Untargeted Metabolomic Approach of Curcuma longa to Neurodegenerative Phytocarrier System Based on Silver Nanoparticles

Curcuma is one of the most famous medicinal and tropical aromatic plants. Its health benefits have been appreciated and exploited in traditional Asian medicine since ancient times. Various studies have investigated its complex chemical composition and demonstrated the remarkable therapeutic properties of curcuma’s phytoconstituents. Oxidative stress is a decisive driving factor triggering numerous pathologies (neurodegenerative, psychiatric and cardiovascular diseases; diabetes; tumors, etc.). Numerous recent studies have focused on the use of natural compounds and nanomaterials as innovative molecular targeting agents as effective therapeutic strategies. In this study, we report, for the first time, the development of a simple target phytocarrier system that capitalizes on the bioactive properties of curcuma and AgNPs. The complete metabolic profile of curcuma was determined based on gas chromatography-mass spectrometry (GC-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). A total of 80 metabolites were identified under mass spectra (MS)-positive mode from 10 secondary metabolite categories: terpenoids, amino acids, diarylheptanoids, flavonoids, phenolic acids, steroids, fatty acids, coumarins, alkaloids and miscellaneous. In addition, the biological activity of each class of metabolites was discussed. A comprehensive characterization (FT-IR, UV-Vis, DLS, SEM, TEM, EDS, zeta potential and XRD) was performed to study the morphostructural properties of this new phytocarrier system. Antioxidant activity of the new phytocarrier system was evaluated using a combination of in vitro methods (total phenolic assay, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and cyclic voltammetric method (Trolox equivalent antioxidant capacity (TEAC) electrochemical assay)). Antioxidants assays showed that the phytocarrier system exhibits superior antioxidant properties to those of its components, i.e., curcuma or citrate-coated-AgNPs. These data confirm the potential to enhance relevant theoretical knowledge in the area of innovative antioxidant agents, with potential application in neurodegenerative therapeutic strategies.


Introduction
Curcuma longa L., a plant that belongs to the Zingiberaceae family, has played an important role in Indian culture since ancient times. The well-known turmeric spice is Direct antioxidants are divided into preventive antioxidants (metal-chelating agents: phytate or curcumin), chain-breaking antioxidants or radical-trapping antioxidants (polyphenolics, phenolic acids and ascorbic acid) and termination-enhancing antioxidants (nonphenolic terpenoid) [45,53]. Their main characteristics are redox activity, as well as the ability to be consumed or undergo chemical changes and be replenished or regenerated.
Antioxidant performance depends mainly on rate constant and thermodinamic parameters of reaction with a specific free radical, as well as a series of physicochemical factors (chemical structure, type of substrate oxidation position, concentration and temperature), the presence of other pro-oxidants or synergists, bioavailability and metabolism pathway [45,54].
The development of a nanocarrier depends on the morphostructural parameters of the nanoparticles (size, shape, surface charge, etc.) and the biological activity of its components [12].
Silver nanoparticles, owing to their unique size and versatility, high surface-to-volume ratio, biocompatibility and mobility, are able to cross the blood-brain barrier (BBB), which represent a major limitation in the current development of active drugs [12][13][14][15][16]20,27,29]. The design of a performant neuroprotective phytoengineered carrier system based on AgNPs represents an innovative alternative that can overcome the limitation of current therapeutic strategies, tracing specific organ and molecular targets linked to neurodegeneration.
To the best of our knowledge, this study is the first to investigate the development of a target-specific nanocarrier delivery system that capitalizes on the therapeutic properties of bot curcuma and AgNPs. We evaluated the morphostructural and antioxidant properties of the proposed phytocarrier system.

Preparation of Phytocarrier System Components
AgNPs synthesis: Citrate synthesis of silver nanoparticles was carried out using the following procedure. An amount of 0.64 g AgNO 3 was added to 1000 mL ultrapure water, followed by the addition of 50 mL of sodium citrate solution (1.6%). The mixture was kept at 90 • C for 90 min and stirred at 800 rpm. Then, 0.8 g ascorbic acid was added, and the obtained suspension was stirred at 90 • C for another 40 min.

Plant Preparation for Chemical Screening
For each analysis, 2.0 g of curcuma powder was subjected to sonication extraction in 35 mL of solvent (methanol/chloroform = 1:1) for 35 min at 37 • C with a frequency of 50 kHz. The solution was concentrated using a rotavapor, and the residue was dissolved in MeOH. The extract was centrifuged, and the supernatant was filtered through a 0.2 µm syringe filter and stored at −18 • C until MS analysis. All samples were prepared in triplicate.

GC-MS Analysis
Gas chromatography was carried on an HP6890 gas chromatograph coupled with an HP5973 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a Bruker Br-5MS capillary column (30 m × 0.25 mm id × 0.25 µm) (Bruker, Fremont, CA, USA) under helium with a flow of 1 mL/min.

GC-MS Separation Conditions
The oven temperature was increased from 50 • C to 300 • C at a rate of 6 • C/min and held at this temperature for 5 min. The temperature of the injector was 280 • C, and the temperature at the interface was 220 • C. The mass of the compounds was registered at 70 eV ionization energy starting after 3 min of solvent delay. The mass spectrometer was source-heated at 230 • C, and the MS Quad was heated at 150 • C. The mass values of the identified compounds were scanned from 50 to 550 amu. Compounds were identified based on their mass spectra, which were compared to the NIST0.2 mass spectra library database (USA National Institute of Science and Technology software, (NIST, Gaithersburg, MD, USA). Retention indices (RIs) were also calculated for each compound based on a C 8 -C 20 alkane standard mixture calibration curve, which was compared to Adams indices in the literature [55].
A percentage area of each compound was calculated by summing all the experimental integrated peak areas (Table 1).

Mass Spectrometry
MS experiments were conducted on a Bruker EIS-QTOF-MS (Daltonics, Bremen, Germany). The full mass spectra were acquired in positive ion mode at the temperature source block temperature (80 • C) and at 25-85 eV collision energy. The scan range of full MS was m/z 100-3000, with a scan speed of 2.0 scans/s. Peak assignment to specific metabolites was based on the NIST/NBS-3 standard library spectral database (National Institute of Standards and Technology/National Bureau of Standards) (NIST, Gaithersburg, MD, USA). The mass spectra of the identified compounds are presented in Table 2.

Phytocarrier System Preparation
For each analysis, 2.0 g of sample was prepared from curcuma powder, and a AgNPs solution was added (curcuma/AuNPs nanoparticles = 1:3) at room temperature (23 • C) and magnetically stirred (450 rpm) for 22 h. The obtained mixture was filtered (F185 mm filter paper) and dried in an oven at 35 • C for 5 h. For TEM analysis, a 0.22 nm filter was used to filter the curcuma powder.

Characterisation of the Phytocarrier System
UV-Vis Analysis: The UV-Vis spectra were obtained using a T90+ UV-Vis spectrophotometer (PG Instruments, UK) with a double beam in the photometric range of 190-900 nm. All absorbance measurements were taken in a 10 mm UV/Vis spectroscopy cell at room temperature, using ethanol as a blank.

Fourier Transform Infrared (FTIR) Spectroscopy
Data collection was performed after 20 recordings at a resolution of 4 cm −1 in the range of 4000-400 cm −1 on a Shimadzu AIM-9000 with ATR devices.

XDR Spectroscoy
X-ray powder diffraction (XRD) was performed using a Rigaku Ultima IV diffractometer equipped with a D/teX ultra-detector operating at 40 kV and 40 mA with monochromatic CuKα radiation (λ = 1.5406 Å) in the 2θ range of 10-80 • , with a scan speed of 5 • /min and a step size of 0.01 • . The XRD patterns were compared with those from the ICDD Powder Diffraction Database (ICDD file 04-015-9120). The average crystallite size and the phase content were calculated using the whole-pattern profile-fitting method (WPPF).

Scanning Electron Microscopy (SEM)
SEM micrographs were obtained with an SEM-EDS system (QUANTA INSPECT F50) equipped with a field-emission gun (FEG) with a 1.2 nm resolution and an energydispersive X-ray spectrometer (EDS) with an MnK resolution of 133 eV.

Dynamic Light Scattering (DLS) Particle Size Distribution Analysis
DLS analysis was perfomed on a Microtrac/Nanotrac 252 (Montgomeryville, PA, USA). Each sample was analyzed in triplicate at room temperature (23 • C) at a scattering angle of 172 • .

High-Resolution Transmission Electron Microscopy (HR-TEM)
The morphology of silver particles and phytocarriers were determined through an ultra-high resolution transmission electron microscope (UHR-TEM) (Libra 200 UHR-TEM, Carl Zeiss, Germany).

Zeta Potential Analysis
The zeta potential was determined on an AMERIGO particle size and zeta potential analyzer (Pessac, France), with six measurements/s, and electrode distance of 5 mm, a temperature of 25 • C, conductivity of 5.10 V, carrier frequency of 8210 Hz, reference intensity of 2660 kcps, applied field of 20.27 V/cm and scattering intensity of 2850 kcps.
The phytocarrier system (0.3 g) and curcuma (0.5 g) samples were dissolved in methanol (9 mL and 15 mL, respectively). The mixtures were stirred at room temperature (23 • C) for 24 h, then centrifuged at 3000 rpm for 5 min. The supernatant was then collected for use in the antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, Folin-Ciocalteu assay (cyclic voltammetry method). The AgNP solution was used without further preparation for the cyclic voltammetry method.

Determination of Total Phenolic Content
The total phenolic content in the phytocarrier system and curcuma samples was determined according to the Folin-Ciocalteu method adapted from the literature [56].
A volume of 0.1 mL of each sample was used for total phenolic content assays. Folin-Ciocalteu reagent 2 N was diluted 1:10 in distilled water. A ratio of sample/Folin-Ciocalteu reagent 0.2 N (1:5) was prepared, vortexed and kept at room temperature (23 • C) for 5 min in the dark. The reaction between samples and Folin-Ciocalteu reagent was allowed to complete, before adding an equal volume of Folin-Ciocalteu reagent and 7.5% sodium carbonate (Na 2 CO 3 ) solution. The obtained mixture was vortexed and kept at room temperature (23 • C) for 1 h in the dark. The samples were scanned by a UV-Vis spectrophotometer (Tecan i-control, 1.10.4.0 infinite 200Pro spectrophotometer with Corning 96 Flat Bottom clear Polystyrol plates, Tecan, Männedorf, Switzerland) for 10 to 40 min after the addition of the sodium carbonate solution and scanned from 400 to 900 nm with intervals of 2 min between each reading to determine the spectra. The absorbance was measured at 725 nm.
The results are expressed in gallic acid equivalents (mg GAE/g sample) using a propyl gallate standard calibration curve between 1 mg/mL and 12.5 µg/mL in methanol.
Sample extract concentrations were calculated based on the linear equation obtained from the standard curve (y = 0.8353x − 0.1207) [57].

DPPH Radical Scavenging Assay
The antioxidant activities of the phytocarrier system, curcuma sample and three antioxidant standards (ascorbic acid, curcumin and beta-carotene) were comparatively evaluated by a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical elimination assay.
A stock solution of 1 mg/mL DPPH and dilutions for a calibration curve with a range of concentrations between 3.12 µg/mL and 0.1 mg/mL were prepared, with the following serial dilutions of standards: ascorbic acid and curcumin were combined with a stock solution of 1 mg/mL, and between 0.1 mg/mL and 0.2 µg/mL was diluted in methanol; β-carotene started from 1.66 mg/mL, and between 0.83 mg/mL and 41.66 µg/mL was diluted in a solvent ratio of 2:1 methanol/dichloromethane. Sample extracts of and the phytocarrier system had dilutions between 0.33 mg/mL and 2.08 µg/mL for curcuma and between 0.33 mg/mL and 4.16 µg/mL for the phytocarrier system. The ratio (v/v) of DPPH to samples was of 1:1.
All solutions were prepared in four replicates in methanol, and 1:1 DPPH 0.1 mg/mL and methanol was used as a control sample.
For β-carotene, methanol/dichloromethane (2:1) was used as a control solvent. All samples were pipetted on 96-well plates and incubated in the dark at room temperature for 30 min. After the reaction was stabilized, the absorbance was measured, and readings were performed at 515 nm. The obtained results were used to calculate the average and the inhibition percentage (Inh%). Inh% was further used to obtain the IC 50 value, which was calculated based on the equation of a calibration curve generated for each sample and standard, with results presented as calculated Inh% versus concentration (µg/mL).

Statistical Analysis
All results were obtained with Microsoft Office Excel 2010. Data were subjected to the average of four replicates for all samples, all calibration curves and concentrations.

Trolox Equivalent Antioxidant Capacity (TEAC) Electrochemical Assays
The stock solutions of ABTS and Trolox were prepared by dissolution in methanol. Further dilutions were achieved with Millipore ultrapure water (18 MΩ·cm).
Voltammetric measurements (cyclic voltammetry, CV) were performed on an Autolab PGSTAT101 apparatus (Eco Chemie BV, Utrecht, The Netherlands) using NOVA 2.2 software in a standard three-electrode electrochemical cell with a carbon paste working electrode (Metrohm, 3 mm in diameter), a platinum wire (CH Instruments) as the counter electrode and an Ag/AgCl reference electrode saturated with 3 M KCl (Methrom, Switzerland). All experiments were run at room temperature.
The TEAC electrochemical assays were performed according to the method reported in the literature [58].
A solution of the stable radical cation ABTS· + was prepared by mixing 7 mM ABTS with 2.45 mM persulfate in a volumetric ratio of 1:1 in methanol. The mixture was kept in the dark for 8 h at 20 • C prior use to ensure the quantitative conversion of ABTS to ABTS· + . The stock solution of ABTS· + was suitably diluted with various amounts of methanol to achieve significant voltammetric outputs during the CV measurements. All working solutions of ABTS· + contained 0.05 M KCl as the supporting electrolyte.
A volume of 250 µL of each sample (phytocarrier system, curcuma and citrate-coated AgNPs) was added to the working solutions of ABTS· + , and the reaction mixtures were left to react for 2-3 min. Several CVs were recorded to evaluate the stability of the reaction mixture and the reproducibility of the datasets. The reaction between ABTS· + and Trolox was used to build the calibration plot. Several aliquots of 1 mM Trolox solution were mixed with ABTS stock solution to yield a final ABTS concentration of 0.12 mM and a Trolox concentration range of 0.02-0.12 mM. For each sample, at least 10 consecutive voltammograms were recorded, and the average response was recorded. The residual standard deviations (RSD) in all assays were below 0.25%. The peak currents were processed with Nova 2.2 software and referenced to the baseline.

Results and Discussions
Plants are an invaluable resource for biomolecules with high therapeutic potential. A considerable variety of secondary metabolites with complex chemical structures has been identified in plants [59,60].
Studies have shown that the stress conditions dictated by the environment in which a particular plant develops influence the production of secondary metabolism and the structural and functional relationships between them [61][62][63][64][65][66].
Moreover, the proportion of volatile compounds and curcuminoids, considered the main bioactive compounds in curcuma, is also influenced by experimental conditions (extraction, etc.) [67].
Accordingly, the biological activity of a specific plant is dictated by the composition and the synergistic and complementary interaction of their secondary metabolites. The combined action of all metabolites in the plant leads to increased biological activity and metabolic stability of an active component [68,69]. Therefore, it is very challenging to establish the interdependence between the therapeutic effect and the chemical composition of a plant. In some cases, in vivo studies do not support the mechanism of action of a particular secondary metabolite isolated from a plant [10].
Given the remarkable economic value of curcuma, studies on turmeric continue to be relevant, especially with respect to its therapeutic potential.
Secondary metabolite screening of curcuma was tentatively carried out via gaschromatography coupled with mass spectroscopy (GC-MS) and electrospray ionizationquadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) analysis.
The GC-MS analysis ( Figure 1) revealed the separation of some low-molecular-weight metabolites from the curcuma sample. The results are summarized in Table 1, including the identification of tentative GC-MS compounds from the curcuma sample. RI-retention index calculated based on a calibration curve of a C8-C20 alkane standard mixture.
The GC-MS analysis revealed the presence of eight major components, accounting The results are summarized in Table 1, including the identification of tentative GC-MS compounds from the curcuma sample. The GC-MS analysis revealed the presence of eight major components, accounting for 89.7% of the total peak area in curcuma samples ( Figure 1).

Mass Spectrometric Analysis of Curcuma Sample
The spectra revealed a complex mixture of low-molecular-weight metabolites molecules, among which only a few were detected. The mass spectra of identified components were compared with those of the NIST/EPA/NIH Mass Spectral Library 3.0 database, in addition to a literature review [67,[79][80][81][82].
The mass spectrum and the phytoconstituents identified by ESI-QTOF-MS analysis are presented in Figure 2 and Table 2, respectively.      The metabolite profile results of curcuma sample performed through GC-MS and mass spectroscopy correspond with the data reported in the literature [1].

Chemical Class
Metabolite Name   Figure 3 presents the metabolite classification chart obtained on the basis of the data analysis reported in Table 3. Miscellaneous trimethylbenzene undecan-2-one dodec-11-en-2-one 2-hydroxy-3-(hydroxymethyl)anthraquinone beta-carotene Figure 3 presents the metabolite classification chart obtained on the basis of the data analysis reported in Table 3. Amino acids and peptides: A total of 11 compounds were identified in the rhizome extract. The essential amino acids (valine, leucine, methionine, phenylalanine and arginine) represent about 45.45% of the total compounds. The non-essential amino acids (proline, aspartic acid, glutamic acid, cysteine, serine, glycine and alanine) represent a slightly higher proportion, accounting for about 54.55% of the total compounds [96][97][98][99][100]. About half of the amino acids identified in the curcuma rhizome sample (arginine, phenylalanine, glutamic acid, methionine, glycine, serine, aspartic acid and proline) act as antitumoral, neuroprotective, antiproliferative and immunomodulant agents [97][98][99][100][101].
Studies have reported that fatty acids exhibit anti-inflammatory, antioxidant, neuroprotective and cardiovascular protective activity [101,102,113].
Coumarin is a secondary metabolite with outstanding therapeutic proprieties, acting as a highly antitumoral and bacteriostatic agent [101,114,115].

Phytoengineered Carrier System
The development of a target-specific phytocarrier system imposes tailored assembled structures with pre-established morphostructural and biological properties able to exert complementary and synergistic therapeutic efficacy and trigger selective controlled delivery, release and activation in specific body regions, in addition to minimizing toxicity [120][121][122][123].
A nanocarrier system that collectively combines the specific features of silver nanoparticles (high biocompatibility, permeability and stability, with unique optical properties) with remarkable antioxidant, neuroprotective and anti-inflammatory effects of both components (curcuma and inorganic components) can increase the response to the specific physiological microenvironment and thus provide high efficiency through enhanced permeability and retention effects [8,[14][15][16][17][22][23][24][25].

FT-IR Spectroscopy
The preparation of a phytocarrier system was investigated by FT-IR spectroscopy to identify the functional groups specific to its two components, curcuma and AgNPs. The individual FT-IR spectra of the curcuma, citrate-coated AgNPs and phytocarrier systems are shown in Figures 4 and 5.    The FT-IR absorption bands identified in the curcuma sample are presented in Table  4. The FT-IR absorption bands identified in the curcuma sample are presented in Table 4. The FTIR peak of AgNPs coated with trisodium citrate (surfactant) (Figure 4a) presents vibrational bands characteristic of a surfactant at 3434 cm −1 (associated with H-OH stretching vibration) and 2925 cm −1 , at 2853 cm −1 (attributed to CH-asymmetric and symmetric stretching vibrations), 1630 cm −1 (associated with COO-stretching vibration) and 1386 cm −1 (assigned to C-H bending) [13].
The obtained results ( Figure 5) confirm the successful development of a phytoengineered carrier delivery system. The obtained spectra of the carrier system include the characteristic peaks of curcuma at 3288 cm −1 , attributed to the -OH group; at 2926.37 cm −1 , assigned to C-H aromatic; at 1627 and 1511.97 cm −1 (C=C and diarylheptanoid heterocyclic ring, respectively); at 1150.89 and 1071.11 cm −1 (C-O and C-O-C bonds, respectively); at 882.44 and 816 cm −1 (C-O and CH vibration of aromatic rings), as well the AgNPs coated with surfactant (trisodium citrate) (Figure 4a,b) [135].
Furthermore, in the IR spectra of the new phytocarrier system, several discernible changes occur compared to the spectrum of curcuma, in particular, in the case of the hydroxyl vibrations (O-H stretching, H-bonded alcohols and phenol groups), which are shifted to lower wavenumbers, indicating that this functional group is involved in the binding of AgNPs [19,[21][22][23][24][25]127,[137][138][139][140].

UV-Vis Spectroscopy
This technique was used to obtain insight into the newly developed carrier system. UV-VIS analysis provides information on the characteristics of AgNPs.
Various studies have reported that the optical properties of AgNPs are influenced by particle size, degree of dispersion, coating and chemical stability [11,13,19,25,141,142].
As shown in Figure 6a, AgNPs present a plasmonic band at 417 nm, which can be assigned to citrate-coated AgNPs, as well as another two weak absorbance peaks at 368 and 970 nm [143,144]. The sharp absorption maximum (λmax) at 417 nm ( Figure 6a) indicates that the size of the synthesized AgNPs is in the 20-120 nm range and dispersed [13,145]. Additionally, a single surface plasmonic peak (SPP) in the UV-Vis spectra suggests that the AgNPs are mostly spherical [145,146].
The shape of the curcuma absorption spectrum (Figure 6b) showed a maximum absorption band at a wavelength of 424.5 nm (attributed to curcuminoids) and a shoulder near 360 and 464 nm (assigned to bet-carotene), as well as a weak absorption band at 261 nm (attributed to diarylheptanoids and terpenocurcumins) [95,130,[146][147][148].
A comparison of the shape of the spectrum of curcuma and that of the new phytocarrier system reveals notable differences (Figure 6b). The surface plasmon band of the plasmon of the new phytocarrier system was detected at 415 nm, with a weak peak at 262 nm from curcuma. These results confirm the achievement of the phytocarrier system.

X-Ray Diffraction Spectroscopy
The XRD analysis was used to investigate the crystalline structure of the carrier system and its components. Figure 7a,b display the XRD patterns of the AgNPs, curcuma sample and the new phytocarrier system. The sharp absorption maximum (λmax) at 417 nm (Figure 6a) indicates that the size of the synthesized AgNPs is in the 20-120 nm range and dispersed [13,145]. Additionally, a single surface plasmonic peak (SPP) in the UV-Vis spectra suggests that the AgNPs are mostly spherical [145,146].
The shape of the curcuma absorption spectrum (Figure 6b) showed a maximum absorption band at a wavelength of 424.5 nm (attributed to curcuminoids) and a shoulder near 360 and 464 nm (assigned to bet-carotene), as well as a weak absorption band at 261 nm (attributed to diarylheptanoids and terpenocurcumins) [95,130,[146][147][148].
A comparison of the shape of the spectrum of curcuma and that of the new phytocarrier system reveals notable differences (Figure 6b). The surface plasmon band of the plasmon of the new phytocarrier system was detected at 415 nm, with a weak peak at 262 nm from curcuma. These results confirm the achievement of the phytocarrier system.

X-ray Diffraction Spectroscopy
The XRD analysis was used to investigate the crystalline structure of the carrier system and its components. Figure 7a,b display the XRD patterns of the AgNPs, curcuma sample and the new phytocarrier system. sorption band at a wavelength of 424.5 nm (attributed to curcuminoids) and a shoulder near 360 and 464 nm (assigned to bet-carotene), as well as a weak absorption band at 261 nm (attributed to diarylheptanoids and terpenocurcumins) [95,130,[146][147][148].
A comparison of the shape of the spectrum of curcuma and that of the new phytocarrier system reveals notable differences (Figure 6b). The surface plasmon band of the plasmon of the new phytocarrier system was detected at 415 nm, with a weak peak at 262 nm from curcuma. These results confirm the achievement of the phytocarrier system.

X-Ray Diffraction Spectroscopy
The XRD analysis was used to investigate the crystalline structure of the carrier system and its components. Figure 7a,b display the XRD patterns of the AgNPs, curcuma sample and the new phytocarrier system.   [139,149].
The mean diameter (D) of the silver crystallites calculated using the Debye-Scherrer formula is about 120 nm.
The XRD pattern of curcuma (Figure 7b) is in the range of 14.8-22.4 • , with large bands and weak peaks characteristic of amorphous phases that can be attributed to curcuma constituents, namely dietary fibers and minerals, such as hydroxides [1,127] There are several notable differences in the XRD pattern for the new carrier system, dispite similarities to that of curcuma. The characteristic bands of the plant in the region between 15.8 and 22.3 are considerable attenuated. Additionally, AgNP peaks can be easily observed at 38.2, 44.1, 64.4 and 78.5 • . The estimated average diameter of the AgNPs from the phytocarrier system is~30 nm, consistent with the SEM results.
The morphology of the curcuma sample ( Figure 9b) displays a surface with a fibrous structure, with irregular, porous areas measuring approximately 6 µm. The presence of pores suggests easy fixation of metal nanoparticles on the surface of the plant sample.
The morphology of the phytocarrier system indicates the presence of metallic nanoparticles and agglomerations of spherical AgNPs with dimensions between 20 and 53 nm, both on the surface of the plant particles and loaded into the pores of herb particles (Figure 9a,b) [127].
The size of these AgNPs within the phytocarrier system (Figure 9a) observed under SEM does not significantly differ from those measured using XRD ( Figure 7b) and according to UV-Vis spectroscopy (Figure 6b) [154].

Scanning Electron Microscopy (SEM)
Based on the SEM micrograph of AgNPs (Figure 8a), the metal particles were welldispersed with a smooth and spherical shape of ca. ~100 nm [13,[150][151][152][153].  The morphology of the phytocarrier system indicates the presence of metallic nanoparticles and agglomerations of spherical AgNPs with dimensions between 20 and 53 nm, both on the surface of the plant particles and loaded into the pores of herb particles (Figure 9a,b) [127].
The size of these AgNPs within the phytocarrier system (Figure 9a) observed under SEM does not significantly differ from those measured using XRD (Figure 7b) and according to UV-Vis spectroscopy (Figure 6b) [154].
However, a considerable variation can be observed between the synthesized AgNPs (about 100 nm) and those from the phytocarrier system (20-53 nm). The significant reduction in the size of AgNPs observed in the phytocarrier system compared to the size of the synthesized AgNPs can be attributed to the specific experimental conditions under which the phytocarrier system was obtained, confirming the variation in the size of the nanoparticles depending on the experimental parameters reported in the literature [155]. On the other hand, it can be assumed that as a result of filtration, the larger silver nanoparticles were better retained at the liquid/filter interface, as larger particles are more However, a considerable variation can be observed between the synthesized AgNPs (about 100 nm) and those from the phytocarrier system (20-53 nm). The significant reduction in the size of AgNPs observed in the phytocarrier system compared to the size of the synthesized AgNPs can be attributed to the specific experimental conditions under which the phytocarrier system was obtained, confirming the variation in the size of the nanoparticles depending on the experimental parameters reported in the literature [155]. On the other hand, it can be assumed that as a result of filtration, the larger silver nanoparticles were better retained at the liquid/filter interface, as larger particles are more prone to form aggregates in the pore region. Additionally, plant pore size was an exclusion parameter for larger silver nanoparticles, allowing only nanoparticles below a certain diameter to enter the pores and be transffered into the herb.

Dynamic Light Scattering (DLS)
DLS analysis was used to obtain information about the particle size and particle size distribution both in the case of citrate-coated AgNPs and for the phytocarrier system.
Ultrapire water was used because the DLS principle is based on the Brownian motion of dispersed particles in a liquid. The intensity of light scattering varies depending on the dispersion of nanoparticles of varying sizes. Figure 10a shows the average mean particle size of citrate-coated AgNPs. The particles have a size of 100 nm, corroborating the UV-Vis and SEM results. Figure 10b presents the DLS analysis of the phytocarrier system. The DLS curve of the phytocarrier system displays two peaks that can be attribut to AgNPs and curcuma nanoparticles.
Superimposition of the individual AgNPs and the phytocarrier system DLS patter (Figure 11) reveals the presence of peaks corresponding to two types of nanoparticles wi varying sizes. The particles have a size of 100 nm, corroborating the UV-Vis and SEM results. Figure 10b presents the DLS analysis of the phytocarrier system. The DLS curve of the phytocarrier system displays two peaks that can be attributed to AgNPs and curcuma nanoparticles.
Superimposition of the individual AgNPs and the phytocarrier system DLS patterns (Figure 11) reveals the presence of peaks corresponding to two types of nanoparticles with varying sizes. The results of the DLS analysis for the phytocarrier system (Figures 10b and 11) suggest that the particle sizes are distributed in a narrow range. The mean diameter of AgNPs from the phytocarrier system is 52 nm. The second mean of the hydrodynamic diameter, attributed to curcuma nanoparticles, is about 220 nm. Because the mean size provided by the DLS analysis refers to the hydrodynamic diameter, the measured nanoparticles dimensions are often seem larger than those obtained by SEM analysis. The AgNPs tend to form aggregates with settled fractions visible from the first day [156,157]. However, this phenomenon was not observed in the case of the phytocarrier system, probably due to the low density of the curcuma.
In addition, well-dispersed particles (AgNPs and curcuma) from the phytocarrier system were identified ( Figure 11). These results suggest high stability of the phytocarrier system without absorbance spectrum modifications [158].

UHR-TEM
TEM analysis provided details about the size and shape of the silver nanoparticles and phytocarrier complex. As shown in Figure 12, the unfiltered AgNPs were polymorphic, with spherical, triangular, prismatic and rod-like shapes and sizes distributed in the range of approximately 35 nm to 150 nm, confirming the SEM analysis. Furthermore, given that the SEM image of curcuma appeared to be less detailed, we decided to doublefilter the curcuma suspension for the UHR-TEM measurements, which allowed only the finest curcuma nanoparticles to pass through. As a result, an approximately 15 nm thick corona of curcuma nanoparticles was formed on the AgNP surface (Figure 12), indicating an increased attraction between the organic and inorganic nanostructures mediated mostly by physical forces. The results of the DLS analysis for the phytocarrier system (Figures 10b and 11) suggest that the particle sizes are distributed in a narrow range. The mean diameter of AgNPs from the phytocarrier system is 52 nm. The second mean of the hydrodynamic diameter, attributed to curcuma nanoparticles, is about 220 nm. Because the mean size provided by the DLS analysis refers to the hydrodynamic diameter, the measured nanoparticles dimensions are often seem larger than those obtained by SEM analysis. The AgNPs tend to form aggregates with settled fractions visible from the first day [156,157]. However, this phenomenon was not observed in the case of the phytocarrier system, probably due to the low density of the curcuma.
In addition, well-dispersed particles (AgNPs and curcuma) from the phytocarrier system were identified ( Figure 11). These results suggest high stability of the phytocarrier system without absorbance spectrum modifications [158].

UHR-TEM
TEM analysis provided details about the size and shape of the silver nanoparticles and phytocarrier complex. As shown in Figure 12, the unfiltered AgNPs were polymorphic, with spherical, triangular, prismatic and rod-like shapes and sizes distributed in the range of approximately 35 nm to 150 nm, confirming the SEM analysis. Furthermore, given that the SEM image of curcuma appeared to be less detailed, we decided to double-filter the curcuma suspension for the UHR-TEM measurements, which allowed only the finest curcuma nanoparticles to pass through. As a result, an approximately 15 nm thick corona of curcuma nanoparticles was formed on the AgNP surface (Figure 12), indicating an increased attraction between the organic and inorganic nanostructures mediated mostly by physical forces.
The curcuma corona appears to be composed of individual nanoparticles of approximately 10-15 nm in diameter. Antioxidants 2022, 11, x FOR PEER REVIEW 24 of 39 The curcuma corona appears to be composed of individual nanoparticles of approximately 10-15 nm in diameter.

Zeta Potential
The zeta potential of the nanoparticles was measured in order to evaluate the surface charge potential, which is an important parameter for evaluation of suspension stability [159].
The zeta potential value of AgNPs was −34.49 mV, indicating the high stability of the suspension. A negative value was expected, as the nanoparticles were stabilized with sodium citrate and ascorbic acid. The zeta potential changed to −22.34 mV for the phytocarrier system, indicating the stability of the solution. This negative surface charge indicates the high biocompatibility of the obtained phytocarrier system [160].

EDS Spectroscopy
The EDS spectra of the phytocarrier system and curcuma sample are shown in Figures 13 and 14, respectively.

Zeta Potential
The zeta potential of the nanoparticles was measured in order to evaluate the surface charge potential, which is an important parameter for evaluation of suspension stability [159].
The zeta potential value of AgNPs was −34.49 mV, indicating the high stability of the suspension. A negative value was expected, as the nanoparticles were stabilized with sodium citrate and ascorbic acid. The zeta potential changed to −22.34 mV for the phytocarrier system, indicating the stability of the solution. This negative surface charge indicates the high biocompatibility of the obtained phytocarrier system [160].

EDS Spectroscopy
The EDS spectra of the phytocarrier system and curcuma sample are shown in Figures 13 and 14, respectively. The curcuma corona appears to be composed of individual nanoparticles of approximately 10-15 nm in diameter.

Zeta Potential
The zeta potential of the nanoparticles was measured in order to evaluate the surface charge potential, which is an important parameter for evaluation of suspension stability [159].
The zeta potential value of AgNPs was −34.49 mV, indicating the high stability of the suspension. A negative value was expected, as the nanoparticles were stabilized with sodium citrate and ascorbic acid. The zeta potential changed to −22.34 mV for the phytocarrier system, indicating the stability of the solution. This negative surface charge indicates the high biocompatibility of the obtained phytocarrier system [160].

EDS Spectroscopy
The EDS spectra of the phytocarrier system and curcuma sample are shown in Figures 13 and 14, respectively.  The energy-dispersive X-ray (EDX) spectrum results of the phytocarrier system revealed strong signals in the silver region, confirming the presence of silver in the analyzed micro area (Figure 14a,b). Figure 15 displays the live map for curcuma and the distribution of the identified elements.
The comparative analysis presented in Figure 16 showing a live map for herb and phytocarrier system (Figure 16) highlights the presence of differences in terms of the proportion of identification elements in the samples resulting from the formation of the new phytocarrier system. The energy-dispersive X-ray (EDX) spectrum results of the phytocarrier system revealed strong signals in the silver region, confirming the presence of silver in the analyzed micro area (Figure 14a,b). Figure 15 displays the live map for curcuma and the distribution of the identified elements.
The comparative analysis presented in Figure 16 showing a live map for herb and phytocarrier system (Figure 16  The comparative analysis presented in Figure 15 showing live map for curcuma and the phytocarrier system ( Figure 16) highlights the presence of differences in terms of the proportion of identification elements in the two samples resulting from the formation of phytocarrier system.
The results of SEM, EDX and SEM live maps confirm the achievement of the new phytocarrier system.  The comparative analysis presented in Figure 15 showing live map for curcuma and the phytocarrier system ( Figure 16) highlights the presence of differences in terms of the proportion of identification elements in the two samples resulting from the formation of phytocarrier system.
The results of SEM, EDX and SEM live maps confirm the achievement of the new phytocarrier system. The comparative analysis presented in Figure 15 showing live map for curcuma and the phytocarrier system ( Figure 16) highlights the presence of differences in terms of the proportion of identification elements in the two samples resulting from the formation of phytocarrier system.
The results of SEM, EDX and SEM live maps confirm the achievement of the new phytocarrier system.

Screening of Antioxidant Activity
The total antioxidant capacity (TAC) of a particular plant results from the collective action of the categories of antioxidants of which it is composed [52].
Generally, in vitro and in vivo methods are used to estimate the total antioxidant capacity of natural compounds.
Despite the availability of a standardized method, the most popular techniques are non-competitive, non-enzymatic assays (Folin-Ciocalteu reaction, DPPH, ABTS/TEAC, CUPRAC, ABTS and FRAP) for in vitro determination of antioxidant capacity in the presence of a chromogenic agent [45,46,52,53].
The antioxidant activity of the phytocarrier system is a result of the complementary and synergistic action of each component (curcuma and AgNPs). More than 80 secondary metabolites from 10 categories were identified in the curcuma sample. Therefore, to reflect the antioxidant properties of the new phytocarrier system more accurately, we used three in vitro, non-competitive, non-enzymatic, distinctive assays (DPPH, Folin-Ciocalteu and cyclic voltammetry).

DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Free Radical Scavenging Assay
DPPH is a simple, fast assay widely used to determine the total antioxidant capacity of a liquid or solid sample. It is based on reduction of DPPH, a stable free radical, through a single electron transfer (ET)-type mechanism [45,46,52,53].
Thus, the antioxidant capacities of the new phytocarrier system and curcumin were evaluated and compared to three antioxidant standards (β-carotene, ascorbic acid and curcumin). These standards were selected based on the chemical screening results, which highlighted the presence of beta-carotene and curcumin in the curcuma sample. Moreover, studies have reported a high content of ascorbic acid in curcuma [161].
The obtained results are presented in Table 5 and Figure 17.  Based on the obtained IC50 values and given that lower IC50 values correspond to higher antioxidant activity, the new phytocarrier system has higher antioxidant activity than that of curcuma, ascorbic acid and curcumin but lower than antioxidant activity than that of the beta-carotene standard. Based on the obtained IC 50 values and given that lower IC 50 values correspond to higher antioxidant activity, the new phytocarrier system has higher antioxidant activity than that of curcuma, ascorbic acid and curcumin but lower than antioxidant activity than that of the beta-carotene standard.
Research has shown that the polarity of solvents significantly influences not only the extraction yield but also their capacity for antioxidant activity [81].
Therefore, only the beta-carotene solvent differed from that of the other samples (curcuma, curcumin, ascorbic acid and the phytocarrier system). Thus, a mixture of solvents with varying polarities (methanol-dichloromethane) was used, owing to its poor solubility in methanol, which can explain the obtained IC 50 value of beta-carotene.

Folin-Ciocalteu Assay
The Folin-Ciocalteu assay measures the reducing capacity of an antioxidant based on electron transfer and is considered one of the most accessible and facile methods for evaluation of the total phenolic content (TPC) of a particular substrate [46,54,162,163].
The total polyphenolic content (TPC) of the phytocarrier system and the curcuma sample was determined; the obtained results are presented in Table 6. According to the obtained data, the total polyphenolic content detected in the case of the new phytocarrier system is more than 50% higher than that of the curcuma sample.
The higher antioxidant capacity of the phytocarrier system compared to the curcuma sample can be attributed to the synergistic action of AgNPs, with a catalytic role played by curcuma polyphenolic compounds [164,165].

Cyclic Voltammetry
The cyclic voltammetry method provides electrochemical measurements of antioxidant characteristics using a redox-based mechanism to determine total antioxidant capacity. This method is reliable, fast, simple and cost-efficient, providing affordable and accurate information on the reducing capacity of active components [52,54,166,167] A cyclic voltammogram provides data on the total antioxidant capacity of a substrate without providing information on the individual contribution of specific antioxidant categories. The total antioxidant capacity is the result of the combined action of two key parameters: the oxidation potential (which reflects the reducing power of a particular compound) and the anodic peak current intensity (Ia) (corresponding to the concentration of the bioactive component). Low oxidation potential values indicate the ability of biomolecules to donate electrons, as well as implicit high antioxidant activity [167][168][169].
The Trolox equivalent antioxidant capacity (TEAC) assay is based on the ability of a given compound with antioxidant properties to scavenge ABTS· + radicals or ABTS 2+ cations [58,170,171].
In the present study, we exploited the ability of Trolox to reduce ABTS 2+ cations according to the following scheme (Scheme 1) [58,171].
The Trolox equivalent antioxidant capacity (TEAC) assay is based on the ability of a given compound with antioxidant properties to scavenge ABTS• + radicals or ABTS 2+ cations [58,170,171].
In the present study, we exploited the ability of Trolox to reduce ABTS 2+ cations according to the following scheme (Scheme 1) [58,171].

Scheme 1. Redox chemistry of the ABTS/Trolox system.
The stability of the ABTS• + radical was monitored through CV measurements at several scan rates ( Figure 18). The absence of redox peaks at 230/231 mV corresponding to the discharge of the ABTS/ABTS + pair suggests that the ABTS working solutions contained only ABTS + species, as confirmed by the appearance of characteristic redox peaks at 489/490 mV.
In the presence of various amounts of Trolox, the CVs of ABTS underwent significant modifications. For Trolox concentrations exceeding 0.02 M, the anodic peak increases Scheme 1. Redox chemistry of the ABTS/Trolox system. The stability of the ABTS· + radical was monitored through CV measurements at several scan rates ( Figure 18). tablishes the catalytic voltammetric currents.
The Trolox equivalent antioxidant capacity (TEAC) assay is based on the ability of a given compound with antioxidant properties to scavenge ABTS• + radicals or ABTS 2+ cations [58,170,171].
In the present study, we exploited the ability of Trolox to reduce ABTS 2+ cations according to the following scheme (Scheme 1) [58,171].

Scheme 1. Redox chemistry of the ABTS/Trolox system.
The stability of the ABTS• + radical was monitored through CV measurements at several scan rates ( Figure 18). The absence of redox peaks at 230/231 mV corresponding to the discharge of the ABTS/ABTS + pair suggests that the ABTS working solutions contained only ABTS + species, as confirmed by the appearance of characteristic redox peaks at 489/490 mV.
In the presence of various amounts of Trolox, the CVs of ABTS underwent significant modifications. For Trolox concentrations exceeding 0.02 M, the anodic peak increases The absence of redox peaks at 230/231 mV corresponding to the discharge of the ABTS/ABTS + pair suggests that the ABTS working solutions contained only ABTS + species, as confirmed by the appearance of characteristic redox peaks at 489/490 mV.
In the presence of various amounts of Trolox, the CVs of ABTS underwent significant modifications. For Trolox concentrations exceeding 0.02 M, the anodic peak increases linearly with the amount of Trolox, whereas the backward catodic peak decreases correspondingly ( Figure 19).
According to Scheme 1, when Trolox is added to the cell, it reacts with the ABTS 2+ cation, the product of electrode oxidation of ABTS· + . Thus, the electrochemical species, ABTS· + , is regenerated and can be reoxidized on the electrode surface. This phenomenon leads to an increase in the anodic current, accompanied by a simultaneous decrease in the cathodic current. Higher concentrations ultimately yield higher oxidation peaks, as more ABTS· + species are regenerated during the voltammetric assay.
∆I p is defined as the difference between the measured oxidation peak currents in the presence of corresponding Trolox (I p,x ) and in the absence of Trolox (I p,0 ).
The optimized volumetric ratio provided the appropriate peak currents for interpolation in the calibration plot. An increase in the oxidation current of ABTS· + was observed, as in the presence of Trolox (Figure 20). linearly with the amount of Trolox, whereas the backward catodic peak decreases correspondingly ( Figure 19). According to Scheme 1, when Trolox is added to the cell, it reacts with the ABTS 2+ cation, the product of electrode oxidation of ABTS• + . Thus, the electrochemical species, ABTS• + , is regenerated and can be reoxidized on the electrode surface. This phenomenon leads to an increase in the anodic current, accompanied by a simultaneous decrease in the cathodic current. Higher concentrations ultimately yield higher oxidation peaks, as more ABTS• + species are regenerated during the voltammetric assay.
The optimized volumetric ratio provided the appropriate peak currents for interpolation in the calibration plot. An increase in the oxidation current of ABTS• + was observed, as in the presence of Trolox ( Figure 20). The electrochemical TEAC assay provided the following results (expressed as mmole/L Trolox): 6.6 ± 0.4, 3.7 ± 0.2 and si 4.2 ± 0.25 (n = 10) for the phytocarrier system, citrate-coated AgNPs and curcuma, respectively.
The value was larger than that suggested by the TEAC data (spectrophotometric method) reported in literature for curcuma extract (TEACcurucma = 0.00292 mmol/L) [172,173].
However, the experimental conditions differed, making the comparison of the results of various studies unrealistic [52].
The TEAC values indicate that the phytocarrier system has a higher antioxidant activity than that of each component (curcuma and AgNPs).
This high antioxidant activity of the phytocarrier system can be attributed to the syn- The electrochemical TEAC assay provided the following results (expressed as mmole/L Trolox): 6.6 ± 0.4, 3.7 ± 0.2 and si 4.2 ± 0.25 (n = 10) for the phytocarrier system, citratecoated AgNPs and curcuma, respectively.
The value was larger than that suggested by the TEAC data (spectrophotometric method) reported in literature for curcuma extract (TEAC curucma = 0.00292 mmol/L) [172,173].
However, the experimental conditions differed, making the comparison of the results of various studies unrealistic [52].
The TEAC values indicate that the phytocarrier system has a higher antioxidant activity than that of each component (curcuma and AgNPs).
This high antioxidant activity of the phytocarrier system can be attributed to the synergistic and complementary action of the antioxidants in curcuma and the antioxidant mechanism of AgNPs (quenched free radicals by donating or accepting electrons) in the presence of the various bioactive compounds in curcuma [165,174].
In addition, the structural characteristics of AgNPs (shape and size) influence the antioxidant activity [175].
Various studies have reported that AgNPs influence the antioxidant activity, triggering ROS production through a reduction in reactive oxygen species [175][176][177].
Furthermore, the antioxidant activity of citrate-coated AgNPs is the result the antioxidant mechanism of the metal nanoparticles, as well as sodium citrate and ascorbic acid from the coating of the nanoparticles [178][179][180].

Conclusions
In this study, a new phytocarrier system with unique optical properties and high antioxidant activity was developed. The metabolite profile of Curcuma longa was determined. The biological activities were discussed for each metabolite category. The achievement of the curcuma-citrate-coated AgNP carrier system was confirmed through UV-Vis FTIR, EDX, XRD, DLS, zeta potential, SEM and TEM studies. The size distribution of AgNPs and curcuma particles was measured through DSL analysis. The stability of AgNPs and the phytocarrier system in aqueous suspensions was determined using zeta potential analysis.
A combination of methods (DPPH, Folin-Ciocalteu and cyclic voltammetry) was used to evaluate the antioxidant properties of the proposed phytocarrier system. The results show a significantly higher antioxidant activity of the phytocarrier than that of curcuma or AgNPs. However, further research is necessary to investigate the biological activity and biocompatibility of the new phytocarrier system. This study motivates future therapeutic research in the fields of innovative neurodegenerative agents.