Secondary Metabolites Evaluation On Medicago Sativa L. Plants Treated By (Fe, Ag, Cu)-TiO2 Nanostructured Materials Towards Sustainable Agriculture.


 The present study analyzed Medicago sativa L. crops irrigated by TiO2 in the anatase phase and TiO2 doped with Ag, Fe, Cu ions at 0.1%w synthesized by the sol-gel method (SG) and the sol-gel coupled with microwaves (Mw-SG). The materials were added to the irrigation water at different concentrations (50, 100, and 500 ppm). Affectation levels were observed by measuring stem morphology, chlorophyll content, secondary metabolite content (total phenolic, flavonoids), and antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl radical assay. The results revealed a reduction in stem and leave size at different concentrations and chlorophyll content, showing no correspondence with the applied dose. Nanostructured materials in the soil generated nitrogen, boron, and potassium deficiencies observed in leaves. No linear effect related to the increase in total gallic acid, rutin content, and antioxidant activity dependent on concentration was determined. The stress factor depended on the dopant type, generating different stress levels at the three organs investigated (leaves, roots, stem). The metabolite augmentation was mainly obtained at 100 and 500 ppm for both synthesis methods.


Introduction
Nanostructured nanomaterials nd many applications in agriculture. They can serve as fertilizers to promote better crop development due to their high selectivity and availability (Zul qar et al., 2019). There is also the possibility of using nanomaterials as pesticidal agents for protection against microorganisms or other detrimental species (Fraceto et al., 2020), or as well as the protection against biotic and abiotic stresses for the development of the plants and the improvement in the production of secondary metabolites, which allows generating added value products (Khan et al., 2021).
Nowadays, the excessive use of nanomaterials is worrying without considering the interactions that they may have with multiple organisms (Millán-Chiu et al., 2020), generating toxic reactions that can compromise the development of plants, microorganisms, and animals. An area such as Nanotoxicology is developed to understand the toxicological mechanisms of various nanomaterials (Ananthi et al., 2020; Zia-ur-Rehman et al., 2018). Which are different from their bulk counterparts, the study and understanding of these mechanisms will make it possible to better use of the nanomaterials, preventing these new products from generating con icts with various organisms.
Titanium dioxide (TiO 2 ) is one of the most produced nanomaterials worldwide (Piccinno et al., 2012). Its applications vary from solar cells to the photocatalytic removal of persistent pollutants (Haider et al., 2019). The increase in its production means a greater risk for introducing nanomaterials (NMs) in the environment without considering the expected applications for agriculture (Klaine et al., 2008). An increasing presence in the environment has meant increased studies related to the toxicological effects that TiO 2 -NMs can have when interacting with living organisms (Ananthi et al., 2020). The interaction mechanisms vary and depend on the nanomaterial characteristics compared to their bulk, making it a complex study (Zia-ur-Rehman et al., 2018). The distribution of sizes, morphology, dose, concentration, and the crystalline phase determines the behavior of the TiO 2 -NMs when they encounter living organisms (Millán-Chiu et al., 2020; Zia-ur-Rehman et al., 2018). With living organisms, modifying the synthesis of a material to obtain new physiochemical characteristics such as a smaller size, higher crystalline quality, or different morphology will differ in the effects when they are exposed, generating a new toxicological pro le (Hossain et al., 2020).
Most of those characteristics are conferred by the synthesis methodology, such as size, which de nes the Based on previous reports, the interaction between plant/TiO 2 -NM can positively or negatively affect the plant product's quality, such as a higher or lower production rate, depending on the plant species (R. Singh et al., 2021). However, one area with potential applications is the stress induction by TiO 2 -NMs. The stress in a plant generates secondary metabolites, which act as defense molecules to mitigate the effects of such stress (Modarresi et al., 2020;Wu et al., 2017). For humans, these molecules derived from secondary metabolism vary from nutrients with antioxidant capacities or products such as medicines. It is known that the capacity for an NM to generate stress in a plant will depend entirely on its physical and chemical characteristics and its introduction path (Ananthi et  . Although to our concern, there is no study relating the exposure effects with such an essential photocatalytic nanomaterial like TiO 2 . This research aims to highlight the effects produced in the morphologic characteristics of the plant and the production of secondary metabolites when it interacts with the pristine TiO 2 as well as with the doped-TiO 2 (F. Huang et al., 2016). Materials And Methods 2.1. Titanium dioxide and doped TiO 2 materials synthesis and characterization Titanium isopropoxide (Sigma Aldrich 97%) was dissolved in isopropanol (J.T. Baker 99%). The solution was stirred for 20 minutes under a nitrogen atmosphere to prevent the oxidation of the titanium precursor.
The hydrolysis process was then performed by adding water into the precursor/solvent solution, and this new solution was then stirred for 1 hour. For the Ag modi ed TiO 2 , the precursor AgNO 3 (J.T. Baker) was used, for the Fe-TiO 2 , the precursor was FeSO 4 •7H 2 O (J.T. Baker), and for the Cu-TiO 2 , the precursor was CuSO 4 •5H 2 O (J.T. Baker), these compounds were added by dissolving them into the water used for the hydrolysis in a 0.1 wt%, the obtained product was dried at room temperature and then calcined at 450°C for 3 hours to promote the anatase crystal phase. For this synthesis, the materials were identi ed as solgel (SG) materials (Esquivel et al., 2013). The TiO 2 samples synthesized by the microwave-assisted solgel method was prepared by the sol obtained after the hydrolysis process. It was transferred into a Te on vessel and placed on a microwave reaction system (Flexiwave Milestone). The process was carried out at a temperature of 180°C for 30 minutes. Once the product is obtained, it was ltered, dried, and calcined at 450 ° C for 3 hours. For this synthesis, the materials were identi ed as microwave-assisted sol-gel (Mw-SG) materials (Hernández et al., 2020).
Morphology and elemental analysis were carried out using a JEOL JSM-6060 LV scanning electron microscope (SEM) operating at a voltage of 15 keV. Elemental analysis was performed by Energy Dispersive X-ray Spectroscopy (EDS Oxford Inca X-Sight coupled to a MT 1000, Hitachi). The crystallinity was determined by X-ray Diffraction analysis (XRD) using a Bruker D8 advanced diffractometer equipped with a Cu seal tube to generate Cu Kα radiation (λ = 1.5406 Å) with angles of 10 < 2θ < 80° in a pitch of 0.01°, Raman analysis was made using a LabRam HR Horiba Scienti c with a NdYGa (λ = 532 nm).

Plant harvest and growth parameters
Alfalfa seeds (Medicago sativa L.) were purchased from a local distributor brand Horta or, Mexico. They were placed in seedbeds using peat moss substrate (Jiffy) with a pH of 5.8, an electrical conductivity of 0.4 mS/cm, moisture fraction of 15%, and particle size of < 10 mm inside a plasticized greenhouse of 68 X 49 X 156 cm of length, wide and height, respectively. For the experiment, three replicates with a population of 6 crops were maintained during development. Sprouts were kept in seedbeds for 15 days before being transferred to plastic containers of 500 mL. Crops were treated by direct soil irrigation with 5 mL solutions of 50, 100, and 500 ppm of TiO 2 and M-TiO 2 (M = Ag, Cu, Fe) with no nutritive solution, during the seedbed development and 50 mL at the bigger container every three days, after completing 80 days of treatment.
Half of the plants were randomly selected for morphological analysis. After harvest, the samples were divided into leaves, stems, and roots immersed in liquid nitrogen to prevent any chemical structural change for future tests. Then the samples were milled and kept under refrigeration at 4°C for further metabolomics quanti cations assays. The greenhouse temperature was recorded using a hygrometer (YASSUN) and obtaining temperature and humidity values at midday. Climatic data were taken from the geo-electromagnetic center of the National Autonomous University of Mexico, Juriquilla campus (longitude: 100º26'48.81" west, latitude:20º42'14.87" north) (Levresse et al., s/f) at noon each day.

Secondary metabolite quanti cation
The extracts' total phenolic and avonoid contents were determined according to the Folin-Ciocalteu spectrophotometric method (Bobo-García et al., 2015) modi ed for 96-well microplate. Total phenol content results were expressed as equivalent mg of gallic acid per gram of fresh sample, and rutin hydrate ( avonoid) is expressed as equivalent mg of rutin per gram of fresh sample and was determined by the 2-aminoethyl-diphenyl borate reagent method (Garcia-Mier et al., 2021).

Antioxidant activity
The extracts' antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method (Parit et al., 2018), and the results were expressed as the percentage of DPPH discoloration (% radical inhibition) named as well as percentage inhibition (IHB), which was calculated with Eq. 1   show aggregates with lesser size than the pure sol-gel method. The internal heat generated through this process helps to form NMs with high crystallinity, small and uniform size (Hernández et al., 2020;Kadam & Park, 2018).
EDS shows the elemental composition of the M-TiO 2 , M = Ag, Fe, Cu NPs, synthesized by the sol-gel method, where the presence of the Ti and O elements can be seen in Fig. 2(a) for the undoped TiO 2 . In Fig. 2(b-d), the elemental mapping for the doped materials is observed, where the presence of the elements Cu ( Fig. 2(b)), Fe (Fig. 2(c)), and Ag ( Fig. 2(d)) are observed. Identical results were obtained for the Mw-SG synthesis method (results not shown). The crystallite sizes for both synthesis methods were calculated using the Scherrer equation showed in Eq. 2, where (D) is the diameter of de crystallite, (λ) is the wavelength of the X-rays, (k) is the Scherrer constant with a value of 0.9 for spherical nanoparticles, (β) is de full width at half maximum obtained from the diffraction signals in the XRD pattern and (ϴ) the peak position.
Crystallite size was also determined using the Williamson-Hall equation (Eq. 3), which considers the structural stress of the crystallite. The equation represents a straight line where (ε) is the slope that provides the strain of the crystallite.
The crystallite size for both synthesis methods is compiled in Table 1. The sol-gel synthesis method gives an average crystallite size of 9 nm, being the undoped TiO 2 the smallest size calculated by the Sherrer equation (Eq. 2). The Williamson-Hall method shows an average size of 17 nm. Since this equation considers the stress of the crystal lattice, either compression or relaxation, the calculated size is bigger than the Scherrer method, which refers to a crystallite with stress in the materials' lattice. The crystallinity degree has values greater than 90%. The crystal phase was also con rmed with Raman spectroscopy, where the spectra of the materials synthesized by the sol-gel method are shown in Fig. 4

Germination and morphological data
The germination of the alfalfa seeds was not affected by the direct irrigation of the TiO 2 materials (with or without doping) at three concentrations for both synthesis methods. For all concentrations except 500 ppm to the doped materials (Ag, Fe-TiO 2 ), the seeds germinated on the 5th day; for this concentration, the seeds germinated on the 6th day. The real leaf was observed for the control on the 11th day, while for the rest of the treatments, the real leaf was observed on the 12th day. For Mw-SG NPs, seed germination and real leaf were also not affected, where for all treatments, germination occurred on the 5th while real leaf appeared on the 11th day.
Nutrient de ciency in the alfalfa crops was observed in leaves based on texture and colors, as shown in undoped NPs treatments showed statistical signi cance compared to control, for Ag-doped 50 ppm caused a 42.9% reduction, 27.78% at 100ppm and 11.18% at 500 ppm. Meanwhile, Fe-doped showed a reduction of almost 25% for all doses with a statistical signi cance. Finally, Cu-doped showed the lowest effect among the NMs with only a reduction of 9% at 50 and 100 ppm, increasing at 500 ppm (17%), where the highest concentration caused a signi cant reduction in stem height.
The measurements obtained from the secondary stem ( Fig. 6(b)) also show a size reduction. For undoped TiO 2 , a 41.16% reduction was obtained at 50 ppm, 55.0% at 100 ppm, and 30 % at 30.3% at 500 ppm, where the three concentrations reached statistical signi cance compared to control. Ag-dope material showed a reduction as concentration was increased, although the three concentrations were statistically similar, obtaining a reduction of 42.9% (50 ppm), 21.6% (100 ppm), and 18.3% (500 ppm). Fedoped material showed a high reduction in the size while the dose is augmenting, showing statistical signi cance among the three concentrations, causing a 37% (50 ppm), 44.3% (100 ppm), and 50.0 % reduction at 500 ppm. Finally, the Cu-doped material also caused a reduction of almost 30% for all three concentrations. Although 50 and 100 ppm caused a reduction, these two treatments showed no statistical difference compared to the control.
In the case of the leaf length ( Fig. 6(c)) measured in the central stem for the undoped TiO 2 , it generates a reduction of about 5% for the three concentrations, while Fe-doped had its maximum reduction at 100 ppm (25.5%). Nevertheless, at 500 ppm, there is an inhibition effect (17.2%). Ag-doped has the same effect with the maximum at 100 ppm (11.9%) and inhibition at 500 ppm. The Cu-doped had no reduction effect at 50 and 100 ppm, while 500 ppm caused a reduction of 28.8%.

Secondary metabolite and chlorophyll content
The UV-visible method of secondary metabolite quanti cation compares the rutin, gallic acid, and inhibition percentage between the NMs used in the treatments at each concentration. After completing 80 days of treatment, the alfa plants were divided into groups containing only leaves, shoots, and roots for a sectional analysis of the metabolite content.
The effect on crop growth was observed in the secondary metabolites content in leaves (Table 2). A statistical analysis using a Tukey assay for comparing data pairs was done to identify signi cantly statistical data. The assay compared the data obtained for gallic acid and rutin with the three doses and the four types of NMs synthesized by the SG method. The results showed no signi cant augmentation of the equivalent grams of gallic acid than control except for the plants treated with the undoped TiO 2 at the materials, even though the equivalents grams of rutin appear to be higher than the control plant. All treatments showed no signi cant effect on this secondary metabolite content in leaves at all concentrations (50, 100, 500 ppm).  Since the NMs were added to the water used for irrigation of the plants, the roots were the primary organ exposed to the NMs, so the highest stress effect related to the production of secondary metabolites should be seen in this organ.
The data showed in Table 4 correspond to the analysis of the roots, where it can observe at rst instance by the Tukey pairwise comparison that the SG synthesized undoped TiO 2 at 50 (70.91%) and 100 ppm (61.01%) showed the signi cative higher effect on the gallic acid production as well for the Cu-doped material at 500 ppm (60.03%) and Ag-doped material at 100 ppm (58.22 %). NMs at speci c concentrations treatments can induce the production of rutin in the roots causes by possible stress.
Plant irrigated with the materials obtained by the Mw-SG method, after 80 days of exposure, the analysis to leaves, stem, and roots were made for metabolite quanti cation. In Table 5 it is presented the quanti cation of gallic acid, rutin, and inhibition % in leaves. Comparing the treatments with the control using a Tukey assay shows that the Mw-SG materials cause an augmentation of gallic acid content compared to the SG materials.  A Games-Howell pairwise comparison was made to a lack of variance between the data for the gallic acid content in roots. The data expressed in Table 7shows that the root's gallic acid content is signi cantly higher for the Cu-doped materials at 100 (70.42%) and 500 ppm (73.97%). In contrast, the rest of the treatments at the different comparisons were statistically equal to the control. The Cu-doped materials at 500 ppm (41.99%) resulted in statistical signi cance compared to the control in the rutin content. Finally, the inhibition percentage calculated in roots is signi cantly higher for the three doses of undoped NMs, copper, and iron-doped titania. Only silver-doped titania at a concentration of 50 ppm was statistically similar to the control. The measurement of chlorophyll by the Konica Minolta SPAD 502 Plus chlorophyll meter revealed a reduction in chlorophyll content in leaves for the plants treated with the sol-gel synthesis method shown in Fig. 8(a) However, all three treatments showed no signi cant difference compared to the control. The chlorophyll content shows interesting results in plants treated with the materials obtained by the Mw-SG synthesis method ( Fig. 8(b)). The lowest dose in undoped TiO 2 , as shown in Fig. 8(b), causes an increase of 7% compared to control. In contrast, the content decreases to about 14% and 25% at higher doses for 100 and 500 ppm, respectively. The Fe-TiO 2 shows a signi cant reduction at 50 ppm (31.5%), and at higher doses of 100 and 500 ppm, there is a reduction of 15%. For the Ag-doped material, there is also an increase of chlorophyll at 50 ppm of about 13%, and for the 100 and 500 ppm treatments, it only increases 15%. Finally, Cu-TiO 2 material caused a reduction of 7.6% (50 ppm), 24.8% (100 ppm), and 30.1% (500 ppm).

Antioxidant activity
DPPH radical inhibition was determined for each treatment in the three organs studied. As shown in Tables 2-7, leave analysis shows an overall augmentation of radical IHB for plants interacting with NMs compared to control. No apparent effect towards IHB% augmentation with NMs concentrations was determined for all treatments. At 50 ppm, the highest radical inhibition was obtained with Ag-doped and undoped NMs. At 100 ppm, the highest effect was determined for undoped NPs when for the rest of the treatments a reduction in %IHB was observed, at the highest concentration Ag-doped and undoped NMs where undoped TiO 2 showed a nal reduction of IHB%. For the stem, the highest reduction at 50 ppm was obtained Ag-doped followed by undoped TiO 2, at 100 ppm highest antioxidant activity was obtained with undoped NMs, at the highest concentration, the same result was obtained where highest undoped TiO 2 gave the highest radical inhibition. In roots, the NMs effect on the inhibition percentage augmentation compared to control was expressed in much more concentrations than in the leaves and stem of the alfa.
Roots exposed to Fe-doped material at the three doses showed a higher inhibition percentage than control. For the Mw-SG treated plant IHB% was also augmented. However, no relation between an augmenting NPs concentration and an increase in radical inhibition was observed. Radical imbibition analysis at leaves ( Where the treatments showed an augmentation in radical inhibition, which is related to an increase in the production of antioxidant compounds which can be found among the phenols and avonoids

Conclusions
It can be showed that alfalfa treated with TiO 2 NMs caused a stress effect on alfalfa crops which resulted in lower growth rate and a higher metabolic content as well as some affectations in chlorophyll content and micronutrient uptake, the results suggest that the degree of stress depends upon on concentrations and doping of TiO 2 NMs, the fact that NMs generated high concentrations of phenols and avonoids could signi cate in an effective mechanism of generating alfalfa crops with higher nutritional content than traditional crops but further assays for determine NMs uptake by roots and translocation are suggested to differentiate NMs size with stress factors as well as safety concerns as well of investigating a higher spectrum of concentrations for achieving an ideal point for secondary metabolite production improvement including a deeper understatement of the toxicological model involved in the NPs-alfalfa interaction, associating it with hormesis curves comprehending the stress generated at low and higher doses which may help in the understanding of other toxicological behavior of photocatalytic materials like TiO 2 .
The NMs of both synthesis methods reduce the chlorophyll content and stem size of treated plants. The presence of dopants was a factor to generate differences in the observed effects. The morphological changes generate differences in the metabolite content. The increase of speci c metabolites and antioxidant capacity in leaves, stems, and roots was marked by the physicochemical characteristics of both synthesis methods and the presence of dopants.
Speci c and complete structural and chemical characterization of NMs is essential for relating the effects observed when interacting with living beings. It is essential to observe that synthesizing methods for obtaining NMs with different structural and chemical characteristics will help understand nanomaterials' toxic effects. Also, more insight toward exposure time, concentration, and dopant content could help to increase secondary metabolite production. Furthermore, without compromising plant health and NMs overuse, based on the results obtained, lower concentrations of titania could help as well to increase chlorophyll content in leaves increasing its photosynthetic activity.  Raman spectra of (a) SG synthesized and (b) Mw-SG synthesized materials.