Artemisia annua Growing Wild in Romania—A Metabolite Profile Approach to Target a Drug Delivery System Based on Magnetite Nanoparticles

The metabolites profile of a plant is greatly influenced by geographical factors and the ecological environment. Various studies focused on artemisinin and its derivates for their antiparasitic and antitumoral effects. However, after the isolation and purification stage, their pharmaceutical potential is limited due to their low bioavailability, permeability and lifetime. The antibacterial activity of essential oils has been another topic of interest for many studies on this plant. Nevertheless, only a few studies investigate other metabolites in Artemisia annua. Considering that secondary metabolites act synergistically in a plant, the existence of other metabolites with antitumor and high immunomodulating activity is even more important. Novel nano-carrier systems obtained by loading herbs into magnetic nanoparticles ensures the increase in the antitumor effect, but also, overcoming the barriers related to permeability, localization. This study reported the first complete metabolic profile from wild grown Romanian Artemisia annua. A total of 103 metabolites were identified under mass spectra (MS) positive mode from 13 secondary metabolite categories: amino acids, terpenoids, steroids, coumarins, flavonoids, organic acids, fatty acids, phenolic acids, carbohydrates, glycosides, aldehydes, hydrocarbons, etc. In addition, the biological activity of each class of metabolites was discussed. We further developed a simple and inexpensive nano-carrier system with the intention to capitalize on the beneficial properties of both components. Evaluation of the nano-carrier system’s morpho-structural and magnetic properties was performed.


Introduction
Romanian phytotherapy has an ancient and very rich tradition based on a very wide diversity of medicinal plants. Thus, in the spontaneous flora of Romania there are about 800 species of medicinal plants. Additionally, plants of the genus Artemisia (Asteraceae) form part of this phytopharmacological treasure.
Artemisia annua (common name: wormwood or năfurica in Romanian) is one of the ancient healing plants recognized in traditional medicine from Europe and Asia. Romanian traditional medicine has exploited its therapeutic properties: antihemorrhagic, antiseptic, antioxidant, digestive, antipyretic, immunomodulatory, antibacterial and antitumoral [1][2][3][4][5]. Artemisia annua is used also in Romania to prepare a digestive wine [1][2][3][4][5]. In traditional Asian pharmacopoeia, it was recommended, especially for the treatment of fevers and colds. The development of highly efficient, selective, simple and inexpensive nano-carrier systems could be an effective method to avoid these risks while ensuring the controlled intake of phytoconstituents with biological activity [40,41].
Modern drug delivery systems based on magnetic nanoparticles could easily accomplish these requirements. Moreover, the latest developments regarding magnetite nanoparticles have demonstrated their benefits and recommend their use in drug delivery and other different biomedical applications (magnetic resonance imaging, tumor therapeutic hyperthermia, etc.) [25,42].
Magnetic/superparamagnetic nanoparticles could represent a more than interesting alternative due to their advantages: their capability for local delivery and the ability to act selectively. However, the possibility of an immune response is the main drawback of these nanoparticles. Therefore, the design of new drug delivery systems based on magnetic/superparamagnetic nanoparticles for use as early detection methods and in the diagnosis, prognosis and monitoring of the evolution of the cancer treatment is required given the social impact of these diseases [25,[43][44][45][46][47][48][49].
Recent studies have shown that nano-carriers based on magnetic nanoparticles lead to a high drug tissue permeability and retention effect and thus enhance the beneficial therapeutic effects [45][46][47].
To our best knowledge, this study investigates the metabolite profile of Artemisia annua grown wild in Romania for the first time. Subsequently, a simple and inexpensive nano-carrier system that capitalizes both the therapeutic properties of Artemisia annua (whole plant) and magnetic Fe 3 O 4 nanoparticles was developed.

Results and Discussion
Extensive research in the field of plants and especially on those with high therapeutic potential has shown that they have a very complex composition of compounds with high biological activity that act synergistically in the body [26,27].
Additionally, a full description of a general metabolic profile for a specific herb is all the more difficult, as significant differences in secondary metabolites was reported among the same plants harvested from various geographic regions of the world. Studies in this area confirmed that the content of specific plant secondary metabolites is the result of several environmental stress factors (climate, soil and biological conditions) which directly influence plant growth, development and topography distribution [28,[50][51][52].
The pharmacological properties of different plant secondary metabolites were extensively investigated. However, their therapeutic benefits have not been completely understood [11,21,22]. Plant metabolites with peptide structures are just an example of this [51].
Bioactive metabolite chemical screening of sweet wormwood (năfurica) was tentatively carried out via gas-chromatography coupled with mass spectroscopy (GC-MS) and electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) analysis.
In addition, the amino acid profile was investigated using GC-MS techniques ( Figure 1). The mass spectra of components identified were determined via comparison of their retention indices and mass spectra with those of NIST/EPA/NIH, the Mass Spectral Library 2.0 data base, as well as by reviewing the literature [32,53]. The results are listed in Table 1, which presents tentative amino acid identification via GC-MS corresponding to Artemisia annua sample [32,54,55].
analysis. However, only a few studies reported the amino acid profile of Artemisia annua [32].
The identified metabolites are listed in Table 2 and classified according to their m/z ratio (mass-to-charge-ratio) (theoretical and measured), chemical name and formula and the related literature.   The identified metabolites are listed in Table 2 and classified according to their m/z ratio (mass-to-charge-ratio) (theoretical and measured), chemical name and formula and the related literature.

Flavonoids
Terpenoids found in the Artemisia annua sample are the one of the major constituents of the total identified metabolites. Previous studies on the therapeutic effect of terpenoids have demonstrated their antimicrobial, antibacterial, antifungal, analgesic and anti-insect activity [74].
Sesquiterpenes, another important class of metabolites from Artemisia annua, were shown to have antitumoral, antiplasmodial, anti-inflammatory and anti-allergic properties. Sesquiterpenes lactones isolated from Artemisia annua are used in antimalaria drugs [75,76].
Terpenoids found in the Artemisia annua sample are the one of the major constituents of the total identified metabolites. Previous studies on the therapeutic effect of terpenoids have demonstrated their antimicrobial, antibacterial, antifungal, analgesic and anti-insect activity [74].
Sesquiterpenes, another important class of metabolites from Artemisia annua, were shown to have antitumoral, antiplasmodial, anti-inflammatory and anti-allergic properties. Sesquiterpenes lactones isolated from Artemisia annua are used in antimalaria drugs [75,76].
Coumarins are metabolites highly relevant to human health. Recent studies on coumarins isolated from plants have shown antioxidant, antimicrobial, antiviral, antifungal, and antiparasitic, anti-diabetic, analgesic, anti-neurodegenerative, and anti-inflammatory activity. Moreover, coumarins have been demonstrated to stimulate the immunologic response and are used in the therapy of different tumors: leukemia, renal and prostate tumors, melanoma and breast cancer [77,78].
Flavonoids were another major category of metabolites identified in the plant sample. A total of 25 different flavonoids were found in the Artemisia annua sample. These compounds exhibit antioxidant, antitumoral, anti-inflammatory, antimicrobial, anti-cholinesterase, neurodegenerative disease (Alzheimer) and atherosclerosis prevention effects .
Fatty acids are involved in neuroprotection and cardiovascular protection mechanisms. Recent studies reported their beneficial role in autoimmune and neurodegenerative diseases, including Alzheimer disease (AD) [85].
Glycosides from herbs showed antitumoral activity, mainly on leukemia and gastric cancer [89].

Nano-Carrier System Based on Magnetic Nanoparticles of Fe 3 O 4
The development of an efficient and selective drug nano-carrier system required an optimal ratio between the herb and magnetic nanoparticles in order to provide the highest biological activity and functionality (selectivity and vectorization). Recent studies regarding types of nano-drug systems have reported the specific bioactive phytochemicals that were loaded into the magnetic nanoparticles [90,91].

FT-IR Spectroscopy
The incorporation of herb phytochemicals into the pores of Fe 3 O 4 nanoparticles was successfully achieved and was confirmed through FT-IR spectroscopy. Figure 4 presents the spectra of the herb, Fe 3 O 4 nanoparticles and the nano-carrier system. sample. A total of 25 different flavonoids were found in the Artemisia annua sample. These compounds exhibit antioxidant, antitumoral, anti-inflammatory, antimicrobial, anticholinesterase, neurodegenerative disease (Alzheimer) and atherosclerosis prevention effects .
Fatty acids are involved in neuroprotection and cardiovascular protection mechanisms. Recent studies reported their beneficial role in autoimmune and neurodegenerative diseases, including Alzheimer disease (AD) [85].
Glycosides from herbs showed antitumoral activity, mainly on leukemia and gastric cancer [89].

Nano-Carrier System Based on Magnetic Nanoparticles of Fe3O4
The development of an efficient and selective drug nano-carrier system required an optimal ratio between the herb and magnetic nanoparticles in order to provide the highest biological activity and functionality (selectivity and vectorization). Recent studies regarding types of nano-drug systems have reported the specific bioactive phytochemicals that were loaded into the magnetic nanoparticles [90,91].

FT-IR Spectroscopy
The incorporation of herb phytochemicals into the pores of Fe3O4 nanoparticles was successfully achieved and was confirmed through FT-IR spectroscopy. Figure 4 presents the spectra of the herb, Fe3O4 nanoparticles and the nano-carrier system.  Withal, the band at 1737 cm −1 is the characteristic absorption peak of Artemisia annua assigned to δ-lactone group [47].
Two main broad metal-oxygen bands are seen in the IR spectra of Fe 3 O 4 nanoparticles (Figure 4) in the range 400-600 cm −1 . The highest vibration band at 576 cm −1 is assigned to the stretching vibrations of M tetra O bond in the tetrahedral voids, and the lowest band at 410 cm −1 (partially visible) corresponds to the stretching vibrations of the M octa O bond in the octahedral void peak [102][103][104].

X-ray Diffraction Spectroscopy
Withal, the band at 1737 cm −1 is the characteristic absorption peak of Artemisia annua assigned to δ-lactone group [47].
Two main broad metal-oxygen bands are seen in the IR spectra of Fe3O4 nanoparticles (Figure 4) in the range 400-600 cm −1 . The highest vibration band at 576 cm −1 is assigned to the stretching vibrations of MtetraO bond in the tetrahedral voids, and the lowest band at 410 cm −1 (partially visible) corresponds to the stretching vibrations of the MoctaO bond in the octahedral void peak [102][103][104].

X-ray Diffraction Spectroscopy
Withal, the band at 1737 cm −1 is the characteristic absorption peak of Artemisia annua assigned to δ-lactone group [47].
Two main broad metal-oxygen bands are seen in the IR spectra of Fe3O4 nanoparticles (Figure 4) in the range 400-600 cm −1 . The highest vibration band at 576 cm −1 is assigned to the stretching vibrations of MtetraO bond in the tetrahedral voids, and the lowest band at 410 cm −1 (partially visible) corresponds to the stretching vibrations of the MoctaO bond in the octahedral void peak [102][103][104].
The spectra of the nano-carrier system (Figure 4) display the characteristic peaks of the herb as well as the metal-oxygen vibration bands at 576 cm −1 and at 410 cm −1 , which confirm the incorporation of the herb into the pores of Fe3O4 nanoparticles [47].   Regarding the diffraction pattern of the herb (Figure 5), in the range of 13-26°, a wide band that is characteristic of some amorphous phases can be observed. This wide band is also found attenuated in the diffraction pattern of the nano-carrier system (Figure 7 and Figure 8). Additionally, in the diffraction pattern of the Fe3O4 nanoparticles (Figure 6), only the peaks of the single crystalline spinel phase Fe3O4 (average crystallite size 10.9 nm) are present.  In the diffraction pattern of the mixture (Figures 7 and 8), crystalline spinel phase Fe3O4 nanoparticles with an average crystallite size of 12.9 nm were identified. A peak at ~26.5° and a band between 13-26° were also present but much attenuated in the spectrum of Artemisia annua.

Scanning Electron Microscopy (SEM)
The SEM micrographs of the herb, magnetic nanoparticles and the nano-carrier system are shown in Figures 9-11. Regarding the diffraction pattern of the herb (Figure 5), in the range of 13-26°, a wide band that is characteristic of some amorphous phases can be observed. This wide band is also found attenuated in the diffraction pattern of the nano-carrier system (Figure 7 and Figure 8). Additionally, in the diffraction pattern of the Fe3O4 nanoparticles (Figure 6), only the peaks of the single crystalline spinel phase Fe3O4 (average crystallite size 10.9 nm) are present.  In the diffraction pattern of the mixture (Figures 7 and 8), crystalline spinel phase Fe3O4 nanoparticles with an average crystallite size of 12.9 nm were identified. A peak at ~26.5° and a band between 13-26° were also present but much attenuated in the spectrum of Artemisia annua.

Scanning Electron Microscopy (SEM)
The SEM micrographs of the herb, magnetic nanoparticles and the nano-carrier system are shown in Figures 9-11. Regarding the diffraction pattern of the herb (Figure 5), in the range of 13-26 • , a wide band that is characteristic of some amorphous phases can be observed. This wide band is also found attenuated in the diffraction pattern of the nano-carrier system (Figure 7 and Figure 8). Additionally, in the diffraction pattern of the Fe 3 O 4 nanoparticles (Figure 6), only the peaks of the single crystalline spinel phase Fe 3 O 4 (average crystallite size 10.9 nm) are present.
In the diffraction pattern of the mixture (Figures 7 and 8), crystalline spinel phase Fe 3 O 4 nanoparticles with an average crystallite size of 12.9 nm were identified. A peak at 26.5 • and a band between 13-26 • were also present but much attenuated in the spectrum of Artemisia annua.

Scanning Electron Microscopy (SEM)
The SEM micrographs of the herb, magnetic nanoparticles and the nano-carrier system are shown in Figures 9-11.  Plants 2021, 10, x FOR PEER REVIEW 14 of 22  As can be seen in the SEM image (Figure 9), the particles of Artemisia anuua shown are in the form of micron-sized fibers and irregular shape particles. The incorporation of herb phytochemicals into the pores of Fe3O4 nanoparticles was also confirmed via the scanning electron microscopy (SEM) images of Fe3O4 nanoparticles ( Figure 10) and Fe3O4 loaded with the herb (Figure 11). The SEM image of Fe3O4 nanoparticles loaded with the herb (Figure 11b) shows a powder that consists of agglomerations of round nanoparticles with dimensions between 5 and 30 nm, as well as irregular shapes with dimensions greater than 60 nm. In the SEM image at high magnification (Figure 11a), irregular shape particles in Fe3O4 nanoparticles' surface modification with herbs can be seen.  As can be seen in the SEM image (Figure 9), the particles of Artemisia anuua shown are in the form of micron-sized fibers and irregular shape particles. The incorporation of herb phytochemicals into the pores of Fe3O4 nanoparticles was also confirmed via the scanning electron microscopy (SEM) images of Fe3O4 nanoparticles ( Figure 10) and Fe3O4 loaded with the herb (Figure 11). The SEM image of Fe3O4 nanoparticles loaded with the herb (Figure 11b) shows a powder that consists of agglomerations of round nanoparticles with dimensions between 5 and 30 nm, as well as irregular shapes with dimensions greater than 60 nm. In the SEM image at high magnification (Figure 11a), irregular shape particles in Fe3O4 nanoparticles' surface modification with herbs can be seen. As can be seen in the SEM image (Figure 9), the particles of Artemisia anuua shown are in the form of micron-sized fibers and irregular shape particles. The incorporation of herb phytochemicals into the pores of Fe 3 O 4 nanoparticles was also confirmed via the scanning electron microscopy (SEM) images of Fe 3 O 4 nanoparticles ( Figure 10) and Fe 3 O 4 loaded with the herb (Figure 11). The SEM image of Fe 3 O 4 nanoparticles loaded with the herb (Figure 11b) shows a powder that consists of agglomerations of round nanoparticles with dimensions between 5 and 30 nm, as well as irregular shapes with dimensions greater than 60 nm. In the SEM image at high magnification (Figure 11a

Magnetic Properties
The magnetic properties of the Fe3O4 nanoparticles and the nano-carrier system were investigated at a low-frequency driving field (50 Hz) by means of an induction hysteresis graph [105]. It was found that both samples exhibit ferromagnetic behavior with narrow hysteresis loops (see Figures 12 and 13).

Magnetic Properties
The magnetic properties of the Fe 3 O 4 nanoparticles and the nano-carrier system were investigated at a low-frequency driving field (50 Hz) by means of an induction hysteresis graph [105]. It was found that both samples exhibit ferromagnetic behavior with narrow hysteresis loops (see Figures 12 and 13).

Magnetic Properties
The magnetic properties of the Fe3O4 nanoparticles and the nano-carrier system were investigated at a low-frequency driving field (50 Hz) by means of an induction hysteresis graph [105]. It was found that both samples exhibit ferromagnetic behavior with narrow hysteresis loops (see Figures 12 and 13).

Magnetic Properties
The magnetic properties of the Fe3O4 nanoparticles and the nano-carrier system were investigated at a low-frequency driving field (50 Hz) by means of an induction hysteresis graph [105]. It was found that both samples exhibit ferromagnetic behavior with narrow hysteresis loops (see Figures 12 and 13).   From the measured hysteresis loops, the saturation magnetization (σ S ), the coercive field (H c ) and the remanent magnetization (σ R ) were determined. The results are presented in Table 4. As expected, the saturation magnetization of the sample Fe 3 O 4 nanoparticles is larger than that of nano-carrier system. Both samples have small values regarding the remnant ratio σ R /σ S (in order of 0.1), which is an indication of the ease with which the magnetization reorients to the nearest easy axis magnetization direction after the removal of the magnetic field. The frequency dependence on the complex magnetic permeability of the samples (Equation (1)) over the frequency range of 1 kHz to 2 MHz was measured at room temperature, and the obtained results are presented in Figure 14 [106].
where µ ( f ) is the real part;µ ( f ) is the imaginary part.
From the measured hysteresis loops, the saturation magnetization (σS), the coercive field (Hc) and the remanent magnetization (σR) were determined. The results are presented in Table 4. As expected, the saturation magnetization of the sample Fe3O4 nanoparticles is larger than that of nano-carrier system. Both samples have small values regarding the remnant ratio σR/σS (in order of 0.1), which is an indication of the ease with which the magnetization reorients to the nearest easy axis magnetization direction after the removal of the magnetic field. The frequency dependence on the complex magnetic permeability of the samples (Equation (1)) over the frequency range of 1 kHz to 2 MHz was measured at room temperature, and the obtained results are presented in Figure 14 [106].
In the investigated frequency range, there are no magnetic relaxation peaks that provide clues about the characteristic magnetization processes. However, given the small sizes of the particles and also the low value of the imaginary component of complex magnetic permeability, it can be assumed that the dominant magnetization mechanism is the Neel process, correlated with the rotation of the magnetic moment inside the particles by overcoming the magneto-crystalline anisotropy barrier [107,108]. The obtained results indicate that the nano-carrier system, wherein the selected ratio of plant:magnetic nanoparticles is 3:1, exhibits magnetic properties. In the investigated frequency range, there are no magnetic relaxation peaks that provide clues about the characteristic magnetization processes. However, given the small sizes of the particles and also the low value of the imaginary component of complex magnetic permeability, it can be assumed that the dominant magnetization mechanism is the Neel process, correlated with the rotation of the magnetic moment inside the particles by overcoming the magneto-crystalline anisotropy barrier [107,108].
The obtained results indicate that the nano-carrier system, wherein the selected ratio of plant:magnetic nanoparticles is 3:1, exhibits magnetic properties.

Materials and Methods
All used reagents were GC grade. Methanol and chloroform were purchased from VWR (Wien, Austria). The Fe 3 O 4 nanoparticles (nanoparticle size: 23 nm) were provided by the National Research and Development Institute for Non-Ferrous and Rare Metals, Pantelimon, Romania. The plant samples (whole plant) were collected in August 2020 from the area of Timis, Romania and were taxonomically authenticated at Victor Babes University of Medicine and Pharmacy, Timisoara, Romania. The plant samples were rapidly frozen in liquid nitrogen (−194 • C), ground and sieved to obtain a particle size lower than 0.5 mm, and they were kept at −80 • C to avoid enzymatic conversion or metabolite degradation. For each analysis, 1.8 g of dried sample was subject to sonication extraction in 25 mL of solvent (methanol/chloroform = 1:1) for 25 min at 40 • 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 analysis.

Nano-Carrier System Preparation
For each analysis, 1.5 g of sample was prepared from dried herb (whole plant, ground and sieved to obtain a particle size lower than 0.5 mm), and Fe 3 O 4 nanoparticles were added (herb/Fe 3 O 4 nanoparticles = 3:1). The obtained mixture was subjected to micronization at room temperature for 5 min.

GC-MS Analysis
Gas chromatography was carried out using ClarusSQ8 GC/MS (Perkin Elmer) apparatus with a ZB-AAA GC column (10 m × 0.25 mm) (Phenomenex, Torrance, CA, USA); carrier gas, He; flow rate, 1 mL/min, following 3.3. GC-MS Separation Conditions (the standard conditions provided with the EZ: faast GC-MS free amino acids kit).

Mass Spectrometry
MS experiments were conducted using EIS-QTOF-MS from Bruker Daltonics, Bremen, Germany. All mass spectra were acquired in the positive ion mode within a mass range of (100-3000) m/z, with a scan speed of 2.1 scans/s. The source block temperature was kept at 80 • C. The reference provided a spectrum in positive ion mode with fair ionic coverage of the m/z range scanned in full-scan MS. The resulting spectrum was a sum of scans over the total ion current (TIC) acquired at 25-85 eV collision energy to provide the full set of diagnostic fragment ions.

Identification of Metabolites
The total ion current (TIC) and selected ion monitoring (SIM) values were compared with those from Phenomenex-EZ: faast amino acid analysis user guide and the results are presented in Table 1. The metabolites were identified via comparison of their mass spectra with those of the standard library NIST/NBS-3 (National Institute of Standards and Technology/National Bureau of Standards) spectral database, and the identified phytoconstituents are presented in Table 2.

FT-IR Spectroscopy
The FT-IR spectrum of the sample was recorded via KBr pellet using a Shimadzu Prestige-21 spectrometer in the range 400-4000 cm −1 , with a resolution of 4 cm −1 .

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

Scanning Electronic Microscopy (SEM)
The SEM analyses were performed using an SEM-EDS system (QUANTA INSPECT F50) equipped with a field emission gun (FEG), 1.2 nm resolution and an energy dispersive X-ray spectrometer (EDS) with an MnK resolution of 133 eV.

Magnetization Experiments
The frequency dependence of the Fe 3 O 4 nanoparticles and nano-carrier system was measured using an Agilent LCR-meter (E-4980A type) at room temperature over the frequency range (1 kHz to 2 MHz) and various values of polarizing field. The duration of the measurement into a constant magnetic field over the entire frequency range was about 40 s.
Complex magnetic susceptibility measurements were made using the short-circuited coaxial transmission line technique at different values of the polarizing field, H, over the range 0-170 kA/m and at the frequency range (100 MHz-6 GHz). The static magnetization measurements for the Fe 3 O 4 nanoparticle sample and the nano-carrier system were performed using a ballistic galvanometer.

Conclusions
In the current study, the complete metabolite profiling of A. annua growing wild in Romania was accomplished. A total of 14 amino acids were identified for the for the first time in plant samples. The biological activities were discussed for each metabolite category. Furthermore, a simple and economical nano-carrier system was developed. A ratio of herb:magnetic Fe 3 O 4 nanoparticles was used, which allowed for the synergic effect of A. annua bioactive compounds and its inorganic component properties to be taken advantage of. The morpho-structural characterization of the nano-carrier system was performed. In addition, the magnetic properties of the nano-carrier were evaluated. Further studies are necessary to investigate the biological properties and the bioavailability of the new nano-carrier system.