Introduction

Nanoparticles are extremely small synthesized structures which have an extremely wide applicability¹ largely due to their unique thermal,² electrical³ and optical⁴ properties. They can be used products ranging from photovoltaics⁵ to biological and chemical sensors⁶ while nanosilver particles are extensively used as an anti-bacterial agent in the health industry⁷ in food storage,⁸ as textile coatings⁹ 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 infections¹⁵; silver nanoparticles also exhibit antifungal, anti-inflammatory, 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

Calotropis procera (family: 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 anticandidal³³ 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 in Europe and North America, and Asia.^41,42,43 Among the Asian Artemisia flora, 150 species have been recorded for China, 50 species have been reported in Japan, and 34 species in Iran, of which the following are probably endemic: A. melanolepis Boiss and A. kermanensis Pold,⁴⁴  A. absinthium⁴⁵, A. annua,⁴⁶ A. dracunculus,⁴⁷ A. aucheri,⁴⁸ A. haussknechtii Boiss, ⁴⁹A. scoparia, A. sieberi⁵⁰ 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.

Materials and Methods

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

Surface morphology of silver 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 brown⁵² and also reported that silver nanoparticles exhibit a yellow-brown 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 well-correlated with previous work where the absorption peak was observed at 475 nm for a silver solution of leaf extracts of Memecylon edule⁵⁴   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

Figure 1: SEM images showing well-dispersed silver nanoparticles formed by extracts of Artemisia sieberi plant Click here to View figure

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 correlated with the variable shapes. Our results are similar to those reported by Elavazhagan and Arunachalam (2011).⁵⁴ Further, Savithramma et al., (2011)⁵⁶ who observed the formation of relatively spherical shaped silver nanoparticles formed with diameter ranging from 30 to 40 nm in Boswellia ovalifoliolata and 40 nm in Shorea tumbuggaia.

Figure 2: SEM images showing well-dispersed silver nanoparticles formed by extracts of Calotropis procera plant Click here to View figure

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.

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