 577 MASL, with an inceptisol-type soil. The study was carried out in already established plantations, with 2 × 2 m

Sample Collection for Foliar Analysis
For the foliar analysis, Moringa leaves were collected from three different regions of Ecuador: the Coastal region, in the province of Esmeraldas at 10 MASL (meters above sea level), with a mollisol-type soil, the Andean region, at 200 MASL (Cotopaxi province, with a selected area with the lowest altitude in this region), with an inceptisol-type soil, and from the Amazonian region, represented by Napo province at 577 MASL, with an inceptisoltype soil. The study was carried out in already established plantations, with 2 × 2 m planting distances and an average age of four years for all studied areas. The collected leaves were taken from the middle third of the trees, out of a total of 60 fully developed compound leaves, collected from 15 trees and transported in the right conditions (paper bags) for analysis. The sampling date was carried out in August 2021. In the laboratory, the leaves were washed (with neutral soap and deionized double-distilled water) and dried for 24 h at 80 • C. After drying, the total N (by micro-Kjeldahl) was analyzed from the Toxics 2022, 10, 661 4 of 14 grounded material. The elements K, Ca, Mg, Fe, Zn, and Cu were analyzed by atomic absorption spectrophotometry, P was identified by a colorimetric method using vanadate molybdate reaction, Bo through a turbidimetric method, and S through photometry. The standards developed by Marrocos et al. [27] were used to compare the results concerning the macronutrients and micronutrients.
To assess the effect of regional variations in the phenolic and flavonoid content of M. oleifera, the samples were collected in the same conditions (time-in the morning hours, and maturity-plants older than three months) from three Ecuadorian regions characterized by different climatic and nutrient characteristics.

Extracts' Preparation
The shade-dried leaves were macerated with methanol for 24 h at room temperature. A rotary evaporator was used for evaporation of the liquid, and the extracts were freeze-dried. The process was repeated 3 times.

Quantification of Total Phenolic Content (TPC)
The method used for TPC quantification was based on the methodology described by Madaan et al. [28], with modifications, consisting of the Folin-Ciocalteau colorimetric method [29]: A portion of the Moringa extract (diluted to 5 mL of Milli-Q water) was added to the 1.5 mL Folin-Ciocalteau reagent, and kept for 5 min for reaction at room temperature (25 • C). After that, 2 mL of a 100 g/L solution of Na 2 CO 3 was added. The absorbance was measured at 760 nm in a spectrophotometer against a blank (without extract) after 30 min. A calibration curve with standard solutions within a range of 50-250 mg/L was obtained using gallic acid, with a correlation coefficient of 0.9968 (DL = 0.7643 mg/L, QL = 2.5477 mg/L (5.9701%), n = 6, p = 3.77 × 10 6 ). The results were expressed as mg GAE/100 g DW.

Quantification of Total Flavonoid Content (TFC)
The method was realized using the Dowd method [30]. A volume of 1 mL of Moringa extract solution (25-200 µg/mL) was added to 0.3 mL of 10% (v/v) AlCl 3 solution in methanol, 0.2 mL (1 M) of potassium acetate, and 5.6 mL of distilled water, and left to rest for 10 min at room temperature. The absorbance was read at 430 nm. A range of 1-100 mg/L of quercetin (QE) was used as a standard for the calibration curve, with a coefficient of 0.9991 (DL = 0.4752 mg/L, QL = 1.5843 mg/L (11.5003%), y = 0.0167x + 0.0245, n = 7, p = 8.79 × 10 9 ). The TFC results were expressed as mg quercetin equivalents/100 g Moringa extract dry weight.

FRAP Assay
The method used for the FRAP assay was described by Benzie et al. [31]. Acetate buffer (300 mM, pH 3.6), a solution of 10 mM of TPTZ in 40 mM of HCl, and 20 mM of FeCl 3 at 10:1:1 (v/v/v) were mixed to obtain the FRAP reagent. The sample was incubated with 2 mL of the FRAP solution at 37 • C. The FRAP solution was prepared the same day by mixing the following reagents: acetate buffer (25 mL), TPTZ solution (5 mL), and FeCl 3 ·6H 2 O solution (10 mL), for 30 min in the dark. The absorbance was read at 593 nm. Six standard solutions of ferric sulfate (Fe 2 SO 3 ) in the range of 0.10-1.00 mmol/L were used for the calibration curve (y = 1.5431x + 0.0004, R 2 = 0.9964, n = 7, p = 3.29 × 10 7 , DL = 0.005939 mmol/L, QL = 0.01979 mmol/L (0.3740%)).

ABTS Assay
The method described by Loizzo et al. [32] was used for the determination of ABTS free radical scavenging activity by mixing ABTS (2 mM) and potassium persulfate (70 mM). The mix was kept at room temperature in the dark overnight. To obtain an absorbance of 0.700 ± 0.005 at 734 nm, the ABTS solution was diluted with 80% methanol, and 100 µL of appropriately diluted samples was added to 2 mL of ABTS solution. The absorbance was recorded after 1 min of incubation at room temperature at 734 nm. TROLOX standard solutions in the range of 10.0-150.0 mg/L were used for the standard curve, with the equation y = 0.5426x − 0.0069 (R 2 = 0.9903, n = 8, p = 0.00102, DL = 0.01796 mg/L, QL = 0.05989 mg/L (0.3250%)).

DPPH Assay
The method presented by Simirgiotis et al. [33] was used to determine the DPPH • radical scavenging activity, consisting in 50 µL of extract added with 2 mL of fresh 0.1 mM solution of DPPH in methanol, kept at room temperature in the dark. The absorbance of the mix was measured after 30 min at 517 nm. The DPPH scavenging ability (%) was calculated with the formula (A control − A sample /A control ) × 100. The IC 50 (concentration of sample required to scavenge 50% of the DPPH free radicals) values were calculated. A low IC 50 value means a powerful antioxidant activity. Then, 10.0-100.0 mg/L of TROLOX standard was used for the calibration curve expressed by the equation y = 0.9081x + 0.08 (R 2 = 0.9542, n = 7, p = 0.000151, DL = 0.00809 mg/L, QL = 0.02698 mg/L (0.2970%)).
Calcium determination was realized by atomic absorption spectrophotometry (AAS) according to the method presented by Allen [34], whereby 0.1 g of sample (dry leaves) was ashed at 525 • C for 8 h and dissolved with 0.1% HCl and 0.5% lanthanum oxide (La 2 O 3 ).
Protein content was estimated by the Kjeldahl method [35] at the nitrogen conversion factor of 6.25, consisting in a mixture of 2 g of dry sample, 7 g of the catalytic mixture, and 15 mL of 96% sulfuric acid. Tubes were placed in a heating block (450 • C) for 1 h. A 4 min distillation was carried out after cooling with the addition of 32% NaOH until the neutralization of the mixture. The distillation product had 0.1 N HCl. The total protein content (%) was calculated by the formula: the total volume (mL) used to neutralize the mix × 0.1 × 1.4 × 5.7/W the weight of the sample Vitamin C content (expressed as mg of ascorbic acid/100 g of fresh weight) was determined using the procedure reported by Xiao et al. [36] based on 2.6-dichloroindophenyl, and the content was measured with the titrimetric method.

Statistical Analysis
For the data analysis of soil and leaf samples, a comparison was made with the works of different authors [37][38][39][40] who have worked with M. oleifera. For the soils, the sufficiency interpretation of the INIAP laboratory was used, and for the determination of the relationships between the cations Ca 2+ , K + , and Mg 2+ , a table of general relationships that predict the possible deficiency of the element in the plant was used. To analyze the phytochemical and antioxidant activity, three replications were used, and the results are expressed as means ± SD (standard deviation). Spearman's correlations were used to determine the relationship between secondary metabolite content and antioxidant capacities. The Kruskal-Wallis test with post-hoc Dun multiple pairwise comparisons was applied to emphasize the differences, taking into account the geographical origin of the samples of secondary metabolites and their antioxidant capacities. The Kruskal-Wallis test is a non-parametric method alternative of one-way ANOVA. The statistical analysis was performed with XL-STAT pro (2013) [41]. Principal component analysis was used to compare the samples using values of secondary metabolites and antioxidant activity.

Soil and Leaf Analysis
The results concerning the nutrients from the soil and foliar samples are presented in Tables 1 and 2.  The Coastal region presented a higher nitrogen content (4.72%) than the other regions (p = 0.00381). The average of the Ecuadorian regions was 4.60, in line with the values identified by Moyo et al. [37] and higher than those of Valdez-Solana et al. [38] (Table 1). For phosphorus, the results were very similar in the three regions, with an average of 0.32%, close to those of Moyo et al. [37] and Witt and Aslam et al. [39,40]. The highest potassium content (2.44%) was found in the Amazonian followed by the Coastal and the Andean regions, respectively. The average of the three zones under study was 2.31%, and these values are directly related to the potassium levels in the soil ( Table 2).
The soil cation ratios K/Mg, (Ca+Mg)/K, and Ca/K for the Coastal and Andean regions indicated a potassium deficiency. In the Amazonian region, the ratio between (Ca+Mg) and K is the only one that shows deficiency for this nutrient. For sulfur, the average in the three areas under study was 0.57%, with the Coastal and Amazonian regions having the highest percentages (Table 3). Calcium is an element that is closely related to the amount of potassium, magnesium, and other cations. In this study, the average for the three studied areas was 2.23%, with the Coastal region having the highest content, followed by the other two regions. This average was lower compared with the results of Moyo et al. [37], Valdez-Solana et al. [38], and Aslam et al. [39], and slightly higher than those of Witt [40].
In the case of magnesium, the average for the three study regions was 0.26%, which is lower than that of the other authors, except Aslam et al. [39]. The Coastal and Amazonian regions have lower Mg content than the Andean region. This can be attributed to the cationic Ca/Mg ratios, which show a probable magnesium deficiency in the soil in the three regions.
In the foliar analysis, the iron value was higher in the Andean region compared to the Coastal and Amazonian regions. The average of the three regions under study was 108.80 mg/kg, a low level compared to the data of Moyo et al., Valdez-Solana et al., and Witt and Aslam et al. [37][38][39][40].
Leaf zinc contents on average were low compared to those of Moyo et al. and Witt and Aslam et al. [37][38][39][40], and higher than those of Valdez-Solana et al. [38]. However, in the soil analysis, zinc cation was found at a high level in the Coastal region and was optimal in the Amazonian region. The high contents of calcium and iron cations inhibit the absorption of zinc cation in the plant.
The copper content in leaves was similar, and the average of the three regions was close to the ranges of Moyo et al. [37], Witt, and Aslam et al. [39,40], and lower than those of Valdez-Solana et al. [38]. This also coincides with the soil values.

Phytochemical Analysis
The total phenolic and flavonoid contents were determined, and the Kruskal-Wallis test showed significant geographical differences in TPC (p = 0.027), with the Dunn post-hoc test (p = 0.007), between the Coastal and Amazonian regions. The same results were found for TFC (p = 0.027), with differences between the Coastal and Amazonian regions (Dunn posthoc test, p = 0.007). The highest TPC values were established in the sample from the Coastal region (152.6947 ± 4.0104 mg/100 g DW) and the lowest (43.6796 ± 4.0477 mg/100 g DW) in the Amazonian region. The same trend was observed for TFC, with the highest content being in the Coastal region (74.133 ± 4.618 mg QE/100 g DW) and the lowest in the Amazonian region (20.7 ± 0.917 mg QE/100 g DW) ( Table 4).  Table 5 shows the antioxidant activity of Moringa samples collected from the three Ecuadorian regions. Significant differences between Coastal and Amazonian regions were observed. All three methods used for antioxidant capacity (FRAP, ABTS, and DPPH assays) showed the same trend, with the highest antioxidant capacity being found in the samples collected from the Coastal followed by the Andean and Amazonian regions (Table 5). On the other hand, in the samples from the Coastal region, it was noticed that TFC was significantly correlated with FRAP activity (p < 0.0001) and DPPH (p < 0.0001). In the Andean region, TFC was correlated with DPPH (p < 0.0001), and in the Amazonian region with FRAP (p < 0.0001). Concerning the TPC, no significant correlation was observed between TPC and antioxidant activity in the studied regions.

Calcium and Protein Content Analysis in Moringa Leaves
The calcium content of the samples varied from 1.78% ± 0.05% in the Andean region to 2.50% ± 0.04% in the Coastal region (Table 4). Significant differences (Kruskal-Wallis test, p = 0.027) were observed between Amazonian and Coastal regions (Dunn post-hoc test, p = 0.007) concerning protein content (%). Leaves from the Coastal region were characterized by the highest protein content, correlated with the highest calcium content. Additionally, leaves from the Coastal region were characterized by highest amount of vitamin C/100 g leaves, followed by the Andean and Amazonian regions ( Table 6).

Multivariate Analysis
Moringa samples collected in the Coastal region showed positive PC1 scores, associated with TPC, calcium, protein, and DPPH ( Figure 2). The samples collected in the Andean region presented high protein and TFC associated with DPPH ( Figure 3). The protein content of samples from the Amazonian region was correlated with Mn, Mg, and Fe levels. Additionally, TPC and TFC were influenced by these macro-elements (Figure 4).

Discussion
The nutritive compounds as well as secondary metabolites depend on different factors (meteorological, geographical, toxicity (excess), and deficiency of macro-and micronutrients in soil) [38]. In general, the nutrient quality of the plants is the same, but is influenced by geographical location and environmental conditions [37]. For this reason, for each region analyzed in our investigation there are different nutrients that influence the accumulation of metabolites, protein, and vitamins in the Moringa leaves. In the case of

Discussion
The nutritive compounds as well as secondary metabolites depend on different factors (meteorological, geographical, toxicity (excess), and deficiency of macro-and micronutrients in soil) [38]. In general, the nutrient quality of the plants is the same, but is influenced by geographical location and environmental conditions [37]. For this reason, for each region analyzed in our investigation there are different nutrients that influence the accumulation of metabolites, protein, and vitamins in the Moringa leaves. In the case of the Coastal region, the metabolites, as phenolic compounds (that also give the antioxidant property of Moringa), are influenced positively by Ca in leaves (calcium uptake from the soil). The Coastal region, based on its climatic characteristics, can provide an efficient absorption of this element due to the help of transpiration (calcium rises to the level of the leaves when the plant transpires). This region is the warmest among all the regions described, and the collection was performed in August when the low humidity made the plant sweat so it could accumulate calcium in the leaves. In the other areas of Ecuador where the climatic conditions do not support the plant's transpiration, the accumulation of calcium in leaves is not supported. Murtadha et al. [42] showed that the absorption of calcium may be slowed down by high temperatures and relative humidity. Ca 2+ has a role in signaling, helping in the regulation of genes for biosynthesis of polyphenols [43]. For the Amazonian region, the protein and secondary metabolite contents in the Moringa leaves were influenced by nutrients such as Mn, Mg, and Fe. These elements were proven to affect plant defense metabolites, having interactive effects on the development of phenols and flavonoids [44], which is a strong reason for secondary metabolites' accumulation in the leaves of Moringa in the presence of these nutrients. The soil nutrients Mn, Mg, and Fe can be found in similar concentrations in Coastal and Amazonian regions and can positively influence the accumulation of desirable compounds, such as phenolics. We can observe a difference between the metabolites' content in these two regions due to the environmental factors characteristic of each area, responsible for fluctuations in Moringa's nutritive qualities and secondary metabolites' accumulation due to different nutrient uptake or nutrient translocation in the plant. The abiotic conditions of the Amazonian region are characterized by an increased relative humidity (RH) and high moisture content in the atmosphere, reducing the nutrient translocation through reduction of the xylem volume. The Coastal area, in comparison, presents a low RH and the atmosphere contains more moisture, and in this way the driving force for transpiration is increased, improving the translocation of minerals in plants, especially the nutrients responsible for metabolite accumulation.
Our results showed that the average values of nutrients from soil samples collected from Ecuadorian regions were similar to those in other works. The average nitrogen and phosphorus contents were closer to the values identified by Moyo et al. [37]. The nitrogen content from the Coastal region is directly related to the protein content. Phosphorus content was very similar in the three regions.
Despite the high (toxic) iron content in the soil from the Coastal region, this element is affected in its absorption by the high contents of calcium, manganese, and zinc present in the soil. Additionally, zinc is found at a high level in the Coastal region and is optimal in the Amazonian region, and the high contents of calcium and iron inhibit the absorption of zinc in the plant.
The so-called miracle tree was already widely used in ancient times for its nutritious and healthy properties due to its diverse pharmacological activity [4]. The present findings provide an insight into the polyphenol and flavonoid contents in M. oleifera leaves depending on the cultivation area. Previous studies reported that M. oleifera extract is rich in phenolic and flavonoid compounds. The quercetin, kaempferol, and rutin described in M. oleifera are associated with environmental and growing factors [45]. Leaves from the Coastal region were abundant in phenols and flavonoid contents. The amounts of phenols found in the leaves collected from this area were similar to those collected in Mexico [46], but lower than those from Southwestern Algeria and Haiti (2813 ± 51 mg GAE/100 g DW) [47]. The case of flavonoid content followed the same trend as phenolic content, with the highest flavonoid concentration expressed in the leaves collected from the Coastal region, and the lowest reported in the Amazonian region. Our study confirmed the importance of growing regions on the synthesis of TPC and TFC, and was compared with other results, where these constituents from lower altitudes were higher than in other regions [46]. In previous reports, flavonoids were represented by quercetin and kaempferol derivates [47].
M. oleifera demonstrates, in all the scientific reports including ours, that it is a plant booming in polyphenols, with strong antioxidant properties, and may decrease the oxidative damage of the tissues by scavenging free radicals [4]. The Moringa genus is characterized by high antioxidant activity caused by its high content of bioactive polyphenols, a fact also demonstrated in our experiment that could be visualized on the leaves, with the highest polyphenolic content in the Coastal region, which also expressed the highest antioxidant capacity compared to the leaves collected from the other Ecuadorian regions. Three different methods (DPPH, ABTS, and FRAP) were used to establish the antioxidant potential of M. oleifera leaves from different geographical Ecuadorian regions. The results of our study indicate a descending order of antioxidant activity from leaves collected in the Coastal region < Andean region < Amazonian region, with the highest values of antioxidant activity being similar to those in some other investigations [48].
The Moringa leaves collected from the three Ecuadorian regions contained relatively high amounts of the examined minerals. Our results showed that the highest value of calcium was in leaves collected from the Coastal region, which was found to be a good source of calcium (2.50%), with the obtained values being similar to others reported in a bibliography about Moringa sp. leaves' calcium content [48]. A diet containing vitamin C helps to maintain the human body's health, being an essential micronutrient for normal functions, supplied by fruits and vegetables. Vitamin C is a potent antioxidant that can eradicate different reactive oxygen species, has a role in stress resistance, keeps membrane-bound antioxidant α-tocopherol in a reduced state, and is a substrate for oxalate and tartrate biosynthesis [49]. In our study, the highest vitamin C concentration could be found in the leaves from the Coastal region, which also coincides with a high antioxidant activity, demonstrating the contribution of vitamin C to this desirable plant property. Our results are in line with earlier findings on reported Moringa oleifera vitamin C content [49]. Proteins are indispensable for the normal functions of the body. The implication of proteins in the growth and maintenance of tissues is well-known. Additionally, proteins are implicated in functions of the body as chemical messengers (such as hormones), aiding the communication between cells, tissues, and organs, providing stiffness and rigidity. Proteins have a vital role in regulating acid and base concentrations in blood and other fluids, helping to form the immunoglobulins, or antibodies, and in this way helping the organism to fight infections [50]. Moringa is recognized to contain proteins, and from this point of view, its inclusion in humans' and animals' diets could represent a curative and therapeutic therapy. The trend is different than in the case of secondary metabolites because although the highest protein content was found in leaves from the same Coastal region, in second place is the Amazonian region, while the Andean region was second highest for secondary metabolites. A possible explanation could be that the highest N content was found in the soils from the Amazonian region, compared to the deficit of this nutrient in the Andean region, as revealed by our experiment. The importance of N for the proteins consists of the use of nitrogen translocated from the soil in both the xylem and phloem for plant protein synthesis [51].
Even if the majority of soil nutrients are in a higher content in the Amazonian region, the Moringa leaves with high biochemical, nutritional, and therapeutic properties were found in the Coastal region. These findings may be explained by the interaction between climatic characteristics (temperature, humidity, etc.) and soil type.
Through this research, we support the traditional idea that Moringa represents a good source for the human and animal diet, providing useful constituents such as vitamins, proteins, and minerals. The effects of abiotic components from three Ecuadorian climatic regions on the biochemical and nutritional properties of Moringa oleifera were studied. Our research represents a general picture of environmental factors and nutrients of three Ecuadorian regions (Amazonian, Andean, and Coastal), responsible for fluctuations in the nutrient and metabolomics composition of M. oleifera. There were variations observed in phytochemical content, antioxidant activity, calcium, protein, and vitamin C content in samples from different regions due to abiotic factors such as relative humidity, which influence the soil nutrients' uptake and translation into the plant, with nutrients such as Mg, Mn, and Fe being responsible for the accumulation of metabolites of interest, such as phenolics. Our results are in accordance with those found in Aloe vera, where Mg, Fe, and Mn levels are directly corelated with pharmaceutical compounds represented by TPC and TFC [52]. Additionally, the authors of [53] underline that the application of compost increases the TPC and TFC.
The highest biochemical and nutritional properties were found in Moringa leaves cultivated in the Coastal region, due to the low relative humidity that triggered the high transpiration rate and implicit minerals' uptake, as well as the low altitude that was demonstrated to have a positive influence on secondary metabolites' accumulation in Moringa. Such information has high importance in the context of developing new areas for the cultivation of plants such as Moringa with a high therapeutic potential. The investigation outlined the importance of the environmental and nutritional conditions (toxicity (excess) and deficiency) to obtain high-quality and high-quantity bioactive compounds, which can increase the nutritive qualities of M. oleifera.