Green Synthesis of Silver Nanoparticles from Salvia aethiopis L. and Their Antioxidant Activity

Salvia species have been used extensively in medicinal and food industries for years due to their significant secondary metabolites contents such as flavonoids and phenolic compounds. Silver nanoparticles capped and stabilized by Salvia aethiopis compounds are expected to reveal considerable biological effects. In this study, Salvia aethiopis L. was heated in distilled water for 2 h. After filtration, water extract was treated with silver nitrate for 2 h at 60 °C to yield the silver nanoparticles (Sa-AgNPs). The structure of silver nanoparticles was elucidated by spectroscopic methods such as Ultraviolet–Visible (UV–Vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Scanning electron microscope (SEM), and zeta potential analyses. The maximum absorption in UV–Vis spectrum was observed at 508 nm. XRD pattern (2θ) at 38.1°, 44.3°, 64.4°, and 77.4° degrees can be assigned to the (111), (200), (220) and (311) Bragg’s reflections of the face-centered cubic crystalline structure. The average size of Sa-AgNPs was found as 74.09 nm by SEM analysis. The characteristic hydroxyl vibration signal appeared at 3222 cm−1. The zeta potential of Sa-AgNPs was found as − 20.3 mV displaying the stability of Sa-AgNPs. Antioxidant activity of extract and Sa-AgNPs were carried out using DPPH⋅, ABTS⋅+ FRAP assay. The Sa-AgNPs revealed a considerable ABTS⋅+ scavenging effect with the value of 4.93 (IC50, µg/mL) compared to BHT (IC50, µg/mL, 8.34). However, Sa-AgNPs displayed a lower DPPH⋅ activity (IC50, µg/mL, 24.37) than that of the standard BHT (IC50, µg/mL, 9.67). The reducing power activity of Sa-AgNPs was found as 4.52 (µmol TE/mg extract) while the standard BHT value was 488 (µmol TE/mg extract).


Introduction
Nanotechnology is a science that has developed rapidly in recent years and has a wide range of applications [1]. Nanoparticles are basic building blocks with different properties due to their large surface area/volume ratio. In recent years, nanoparticles have formed the basis of modern materials science [2]. Silver nanoparticles (AgNPs) gain great interest in biology, biomedical, drug delivery, medicine, agriculture, food industries, textile industries, and electronics [3].
Electrochemical biosensors derived from carbon nanomaterials have sensitivity, selectivity, low limits of detection to detect the compounds [4]. The nanostructures of SnO 2 have been employed for modification of electrodes to improve the sensor properties such as sensitivity, stability, detection limit range. The nanoparticles with thermal, electrical, and mechanical properties make them suitable materials for sensors [5]. Zinc oxide nanostructures propose a wide range of applications due to their unique properties such as chemical stability, photostability, a broad radiation absorption. ZnO is a nontoxic, biodegradable, and biocompatible material that makes it appropriates for biomedical, drug delivery, and bioimaging areas [6].
Several synthetic routes have been developed to produce AgNPs including electrochemical, radiation [7], and photochemical [8]. However, these methods lead to environmental contamination, toxic residue, and high cost. Therefore, natural products gain great interest for the synthesis of nanoparticles due to their eco-friendly, low cost, high efficiency, and scale-up properties [9,10].
Natural products such as plant extract, microorganisms, algae, oilcake, vegetable waste, seaweed, enzymes, arthropods have been used to produce AgNPs. It has been accepted that plant-based materials are the promising substrate for AgNPs synthesis due to the corresponding advantages [11]. The biological effects of AgNPs depend on some crucial factors such as surface chemistry, size, shape, particle morphology, particle composition, coating/capping, agglomeration, and dissolution rate, particle reactivity in solution, the efficiency of ion release, cell type, the type of reducing agent [12]. The high concentration of capping agents can lead to the agglomeration of nanoparticles [13]. In addition, the synthesis of size-controlled, and high yield nanoparticles can be achieved by the agglomeration of AgNPs. AgNO 3 was reduced by sodium ascorbate in the presence of a low concentration of poly(vinylpyrrolidone) which is the longchain polymer that controls the AgNPs agglomeration [14].
Free radicals are called reactive oxygen species including hydroxyl (OH ⋅ ), peroxyl (ROO ⋅ ), superoxide (O 2 ⋅− ), peroxynitrite ( ⋅ ONOO − ) radicals that were produced throughout oxidation within the mammalian body [22]. The human body has many protection systems against oxidative stress. The natural antioxidants become insufficient in some situations and then, the excess radicals can damage to cell membrane resulting in diseases [23]. Therefore, food including antioxidants should be consumed to cope with this situation. An antioxidant is defined as a substance that inhibits the oxidation of the substrate [24]. Accordingly, phenolic compounds are produced from the secondary metabolism of plants and are considered natural antioxidants because they protect many organs from oxidation [25]. There has been an increase in the use of natural antioxidants due to the benefits provided by the aromatic herbs of extracts, essential oils, and spices [26]. Herbal-based products contain phenolic phytochemicals, one of the most powerful antioxidants, and contribute to body defense against oxidative damage. These compounds protect against deterioration and provide antioxidant substances to the human body because of their consumption [27][28][29].
Salvia L. species have been used since ancient times due to their antioxidant, natural preservative, spice, aromatic substance, and medicinal properties [30]. Salvia is an important genus belonging to the Lamiaceae (formerly Labiatae) family. Around the world, 1000 species of Salvia are used as herbal tea and flavouring, as well as in the cosmetics and pharmaceutical industries. Salvia species have been used in the treatment of colds, coughs, toothache, gastrointestinal problems, coronary heart disease, hepatochirosis, hepatitis, cerebrovascular disease, chronic renal failure, dysmenorrhea, and neurasthenic insomnia as traditional medicine [31]. In addition, Salvia herbs are known to have a wide variety of pharmacological activities such as antimicrobial, antioxidant, anti-inflammatory, anticancer, hypoglycemic, hypolipidemic, antinociceptive, memory-enhancing effects. Salvia genus is rich in polyphenols, especially phenolic acid, and flavonoids [32,33].
This study aims to synthesize silver nanoparticles using Salvia aethiopis and investigate the antioxidant activity. Due to the importance of AgNPs in many technological fields, the AgNPs capped and stabilized by the flavonoids found in S. aethiopis could be promising materials for the food and pharmaceutical industry.

Plant Materials
Salvia aethiopis L. was obtained from Tokat Gaziosmanpasa University Aromatic and Medicinal Plant Field.

Synthesis of Silver Nanoparticles
Salvia aethiopis leaves were powdered by grinder and powder material (50 g) was heated with distilled water (200 mL) at 50 °C for 2 h. After filtration with Whatman filter paper, silver nitrate distilled water solution (0.037 mM, 200 mL) was added to the extract solution slowly. The reaction mixture was heated at 60 °C for 2 h. The colour change from yellow to brown was observed. After completion of the reaction, Sa-AgNPs were obtained by repeated centrifugation at 5000 rpm for 20 min then washed thoroughly with distilled water. The Sa-AgNps were dried by lyophilisation [10].

Characterization of Silver Nanoparticles
The UV-Vis spectra were recorded on Hitachi U-2900 spectrophotometer. The maximum absorption was detected at 508 nm. XRD measurement was carried out on an Empyrean, Malvern Panalytical diffractometer, the operation voltage of 45 kV at a 40-mA current strength. The crystallographic structure of Sa-AgNPs was determined by the XRD pattern. The diffracted intensity was carried out in the region of 2θ from 20º to 90º. The particle size was calculated by dynamic light scattering (DLS) on a Delsa Nano C instrument. The Sa-AgNPs properties were determined by Scanning Electron Microscope (SEM) on Quanta Feg450. EDAX detector and EDX were used to determine the elemental analysis. quanta 450 FEG was used for surface and point analysis. Zetasizer Nano ZSP (Malvern instrument) was employed for zeta potential measurement.

DPPH ⋅ Free Radical Scavenging Assay
DPPH ⋅ free radical scavenging effect of S. aethiopis mediated silver nanoparticles and the extract was carried out according to the procedure described in the literature [29]. DPPH ⋅ radical (1.0 mL, 0.26 mM) was treated with the Sa-AgNPs (3.0-30 µg/mL) at room temperature (rt) for 20 min. During the reduction, the solution color fades, and the absorbance decreases. BHT, BHA, and Trolox were used as standard compounds. The equation was used for the calculation of DPPH ⋅ scavenging effect (1) A 1 is the absorbance of the control and A 2 is the absorbance of the sample.

ABTS ⋅+ Radical Cation Activity
ABTS ⋅+ radical cation assay is based on the ability of antioxidants to reduce ABTS ⋅+ (blue/green) to ABTS −2 (colorless). ABTS radical cation solution was produced by the reacting of 7.0 mM ABTS with K 2 S 2 O 8 (2.45 mM) at a ratio of 2/1 (v/v), the mixture was kept in the dark at rt for 12 h. After adjusting pH by treatment of ABTS ⋅+ solution with phosphate buffer (0.1 mM, pH 7.4), Sa-AgNPs were treated with ABTS ⋅+ (1.0 mL) at several concentrations (3.0-30 µg/ mL). The absorbance measurement was executed at 734 nm and ABTS concentration was calculated by the calibration curve with the given Eq. (2): in which, A 1 is ABTS ⋅+ initial concentration and A 2 is ABTS ⋅+ remaining concentration in the sample [30].

Reducing Power
Reducing power of extract and Sa-AgNPs were measured according to previously published method [22]. Reducing power was calculated from the calibration curve of ascorbic acid and presented as µg/mL of extract or silver nanoparticles. The samples (extract and Sa-AgNPs) were mixed with 200 mM of sodium phosphate and volume was adjusted to 1.25 mL with water, followed by the addition of potassium ferricyanide, K 3 Fe(CN) 6 (1.25 mL, 1%). Later, the mixture was incubated for 20 min at 50 °C, and then 1.25 mL of 10% trichloroacetic acid was added and then thoroughly vortexed.
An aliquot (1.0 mL) was mixed with water (1.0 mL) and ferric chloride (0.5 mL, 0.1%) and then vortexed. The absorbance was measured at 700 nm against a blank using a spectrophotometer [31]. The high absorbance value revealed the high reducing activity.

Statistical Analysis
GraphtPad Prism software (version 8.0.1), one-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis. The results were stated as mean values ± standard deviation (P < 0.05).

Synthesis of Silver Nanoparticles
The silver nanoparticles were synthesized using Salvia aethiopis leaves. The plant material was heated in distilled water, after removal of the solid part, the extract solution was treated with the silver nitrate solution. The secondary metabolites in the water solution reduced the Ag + to Ag 0 . Afterward, Ag atoms were capped and stabilized by secondary metabolites that the plant synthesized. The colour change of the reaction mixture from dark yellow to dark brown confirmed the formation of Sa-AgNPs (Fig. 1).
In the reaction mechanism (Fig. 2), the silver ions were reduced by bioactive compounds that oxidized. After the stage of ion reduction, clustering, and growth of nanoparticles, the silver nanoparticles formed. Since Salvia species include luteolin, the reaction mechanism was showed for this compound.

UV-Vis Spectral Analysis
The maximum absorption at 508 nm at UV-Vis spectrum revealed the formation of the silver nanoparticles (Fig. 1). The UV-Vis spectroscopy is mostly used for the identification of silver nanoparticles. Free electrons in metal nanoparticles yield a surface plasmon resonance absorption band. The peak revealed the typical surface plasmon resonance of Sa-AgNPs. The UV-Vis spectrum showed the presence of bioactive compounds in the S. aethiopis leaves for the formation of silver nanoparticles. The particle size and shape have a significant effect on the wavelength shift. The various metal nanoparticles in the size range from 2 to 100 nm were identified at 300-800 nm wavelengths.

Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR spectrum showed the functional groups responsible for the silver ion reduction and stabilization of Sa-AgNPs. The signal that appeared at 3211 cm −1 could be attributed to the hydroxyl group and the peak at 2933 might be due to the CH stretching. The peak at 1587 cm −1 could be assigned to the NH bending. The other signals at 1382 cm −1 , 1257 cm −1 , and 1153 cm −1 belonged to the OH bending, C-O stretching, and C-O stretching, respectively. The peak at 1019 could be assigned to sulfoxide stretching (Fig. 3).
The average particle size was found as 74.09 nm by Debye-Scherrer's Eq. (3) in which D is the crystal size, λ is the wavelength of x-ray, θ is the Braggs angle in radians and β is full of half maximum of the peak in radians.

Scanning Electron Microscope (SEM) Analysis
The surface morphology was found a spherical nature with an average size of 74.09 nm (Fig. 5). The energy dispersive analysis of x-rays (EDX) indicated the formation of Sa-AgNPs (Fig. 6). The intense peak of AgNPs in the EDX  The metallic silver nanoparticles show the typical strong signal peak at 3.0-3.3 keV due to the surface plasmon resonance [36]. Elemental compositions of the Sa-AgNPs were given in Table 1.

Zeta Potential Analysis
Zeta potential is defined as electrical potential that forms at the solid-liquid interface due to the motion of the nanoparticle and the solvent. The Sa-AgNPs stability depend on the electrical potential and surface charge. The surface potential of charged particles rises with the rise of zeta potential. The zeta potential of Sa-AgNPs was measured as − 20.3 that revealed the stable of nanoparticles as well as repulsion among the particles (Fig. 7). Moreover, surface of the Sa-AgNPs was negatively charged that dispersed in the medium.

Antioxidant Activity
Aromatic and medicinal plants have been used extensively for medicinal purposes for years due to their bioactive contents [37][38][39][40]. After the development of spectroscopy in the nineteenth century, the isolation and identification of secondary metabolites from corresponding plants gained great interest and it became the focus of science [33,41]. Therefore, these plants have been used for the synthesis of nanoparticles that exhibited a broad spectrum of biological activities [42]. Antioxidant activity of Sa-AgNPs was carried out using DPPH ⋅ (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay, ABTS ⋅+ [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation] scavenging assay, and ferric reducing antioxidant power (FRAP) assay (Fig. 8). In DPPH ⋅ assay, Sa-AgNPs revealed a significantly higher activity (IC 50 , µg/mL, 24.37) than the extract (IC 50 , µg/mL, 45.41). But the activities of both extract and Sa-AgNPs were lower than that of the standards. In ABTS ⋅+ assay, Sa-AgNPs displayed excellent activity with the value of 4.93 (IC 50 , µg/mL) compared to the BHT (8.34) and Trolox (5.71). In concern to the reducing power assay, the Sa-AgNPs possessed a higher activity than extract but lower than BHA. Consequently, the silver nanoparticles synthesised from S. aethiopis revealed good antioxidant activity. Hence, they have the potential to be used in the pharmaceutical and food industries. This is the first report for synthesis of AgNPs using S. aethiopis.
Some Salvia species were employed for the green synthesis of nanoparticles. Silver nanoparticles were synthesised from Salvia leucantha that revealed considerable antibacterial activity [43]. Moreover, Salvia officinalis, well known of Salvia genus was used for the nanoparticles synthesis that revealed the significant antibacterial [44], antiplasmodial [45], antioxidant and anti-inflammatory [46], antileishmanial effects [47]. Hence, it was expected that the Salvia genus, Salvia aethiopis which was the subject of present research would display considerable antioxidant activity.
Aromatic and medicinal plants have been employed comprehensively for silver nanoparticles synthesis. Rosa brunonii Lindl was used for synthesis of silver nanoparticles that revealed the strong antioxidant, antimicrobial, and photocatalytic activities [48]. Another scientific work indicated that silver nanoparticles synthesised from rhizome of Rheum australe had good electrical conductivity of the mixture of AgNPs with DPPH. Thus, AgNPs from corresponding plant had a potential usage in sensors [49].  The particle size of the nanoparticles influences the antioxidant activity. In a research, size-dependent antioxidant activity was generated using nine different sizes of TiO 2 nanoparticles . The highest antioxidant activity was observed for 30 nm particles, and it was constant above 30 nm. The activity was observed to decrease as size decreased from 30 to 10 nm, and again constant for particles smaller than 10 nm [50].

Conclusion
Due to the increasing interest in natural products in the food and pharmaceutical industry, the silver nanoparticles green synthesised from S. aethiopis could be an antioxidant agent. The synthesis of silver nanoparticles using S. aethiopis was an easy, eco-friendly, and economical process. This synthesis did not require the use of any hazardous chemicals and reagents for reducing and processing. The XRD pattern displayed that the AgNPs have a face-centered cubic (fcc) lattice structure. The nature of AgNPs was found to be spherical shape. The further scientific study should be executed to show the anticancer potential of Sa-AgNPs.