Synthesis of Silver Nanoparticles from Artemisia sieberi and Calotropis procera Medical Plant Extracts and their Characterization using SEM Analysis

The synthesis, characterization and application of biologically synthesized nanomaterials have become important research areas in nanotechnology, and the green synthesis of nanoparticles using plants is being increasingly studied largely because this approach is considered to lack the problems associated with conventional synthesis. Here we report the synthesis and characterization (using a scanning electron microscope) of silver nanoparticles (AgNPs) obtained using extracts of leaves of the medicinal plants, Artemisia sieberi and Calotropis procera. Scanning electron microscopy (SEM) studies revealed the characteristics of the synthesized nanoparticles which were confirmed by analyzing the excitation of surface plasmon resonance (SPR) using UV–vis spectrophotometer at 482 nm. SEM analysis of the synthesized Ag NPs clearly showed that the particles were predominantly spherical in shape, mostly aggregated and having a size around 8–20 nm. Finally, we consider that the nanoparticles synthesized in this study have potential for wide application in nanotechnology and nanomedicine.

Nanoparticles are extremely small synthesized structures which have an extremely wide applicability 1 largely due to their unique thermal 2 , electrical 3 and optical 4 properties. They can be used products ranging from photovoltaics 5 to biological and chemical sensors 6 while nanosilver particles are extensively used as an anti-bacterial agent in the health industry 7 in food storage 8 , as textile coatings 9 as well as in a range of environmental applications 10 . Currently nanosilver is prepared using methods which include electrolysis, and physical, chemical, and biological methods 11,12 . The bioreduction of a precursor salt of silver by a bioactive compounds present in plants is a so-called biomimetic method in which; natural products are used as reducing agents to produce nanoparticles 13 .
Silver nanoparticles play an increasingly important role in biology and medicine due to their attractive physiochemical properties The most important application of silver and silver nanoparticles is in medical industry, where they are used for example, in topical ointments to prevent infection against burn and open wounds 14 . Silver products have long been known to have strong inhibitory and bactericidal effects, as well as a broad spectrum antimicrobial activities, which has been used for centuries to prevent and treat various diseases, particularly bacterial infections 15 ; silver nanoparticles also exhibit antifungal, antiinflammatory, antiviral, antiangiogenesis, and antiplatelet activity 16 . Nanoparticles can be synthesized using a variety of chemical, physical, and biological approaches. Although chemical synthesis requires short time periods for the synthesis of large quantities of nanoparticles, this method the need requires capping agents for size stabilization of the nanoparticles. The chemicals used in this approach to nanoparticle-synthesis and stabilization are also toxic and the process leads to non-eco-friendly by-products. For environmentally nontoxic nanoparticle synthesis has led to the development of a number of biological approaches, which largely avoid the use or production of toxic chemicals. As a result, there is an increasing demand for "green nanotechnology." A number of biological approaches for both extracellular and intracellular nanoparticles synthesis have been reported to date using microorganisms including bacteria 17 , fungi 18,19 and plants 20,21,22 . Plants generally provide a better platform for nanoparticle synthesis as they are free from toxic chemicals and contain natural capping agents. Moreover, the use of plant extracts reduces the cost of the isolation and culture of micro-organisms thereby increasing the cost effectiveness of nanoparticle synthesis 23 .
C a l o t r o p i s p r o c e r a ( f a m i l y : Asclepiadaceae) (C. procera) is a cultivable wild xerophytic shrub which is found across Africa, Asia and South America 24 . It produces milky white latex which has a range of curative properties 25,26,27 . Latex is found in special branching tubes called latex tubes 28,29 , and has been the subject of interest due to its biological activities including, antibacterial 30 , antifungal 31 , antiviral 32 , anticandidal 33 and anticarcinogenic propertoes 34,35 . More than 80% of the dry mass of the crude latex corresponds to rubber and the rest 20% covers soluble fractions rich in protein including antioxidant enzymes, cysteine protease with free thiol group and tryptophan 36,37 .
The genus Artemisia has always been of considerable pharmaceutical interest and is used in traditional medicines to treat a variety of diseases 38,39,40 . The genus includes small herbs found in Northern temperate regions and belonging to the important family Compositae (Asteraceae), which comprises about 1,000 genera and over 20,000 species. Within this family, Artemisia is included into the family Anthemideae, which is itself, made up of over 400 species and is found  50 and A. sieberi Besser 51 . Here, I described a simple one step method for the synthesis of silver nanoparticles using leaf extracts of Artemisia sieberi and Calotropis procera; the nanoparticles were then characterized by SEM analysis.

Plant extract
Fresh leaves of Artemisia sieberi and Calotropis procera were collected from Saudi Arabia, Riyadh and washed three times with tap water and then distilled water and then dried at room temperature. In order to obtain leaf extracts, five grams of air dried leaves were cut into fine pieces and boiled for 10 min in microwave oven. The extract was then cooled at room temperature and filtered by using Whatman filter paper No. 1; the filtered plant extract was then used to synthesize silver nanoparticles.

Silver nitrate solution
Silver nitrate (Merck) solution of 4 mM was prepared by dissolving appropriate amount of silver salt in distilled water and storing in an amber coloured bottle.

Synthesis of silver nanoparticles
For the synthesis of silver nanoparticles, 100 ml of leaf extract was mixed with 100 ml of silver nitrate aqueous solution in an Erlenmeyer flask and stored at room temperature. A change in colour was observed after mixing plant extract and silver solution. The reduction of silver in the colloidal solution was monitored by UV-Visible spectroscopy analysis. The mixture was then centrifuged at 15,000 rpm for 5 min and the supernatant was subjected to characterization by the use of a UV-visible spectrophotometer and by scanning electron microscopy. A further two flasks were used as controls where one flask contained only an aqueous plant extract, while the second contained a silver solution.

Characterization of Silver Nanoparticles
The synthesized silver nanoparticles were characterized using the following techniques:

UV-Visible spectroscopy
The reduction of silver ions in the colloidal solution was confirmed by UV-Visible spectroscopy. A small aliquot of sample was taken in a quartz cuvette and observed for wavelength scanning between 300-700 nm with control samples as a reference. PerkinElmer Lambda 950 UV/Vis spectrometer was used for UV Visible spectroscopy.

Scanning electron microscopy
S u r f a c e m o r p h o l o g y o f s i l v e r nanoparticles was characterized by scanning electron microscopy. The sample was prepared by centrifuging colloidal solution after 6 h of reaction at 14,000 rpm for 4 min. The pellet was the re-dispersed in deionized water and again centrifuged. The process was repeated three times and the material was finally washed with acetone. The purified silver nanoparticles were sonicated for 10 min for making the suspension and then a drop from the suspension was placed on the carbon coated copper grid. The sample was kept under lamp until completely dry. The prepared sample was subjected to SEM analysis by using Jeol JSM-6490A Analytical Scanning Electron Microscope at king Saud University, Riyadh.

RESULTS AND DISCUSSION
In this study, when the leaf extract was mixed with silver nitrate solution its colour started to change which is important since silver nanoparticles in aqueous solution exhibit dark brown colour. A change in colour occurred because of excitation in surface Plasmon resonance which indicates the formation of silver nanoparticles. These findings are similar to those reprted previously where the colour of fresh suspension of Vitex negundo and a silver nitrate solution was also seen to be dark brown 52 and also reported that silver nanoparticles exhibit a yellowbrown colour in aqueous solution due to the excitation of surface plasmon vibrations in silver nanoparticles 53 . Confirmation of the presence of silver nonoparticles in our work as done using UV-Visible spectroscopy. A small aliquot from the reaction mixture was taken in a quartz cuvette and observed for absorption spectrum. It was noted that a colloidal solution after 6 h of reaction showed absorption peak at 482 nm, confirming the presence of silver nanoparticles in the solution. The result of the UV-Visible spectrum was wellcorrelated with previous work where the absorption peak was observed at 475 nm for a silver solution of leaf extracts of Memecylon edule 54 and also similarly reported that the absorption spectra of silver nanoparticles formed in the reaction media have an absorbance peak at 430-440 nm. 55 The synthesized silver nanoparticles were further characterized by SEM analysis where it was noted that the particles were predominantly spherical in shape, although other than the spherical shaped particles were also present. The particle size ranged from 8 nm to 20 nm ( Fig. 1  and 2). The different sizes of particles may be  In the present study, the biosynthesis of silver nanoparticles was successfully obtained using a green method of preparation. This method used showed that select plants can be used as an effective stabilizing reducing agent for the synthesis of AgNPs. The methodology employed here is very simple, easy to perform, inexpensive, eco-friendly and provide an improved alternative to chemical synthesis. The formed-AgNPs are highly stable spherical shaped particles, (when observed under the SEM). It is hoped that novel and improved silver nanoparticles will be produced in further studies.

CONCLUSION
In conclusion, leaves of Artemisia sieberi and Calotropis procera were found to be excellent sources for synthesis of silver nanoparticle. Silver nanoparticles were synthesized by applying an environmentally safe method which minimizes the addition of hazardous wastes in the environment. The synthesized nanoparticles were spherical, 8 -20 nm in size, crystal in nature and showed absorption spectrum at 482 nm characterized by using different techniques. Important outcomes of the study are likely to be in the further development of value added products from the medicinal plants, Artemisia sieberi and Calotropis procera for use in biomedical and nanotechnology based industries.