Stachytarpheta cayennensis Aqueous Extract, a New Bioreactor towards Silver Nanoparticles for Biomedical Applications

This study reports the preparation and characterization of silver nanoparticles synthesized by the mediation of the plant weed Stachytarpheta cayennensis through solution method. Ultraviolet visible spectroscopy (UV-Vis) determines the presence of nanoparticles in the solution. Infrared spectroscopy (IR) proves organic molecules at the particles interface. Powder X-ray diffraction (PXRD) provides phase composition and crystallinity. Shape was showed by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) demonstrated the elemental mapping of the silver nanoparticles. Hydrogen peroxide scavenging and phosphomolybdenum antioxidant assays, egg albumin denaturation particles could be considered as a potential source for biomedical applications.


Introduction
One of the most direct effects of reducing the size of materials to the nanometer range is appearance of quantization effects due to the confinement of the movement of electrons, which leads to discrete energy levels depending on the size of the structure. Control over dimensions as well as composition of structures makes it possible to tailor material properties to specific applications which are influenced by quantum confinement effect (QCE) [1]. Hence, quantum confinement (QC) in semiconductors results from the geometric confinement of electrons, holes or excitons (electron hole bound pair). When an electron-hole pair is squeezed below the dimensions approaching excitons Bohr radius, quantum confinement effects become prominent in the structure and the effective band gap increases. The smaller the nanostructure, the larger the effective band gap and the greater the energy of the optical emission, which changes the optical and the electronic properties, when the sample size is sufficiently smaller than 10 nm [2]. Nanoparticles are considered to be the future building blocks of advanced materials and devices that can satisfy several promises of nanotechnology [3]. Nanoparticles with particular shapes and dimensions have been obtained by various techniques including physicochemical and biological routes. Material scientists are developing research towards novel materials with better properties, more functionalities and lower cost than the existing ones. In this light, methods are employed to improve the nano-entity performance to gain better control over particle size, distribution and morphology [4]. The physical and chemical processes of nanoparticles production are typically expensive and require hazardous chemicals while the "green synthesis" approach is environmentally benign and cost-effective. Plants represent the most explored group of living organisms for the green synthesis of nanoparticles [5]. The use of natural products can also lead to capping groups that are biocompatible, circumventing the need for ligand exchange prior to biological applications [6]. However, greener, more sustainable or biogenic methods do not guarantee the fabrication of nanomaterials with improved or even identical properties to those produced by traditional methods [6]. One of the critical goals is the satisfactory reproducibility of synthesized materials.
Weeds, the unwanted plants in the agriculture fields can be used in the synthesis of nanoparticles. Silver nanoforms have been described with plant weeds F. E. Meva et al. such as Parthenium [7], Ipomoea aquatica, Enhydra fluctuans and Ludwigia adscendens [8].
Stachytarpheta cayennensis (L. C. Rich) Vahl is a ligneous weed 0.5 -1 m high. The plant leaves are used in folk medicine in infusion as a stomachic and febrifuge to treat chronic liver diseases, flues, cough and arthritis and also as a diuretic and sudorific [9]. The leave extracts have been reported for hypoglycemic, antimicrobial and antispasmodic activity [10] [11]. Phytochemical screening revealed the presence of saponins, carbohydrates, flavonoids and terpenoids [11].
In the current study, we report the first use of Stachytarpheta cayennensis plant extract for silver nanoparticle synthesis. In-vitro antioxidant and anti-inflammatory experiments are conducted considering the potential pharmacological use of the nanoparticles.

Plant Material and Extract Preparation
Whole plants of Stachytarpheta cayennensis (L. C. Rich) Sehau ( Figure 1) were procured from the University of Douala, Cameroon. Plant identification was confirmed at the National Herbarium of Cameroon under number specimen 21136/SRF Cam. The organic material was surface cleaned with running tap water followed by de-ionized water to remove all the dust and unwanted visible particles. Aqueous plant extracts were prepared by boiling 10 g of organic material in 200 mL of de-ionized water for 5 min at 80˚C. The extract was filtered through Whatman No. 1 filter paper to remove particulate matter. The obtained clear solution was then stored at 4˚C for further use, being usable for one week due to the gradual loss of plant extract viability for prolonged storage [12].

Synthesis of Silver Nanoparticles
Silver nitrate (AgNO 3 ) was obtained from Sigma-Aldrich chemicals Germany. De-ionized water was used throughout the reactions. All glass wares were washed with dilute nitric acid (HNO 3 ) and de-ionized water, and then dried in hot air oven. A solution of AgNO 3 10 −3 mol/L was prepared in de-ionized water. As a standard procedure, 10 mL of organic material extract was added to 50 mL of 10 −3 mol/L aqueous AgNO 3 solution, hand shaken during 1 min, and incubated at room temperature in the dark to minimize the photoactivation of silver nitrate. The reactions were made under static conditions.

Ultraviolet-Visible Spectroscopy (UV-Vis)
The reaction was monitored by ultraviolet visible spectroscopy of the reaction suspension (2.5 mL) using an UV-visible Uviline 9100 spectrophotometer operating at 1 nm resolution with optical length of 10 mm. Concentrations were determined after 24 hours of incubation by centrifugation at 6000 rpm during 1 hour. UV-Vis analysis of the reaction mixture was observed for a period of 300 s.

Infrared Spectroscopy (IR)
The Fourier Transform Infrared spectrum (FTIR) of copper nanoparticles was recorded with a Nicolet iS5 FT-IR Spectrometer. The sample was prepared at 0.25 mm thickness as KBr pellets. The spectrum was measured between 400 and 4000 cm −1 at a resolution of 0.4 cm −1 .

Powder X-Ray Diffraction (PXRD)
Powder X-ray diffraction measurements were carried out at ambient temperature using a BRUKER D2 Phaser diffractometer (Cu K-Alpha [Å] 1.54182, 35 kV) by preparing a film of the silver-organic nano-powder on the flat, low background silicon sample holder. The peaks and phase were identified using the profile matching routine of MATCH Version 3 program [13] and the Rietveld refinement was carried out using the Fullprof 2000 software package [14].

Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX)
Scanning electron microscopy images and energy dispersive X-ray spectrometric measurements where done on a Jeol scanning electron microscope JSM-6510 with a tungsten cathode and an EDX unit. The samples were coated with Au for 20 s at 30 mA by using a Jeol JFC-1200 sputter coater (JSM-6510). Microscopy provides detailed characterization of the distribution and morphology of the nanoparticles and the presence of nano-silver elements was confirmed by EDX at 20 keV.

Hydrogen Peroxide Scavenging Assay
The experiment was carried out using the modified methods of Bhakya et al.

Phosphomolybdenum Assay
The antioxidant capacity was evaluated by the phosphomolybdenum method [16], with modifications. The phosphomolybdate reagent solution was prepared by adding 1 mL of each 0.6 M sulphuric acid, 28 mmol/L sodium phosphate and 4 mmol/L ammonium molybdate to 20 mL of distilled water and the volume brought to 50 mL by adding distilled water. Different concentrations of nanoparticles ranging from 50 µg/mL to 3 µg/mL were added to each test tube individually containing 3 mL of distilled water and 1 mL of molybdate reagent solution. These tubes were kept incubated at 95˚C for 90 min. After incubation these tubes were normalized to room temperature for 20 -30 min and the absorbance of the reaction mixture was measured at 695 nm. Ascorbic acid was used as positive reference standard.

Anti-Inflammatory Activity
Egg albumin denaturation assay: The reaction mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen's egg), 2.8 mL of phosphate buffered saline (PBS, pH 6.4) and 2 mL of varying concentrations of extract so that final concentrations become 50, 25, 12.5, 6.25, 3.125 μg/mL. Similar volume of double-distilled water served as control. Then the mixtures were incubated at (37˚C ± 2˚C) in an incubator for 15 min and then heated at 70˚C for 5 min. After cooling, their absorbance was measured at 660 nm by using vehicle as blank [17] [18]. Ibuprofen at the final concentration of 50, 25, 12.5, 6.25, 3.125 μg/mL, was used as reference drug and treated similarly for determination of absorbance. The percentage inhibition of protein denaturation was calculated by using the formula 1.

Statistical Analysis
The experiments were carried out in triplicate and the results are given as mean ± standard deviation (SD). A sample analysis test was used for the comparison between the means of samples and standard. Values with p < 0.05 are considered significant. Correlation analysis was carried out for the activities using Excel-2016.

Ethical Considerations
All experiments were carried out according to the approved protocol by the In-

UV-Vis Spectroscopy
The absorption spectra of the synthesized silver nanoparticles are recorded against water in order to monitor the formation and stability of silver nanoparticles. The visual observation of the colour change of the mixture solution plant extract and silver ion is the preliminary evaluation. Colours of silver nitrate, Stachytarpheta cayennensis leaf extract and silver nanoparticles solution is shown in Figure 2. The sharp surface plasmon resonance absorbance band has been obtained at 449 nm ( Figure 3).

PXRD Analysis
where Dv is the average crystalline size; K is a dimensionless shape factor, with a value close to unity (0.9); λ is the wavelength of Cu Kα; β is the full width at half-maximum of the diffraction peaks, and θ is Bragg's angle.
The Williamson-Hall analysis relies on the principle that the approximate formula for the size broadening and the strain broadening vary quite differently with respect to the Bragg angle [19]. The crystallite size and strain can be obtained from the intercept at y-axis and the slope of line, respectively.
where β is FWHM in radian, t is the grain size in nm, ε is the strain, λ is the X-ray wavelength in nanometers, and C is a correction factor taken as 0.94 [20].

SEM and EDX Morphological Studies
Scanning electron microscopy is one of the powerful tools to identify the morphology of the synthesized nanoparticles. Figure 7 shows an SEM image of the green synthesized silver nanoparticles obtained after 24 h of incubation using 1 mmol/L silver nitrate solution. The energy dispersive X-ray analysis (EDX) is shown in Figure 8. EDX qualitative spectrum shows a strong silver peak (3 kev) along with chloride, oxygen, carbon as main elements.

Formation Mechanism of Silver Nanoparticles
The formation mechanism is schematized in Figure 9. It involves a simultaneous formation of pure silver nanoparticles and at the same time silver chloride nanocrystallites.

IR Spectroscopy
Infrared spectroscopy appears as one of the key methods to study the nanopar-   generally arise from ring stretching or C−H groups in plane bending [22]. Bands lower than 1000 cm −1 are generally attributed to inter-atomic absorption vibrations of metals in fingerprint region.

Antioxidant Activity
The silver nanoparticles percentage inhibition of superoxide radical increases with the quantity of sample ( Figure 11). The maximum inhibition of superoxide radical scavenging activity is 46% for Ag-Stachytarpheta cayennensis at the concentration of 50 µg/mL.
The maximum phosphomolybdenum production was 68% for Ag-Stachytarpheta cayennensis at the concentration of 50 µg/mL ( Figure 12).  The assay involves an electron transfer (ET) mechanism [23] and is dose-dependent ( Figure 10). Values with p < 0.05 were considered as significant.

Anti-Inflammatory Activity
The nanoparticles on this report exhibit moderate inhibition of egg albumin denaturation by opposing to self-aggregation of proteins ( Figure 13). The effect was compared to ibuprofen. The calculated anti-inflammatory action was found to be 30% for Ag-Stachytarpheta cayennensis at 50 µg/mL.

Discussion
A visual observation gives first evidence of silver nanoparticles formation. The solution colour change within seconds to yellowish brown and deep brown due to formation of plasmons at the colloid surface. After 48 h of reaction, precipitation occurs indicating that the plant secondary metabolites are not able to stabilize the nanoparticles in solution by disrupting the electrostatic attractions and or the deficiency of molecules of leaf extract to act as capping agents. Thus, aggregation is not reduced by decreasing the concentration of silver salt used for the synthesis. For nanocrystals, the enhanced overlap of the electron and the hole wavefunctions in the spatially confined structure increases the excitonic binding energy, thereby making observation of the excitonic peak possible, even at room temperature. The UV absorption spectrum can also provide a qualitative indication of the size distribution. For example, sharp excitonic peak in the absorption spectra in the case of small nanocrystals (see Figure 3) is indeed indicative of the narrow size distribution of the nanocrystals in the sample [24]. A signal is recorded in the ultraviolet visible spectra due to surface plasmon resonance of Ag-nanoparticles [25]. Figure 13. Albumin denaturation assay. Blue: Ag-Stachytarpheta cayennensis, orange: Ibuprofen. Error bars are standard deviation.

F. E. Meva et al.
Powder diffraction is the key method for phase and nature identification and is widely used in nanoparticles characterisation. Match identification software confirm presence of Ag and AgCl nanomixture. A similar observation was made using leaf extracts of Corchorus olitorus and Ipomea batatas or flowers extract of Albizia julibrissin [11] [26]. No other characteristic peaks were found in the XRD spectra, indicating the high purity of the prepared Ag and AgCl nanoparticles. To calculate the average crystalline particle size of the synthesized nanoparticles, the most intense peaks of Ag and AgCl were preferred [27]. Therefore, we've selected the (111) and (200) lattice planes of Ag and AgCl respectively. The calculated average crystalline particle sizes were found to be 13 nm and 20 nm for Ag and AgCl respectively. The intense and narrow diffraction peaks revealed the crystalline nature of the synthesized nanoparticles [28]. Therefore, in the nanoparticle system AgCl is less crystalline than Ag.
The  Figure 4 indicates that micro-strains cannot be a dominant source of broadening or proves that the strain is compressive while positive value indicates an expansion of the strain [29]. The low values indicate the occurrence of lattice relaxation within nanocrystallites [19]. The nonlinearity between βcosθ and sinθ can be due to low crystallized powder samples [29].  [26]. In the reaction mechanism in this work described, there is a competition in the growing of silver and silver chloride nanoentities. Silver chloride is formed by the competitive reaction of free chloride. This mechanism considers that chlorine is an essential micronutrient for higher plants and is present mainly as Cl − counter ions, although plants do contain compounds with covalently bound Cl. Chloride is a major osmotically active solute in the vacuole and is involved in both turgorand osmoregulation [30]. Free silver ions react readily with chloride to give insoluble silver chloride which poses environmental issues. It is unclear if the fast agglomerating structure is a core shell, simple mixture or a cluster. This ag-  [32]. This fact originates from coordinatively unsaturated surface atoms which are energetically less stable than well-ordered and fully coordinated atoms in the bulk [33]. The assembly contains silver nanoparticles with reducing and capping metabolites. FTIR spectral studies suggest that the amide and hydroxyl groups found in proteins and polyphenols have a stronger affinity to bind with metal and facilitate the formation of a coat over the nanoparticles.
The development of nanoparticles used as therapeutic agents has introduced new opportunities for the improvement of medical treatment [34]. Traditional therapy often fails to treat inflammatory diseases due to lack in the bioavailability of the drug, harmful side effects and development of drug resistance [35].
Most of the semiconductor and metallic nanoparticles have immense potential for cancer diagnosis and therapy on account of their surface plasmon resonance enhanced light scattering and absorption [36]. Antioxidants can play a protective role in a number of diseases such as cardiovascular and neurodegenerative diseases in which oxidative stress and free radicals are the major contributors [37]. Therefore, silver nanoparticles perform well as cancer therapeutics because they can disrupt the mitochondrial respiratory chain, which induces the generation of reactive oxygen species and ATP synthesis, which can induce DNA damage [38] [39]. The superoxide anion radical is recognized to be very injurious to cellular components as a herald of more reactive oxygen species, contributing to tissue damage and various diseases [40]. with free radicals to convert them to more stable products, which can terminate radical chain reaction. Moreover, the reducing power of Ag nanoparticles correlated well with the radical scavenging activity [42].
Reaction of albumin with nanoparticles results in a physical adsorption of protein on the nanoparticles surface. A complex is formed which is usually isolated by centrifugation. In that reaction albumin is the reducing agent and a stabilizer [43]. The study of the impact of nanoparticles on protein aggregation is an area of considerable research considering their use as anti-inflammatory or anti-neurodegeneratives agents. In related diseases, solubles proteins and peptides are converted into highly organized fibrillar aggregates. The accumulation of abnormal protein and peptide aggregates exerts toxicity by disrupting intra-F. E. Meva et al. Journal of Biomaterials and Nanobiotechnology cellular transport, overwhelming protein degradation pathways and/or disturbing vital cell functions [3]. Thermal denaturation of albumin leads to the exposure of hydrophobic residues, which increases hydrophobic attraction that overcomes electrostatic repulsion and triggers the aggregation of amorphous aggregates [3]. Protein denaturation is a process in which protein tertiary or secondary structure is disturbed by the application of external stress. It is a well-known cause of inflammation [4]. 30% antiinfmammation action for Ag-Stachytarpheta cayennensis was found at 50 µg/mL. These biogenerated nanoparticles may find application in specific and systemic inflammation as well as degeneration in in-vitro models and in-vivo preclinical models or in drug synergy.

Conclusion
We have described a simple green method for silver nanoparticle synthesis using