RSC Advances

Silver and gold nanoparticles from Sargentodoxa cuneata:

synthesis, characterization and antileishmanial activities

Journal: RSC Advances

Manuscript ID: RA-ART-07-2015-013206.R1

Article Type: Paper

Date Submitted by the Author: 18-Aug-2015

Complete List of Authors: Ahmad, Aftab; Beijing University of Chemical Technology , College of Life
Science and Technology State key LaboratoryChemical Resource
Engineering
Syed, Fatima; University of Peshawar, institute of chemical sciences
Shah, Akram; University of Peshawar, Zoology department
Khan, Zahid; University of Peshawar, institute of chemical sciences
Yuan, Qipeng; Beijing University of Chemical Technology, College of life
science and technology
Khan, Arif Ullah; Beijing University of Chemical Technology, College of life
science and technology
Tahir, kamran; Beijing university of chemical technology, chemistry
Page 1 of 44 RSC Advances

Graphical abstract

Promising antileishmanial properties were observed with Sargentodoxa cuneata mediated

Ag and AuNPs. This study opens a platform for the synthesis new leishmanicidal agents
 RSC Advances Page 2 of 44

Silver and gold nanoparticles from Sargentodoxa cuneata: synthesis,

characterization and antileishmanial activities
Aftab Ahmad a, Fatima Syed b, Akram Shah c, Zahid Khan b, Kamran Tahir a, Arif Ullah

Khan a, Qipeng Yuan a*

a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical
Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing 100029, China
b
Institute of Chemical Sciences, Biochemistry Section, University of Peshawar (25120) Pakistan
c
Department of Zoology, University of Peshawar (25120) Pakistan

*Authors to whom correspondence should be addressed;

[email protected], [email protected] ,

Ph: +86 10 64437610 , Fax: +86 10 64437610

Page 3 of 44 RSC Advances

Abstract

Leishmaniasis remains one of the fatal diseases worldwide and the conventional antileishmanial

therapies are associated with several drawbacks. Therefore, there is a need to develop new

antileishmanial strategies. Biogenic silver and gold nanoparticles possess broad-spectrum

antimicrobial activities and could be future alternative to current antimicrobial agents. In this

report, we present a simple and green approach to synthesize silver and gold nanoparticles with

efficient biological activities. Phytochemicals from Sargentodoxa cuneata were used to reduce

and stabilize the silver and gold ions into metallic nanoparticles. The synthesized nanoparticles

were characterized by UV-visible spectroscopy (surface plasmon resonance), X-ray diffraction

analysis (crystallinity), High-resolution transmission electron microscopy (size and morphology),

energy dispersive X-ray (elemental composition) and FTIR (surface functionalities). Under the

optimized conditions, the synthesized silver nanoparticles were spherical in shape, small size (3-

8 nm) and well dispersed. However, the gold nanoparticles were mostly hexagonal in shapes

with approximate size from 15 to 30 nm. Promising antileishmanial activity was shown by silver

and gold nanoparticles with an IC50 value of 4.37 and 5.29 µg/mL respectively. Silver

nanoparticles also exhibited significant antibacterial activity against Staphylococcus aureus (32 ±

3 mm), Pseudomonas araginosis (16 ± 2 mm), and Bacillus subtilis (18 ± 2 mm). The depicted

biological activities of nanoparticles are not simply due to the capped silver and gold atoms but

also to their surface macromolecules. Thus, the use of Sargentodoxa cuneata as reducing and

capping agent will retain its biological activities even after the depletion of maintained silver and

gold. The findings of this study indicate that, these nanoparticles could be an alternative, safe,

and effective source of antileishmanial agents.

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Keyword: Sargentodoxa cuneata, silver gold nanoparticles, Leishmania tropica, bacteria,

antimicrobial activities

1. Introduction

Leishmaniasis is a tropical disease caused by parasites of the genus Leishmania. The disease is

transmitted by the bite of a female phlebotomine sand fly and has several clinical symptoms that

range from self-healing cutaneous to fatal visceral form. World health organization (WHO) has

declared leishmaniasis as category 1 disease (most emerging and uncontrollable) affecting

approximately 88 countries with annual incidence of cutaneous leishmaniasis around 1.5 million

worldwide. The visceral form of leishmaniasis is endemic in the south East Asian region with

300,000 cases in 2006 1. Furthermore, an increase rate of leishmaniasis has been reported around

the globe, which has been associated with a possible increase of the disease vectors due to global

warming 2. Severe toxicity and resistance to the current antileishmanial drugs has also been

observed in these parasites 3. Therefore, an urgent need exists to search for more effective and

selective therapeutic agents with broad-spectrum antimicrobial activities. Screening of medicinal

plants for the synthesis of metal nanostructures could be a natural alternative for the discovery of

new, more effective, less toxic and economical antileishmanial agents.

Nanobiotechnology is an emerging field that is dedicated to create and improve the utility of

nanoscale materials for a wide range of applications in advanced biotechnology 4. These

nanostructures have unique characteristic features that arise from their extremely small sizes and
5, 6
large surface area, which are significantly different from those of bulk masses . The nano

forms of silver and gold metals are of special interest in various disciplines, including catalysis,

disease diagnoses, gene expression, household consumable products, pharmaceuticals, and

cosmetics 7, 8. Gold nanocrystals have been used in immunoassay, cancer cells detection, protein
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9-12
assay, and capillary electrophoresis . These nanostructures upon cellular uptake behave as

thermal scalpels to kill the infected cell 13,14.

Silver is a promising agent possessing broad spectrum antibacterial activity with minimum

chance of bacterial resistance to it 15. It has been published that silver ions interfere with bacterial

DNA replication, disrupt cell membrane, inhibit critically important enzymes and damage
16-18
bacteria by a process called respiratory burst mechanism . Furthermore, silver and gold

nanoparticles have the ability to produce reactive oxygen species (ROS), which play an

important role in killing pathogenic microbes. It has been reported that leishmania parasites are
19
highly sensitive to ROS . In order to kill leishmania parasite by a treatment that involve

reactive species, a continuous supply of these oxygen species can be ensured with the use of

noble metal nanoparticles. Moreover, small amount of metal nanoparticles are not toxic to

human cells and therefore, could be an alternative, safe, and environmentally acceptable

antimicrobial agent.

Several physical and chemical methods have been employed to synthesize nanoparticles;

however, all those methods are associated with several shortcomings such as the use of toxic

chemicals and intensive energy and capital consumption; thus making these synthesis procedures

economically expensive and environmentally not friendly. The synthesis of nanoparticles with

phytochemicals is a greener, non-toxic, and environmentally acceptable procedure. Plant

kingdom represents a renewable source of biologically active phytochemicals, which could be

successfully utilized for the synthesis of metal nanoparticles 20. The biomolecules of plants may

act as both reducing and stabilizing agents in the synthesis of metal nanoparticles 21, 22.

The use of medicinal plants for the synthesis of nanoscale materials will effectively enhance their

biological activities and would remain active even after the depletion of the capped metal.
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In the present study, we report on the biogenic synthesis of Ag and Au nanoparticles using

phytochemicals from a Chinese medicinal plant "Sargentodoxa cuneata". To the best of our

knowledge, this is the first report describing the antileishmanial activities of biogenic gold and

silver nanoparticles. The prepared Ag and Au nanoparticles were also evaluated for the

antibacterial activities.

2. Experimental section

2.1. Preparation of plant extract

Powdered plant material (10 g) was extracted in 200 mL de-ionized water. The suspended plant

material was initially heated at 60 ºC for 10 min to avoid any possible degradation of the active

biomolecules. The suspension was then stirred for 1 h at room temperature (24 ºC). The extracted

biomass was centrifuged at 5,000 rpm for 5 min to remove the bulk plant material. Finally, the

supernatant obtained was filtered through Whatman no.1 filter paper. The clear extract obtained

was used for the synthesis of nanoparticles.

2.2. Extraction of phenolic compounds

Dried and finely ground plant material (5 g) was extracted twice with 100 mL aqueous (70%)
23
acetone . The suspended plant material in aqueous acetone was subjected to ultrasonic

treatment for 20 min at room temperature. The extracted biomass was then filtered and

centrifuged (4 ºC) at 5000 rpm for 10 min. The supernatant obtained was used as a source of total

phenolic contents. Tannin was precipitated from the aqueous extract of Sargentodoxa cuneata

with polyvinyl polypyrrolidone (PVPP). Both the aqueous extracts after total phenolic and tannin

extraction were separately used for the synthesis of silver nanoparticles.

2.3. Synthesis of silver and gold nanoparticles

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Silver and gold nanoparticles were synthesized employing the aqueous extract of Sargentodoxa

cuneata as a reducing and stabilizing agent. To synthesize silver and gold nanoparticles, different

concentrations of the plant extract (1-10 mL) were mixed separately with 30 mL of silver nitrate

and HAuCl4.4H2O (2 mM) solutions. The synthesis procedure was carried out at 30 ºC under

vigorous stirring. The progress of synthesis was examined by visual observation of the color

change from light yellow to deep brown for silver and purplish for gold nanoparticles. At the end

of the process, the colloidal suspensions were centrifuged at 10,000 rpm for 10 min. The pellets

obtained were washed thrice with distilled water, followed by freeze-drying under vacuum and

were finally stored at 4 ºC for further use. One of the problems associated with the biogenic

synthesis of metal nanoparticles is their reproducibility. In order to address this problem, we

performed the biosynthesis of nanoparticles in triplicate under the same experimental conditions.

In another experiment, silver nanoparticles were also synthesized by chemical method using

NaBH4 as reducing agent and sodium citrate as capping agent.

2.4. Optimization of nanoparticles synthesis

Various parameters that affect the synthesis of nanoparticles were optimized to get optimal

product. The study parameters involved in the optimization process were extract concentration

(1-10 mL), metal ions concentration (1-4 mM), pH (4-10), temperature (25-70 ºC) and reaction

time (0-300 min). The optical density of colloidal suspensions was measured at 428 and 536 nm

for silver and gold nanoparticles respectively.

Characterization of nanoparticles

2.5. UV visible spectroscopy

UV-visible spectroscopy was used to monitor the rate of metal reduction and progress of

nanoparticles biosynthesis. Aliquots taken from the reaction mixture were scanned at

wavelengths between 200 to 800 nm (UV spectrophotometer Shimadzu Japan- 2450).

 RSC Advances Page 8 of 44

2.6. High resolution transmission electron microscopy (HRTEM)

High-resolution transmission electron microscope (JEOL-JEM 3010 transmission electron

microscope) was used to study the surface morphology, size, and dispersities of the synthesized

nanoparticles. Samples for HRTEM were prepared by dissolving 2 mg (sonication) of the

nanoparticles in 10 mL methanol. Two drops of the prepared solution were placed on the carbon

supported copper grids and were allowed to evaporate the solvent. Particle size distribution was

determined from a histogram considering more than 300 particles measured using multiple TEM

micrographs.

2.7. XRD analysis

X-ray diffraction analysis was used to determine the crystalline nature and average particles size

of the synthesized particles. XRD analysis was performed at 2 θ in the range of 20 to 80 degrees

using powder X-ray diffractometer (D8 Advance diffractometer).

2.8. Antibacterial assay

The antibacterial activity of the synthesized nanoparticles was tested against four bacterial strains

viz E. coli, staphylococcus aureus, Pseudomonas araginosis and Bacillus subtilis. Nutrient agar

was autoclaved at 121 ºC for 1 h and poured into sterilized Petri plates. The autoclaved agar plats

were inoculated with bacterial cells suspension. Wells (6mm) were made in each agar plat using

sterile metallic borer. The colloidal suspension of nanoparticles in DMSO was used as test

sample while streptomycin and DMSO were used as positive and negative control respectively.

In order to assess the antibacterial activity of capped biomolecules, we performed a control

experiment with chemically synthesized silver nanoparticles. The plates were incubated at 37 ºC

for 24 h and the antibacterial activity of the selected nanoparticles was measured as zone of

inhibition in mm.
Page 9 of 44 RSC Advances

2.9. Antileishmanial activity

Leishmania tropica strain was cultured in medium 199 containing 10% inactivated Fetal Bovine

Serum (FBS). The antileishmanial activity was performed according to the procedure described

by of Nabi et al. 2012 with slight modification 24. The antileishmanial activity of silver and gold

nanoparticles was performed against Leishmania tropica promastigotes. Every assay tube

contained 3 mL of medium with 1 × 105 parasites/mL of L. tropica promastigotes. 5 µL (4

mg/mL) of silver and gold nanoparticles was loaded to each well and incubated at 28 ºC.

Parasites in both the control and treated samples were counted by hemocytometer at 24, 48, 72

and 96 h of incubation and the activity was expressed as percent inhibition.

2.10. Cytotoxicity assay

The cytotoxicity assay of the phytosynthesized silver nanoparticles was measured using murine

macrophages (J774 cell line) by MTT test 25. The cells were seeded in 96 well culture plates at a

density of 1 × 106, allowed to attach for 24 h and treated with different concentration (10-1000

µg/mL) of biosynthesized silver nanoparticles. The treated cells were incubated for 48 h for

cytotoxicity analysis. Finally, the cells were subjected to MTT assay. MTT stock concentration

(5 mg/mL) was prepared in PBS, and 100 µL of this solution was added to each wells (AgNPs-

treated) and incubated for 4 h. Then, 100 µL of dimethyl sulphoxide (DMSO) was added to each

well (to dissolve the formed purple formazan crystals), and the absorbance values were

determined at 590 nm in a multi-well ELISA plate reader (Eliza MAT 2000, DRG Instruments,

GmbH). Results were expressed as % cell viability.

       
% viability =       
× 100

3. Results and discussion

3.1. UV visible spectroscopy

 RSC Advances Page 10 of 44

Formation of silver and gold nanoparticles was confirmed by using UV-Vis spectral analysis.

The reduction of gold and silver ions into metallic nanoparticles show a characteristic localized

surface plasmon resonance (LSPR), where metal electrons in the conduction band collectively
26
oscillate in resonance upon interaction with light of specific wavelength . Localized surface

plasmon resonance varies with particle size, shape and the reaction medium. The appearance of

such characteristic LSPR bands is used to detect the synthesis of metal nanoparticles. Fig.1

illustrates the time dependent evolution of UV-Vis spectra of silver and gold nanoparticles under

different experimental conditions. The formation of nanoparticles was controlled by varying the

amount of plant extract and metal salts concentration. Initially, the formation of nanoparticles

was carried out at 3 mM silver, 2 mM gold and 5 mL plant extract (pH = 7, 30 ºC). It was

observed that AgNPs revealed a localized-SPR peak of low intensity at 436 nm when 2 mL of

the plant extract was mixed with 30 mL (3 mM) silver nitrate solution (Fig 1 a), indicating the

synthesis of small amount of nanoparticles. However, when 10 mL of the plant extract was used,

the SPR peak blue shifted from 436 to 432 nm with the corresponding increase in peak intensity.

This combine phenomenon indicates a faster rate of silver reduction with a narrow size particles

distribution (Fig. 1b). The intensity of localized-SPR peak is directly proportional to the

concentration of nanoparticles in the solution. However, a red shift in the LSPR pattern (from

432 to 450 nm) was noted with further increase in the aqueous extract (10 mL) of Sargentodoxa

cuneata (Fig 1 c), suggesting a possible increase in the particle size. Similar variation in the

LSPR pattern was also shown by gold nanoparticles synthesized under different plant

concentrations. LSPR peaks red shifted from 536 to 562 nm when plant concentration was

increased from 5 mL to 10 mL (Fig 1 e, f), suggesting the probability of increasing particle size.

An increase in particle size induces a red shift of LSPR from shorter to longer wavelength, which
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10

is in agreement with the HRTEM results (Fig. 4). High concentration of the plant extract

introduces more reducing agents, which result a secondary reduction process on the surface of

preformed nuclei and additional interaction between the surface biomolecules. The two

phenomena giving rise to bigger particles.

The rate of gold reduction was much faster than silver ions, as was observed from the abrupt

color change and rapid saturation of peak intensity in 20 min. This is due to the fact that standard

reduction potential of Au3+/Au is higher than Ag+/Ag 27. Furthermore, no detectible change in the

peak intensity and blue/red shift pattern was observed after 20 min of incubation, suggesting a

nano size distribution of gold nanoparticles and completion of the reaction. The observed

uniform symmetry of LSPR bands (Fig. 1) indicates the mono-dispersion of nanoparticles 28.

Different concentrations of silver and gold ions were used to get the optimum level of these

metals. Both the metals at their low precursor concentrations showed localized-SPR bands with

relatively low intensities. However, maximum absorbance was observed for silver and gold

nanoparticles at 432 and 536 nm when 3 and 2 mM of silver and gold salts were used (Fig. 1 b,f).

Further increase in the salts concentrations resulted visible precipitation with corresponding

decrease in absorbance (428 nm) and red shift in the LSPR pattern (Fig.2 b, d). Our results

indicated that better size control and maximum production of silver and gold nanoparticles could

be achieved at 3 mM AgNO3 and 2 mM AuHCl4. 4H2O respectively. Similarly, plant extract

concentration was optimized by keeping the silver and gold concentration at their constant levels

(3 and 2 mM). Results shows that 5 mL of the plant extract demonstrated the best result out of

the tested concentrations. Subsequent optimizations of other parameters were carried out by

keeping the plant and salt concentrations at their optimum levels. Fig. 3 a represents the effect of

pH on the biosynthesis of Ag and AuNPs. Results showed that pH of the reaction media have
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strong influence on the biosynthesis of nanoparticles. In both the cases, alkaline pH favored the

synthesis of nanoparticles. In the case of silver nanoparticles, alkaline pH resulted an increase in

the LSPR peak intensity along with a blue shift, indicating a significant reduction of the silver

ions in metallic nanoparticles (Fig. 1d). Previous study reveals that silver ions are efficiently
29
reduced at higher pH . This observation suggests that higher pH induces more nucleation and

growth of metal nanoparticles, which may be due to the activation of the phytochemicals

involved in their synthesis in alkaline conditions. Kajani et al. also reported that alkaline pH is

suitable for the synthesis of silver and gold nanoparticles 21.

The effect of reaction temperature on the biogenic synthesis of gold and silver nanoparticles is

shown in Fig. 3 b. A gradual increase in the absorbance at 428 and 536 nm was noted for both

silver and gold nanoparticles, indicating that metal reduction is accelerated in the presence of

plant extract at elevated temperature. However, it was observed that a slight decrease in the SPR

band intensity occurred beyond 50 ºC in the cases of gold and 60 ºC in the case of silver

nanoparticles. This indicates reaction saturation and may presents a tendency for precipitation at

elevated temperature.

The progress of nanoparticles synthesis was monitored for 300 min. The rate of silver reduction

was relatively slow and maximum localized SPR intensity was observed after 180 min (Fig. 3 c).

On the other hand, the rate of gold reduction was faster than silver ions as evident from the

appearance of maximum LSPR intensity within 20 min. No major increase in the LSPR intensity

was observed after 20 minutes indicating the completion of reaction. Under the optimized

conditions (5 mL extract, 3 mM silver, 60 ºC and pH 8), the prepared silver nanoparticles

revealed maximum SPR intensity (Fig.1d). Similarly, gold nanoparticles shoed maximum SPR

peak at 5 mL extract, 2 mM gold solution, 50 ºC and pH 8 (Fig 1 i).

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The stem extract of Sargentodoxa cuneata is rich in various phytochemicals like phenolic

compounds and flavonoids. These phytochemicals play an important role in the synthesis of

nanoparticles by reducing and stabilizing the metal ions into metallic nanoparticles. The surface

adhered biomolecules form a covering on the nanoparticles and hence protect them from

aggregation.

3.2. XRD analysis

XRD pattern of the synthesized nanoparticles (Fig. 4) indicates four diffraction bands at 38.175ᵒ,

44.093ᵒ, 64.431ᵒ and 77.373ᵒ which can be attributed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) Bragg

reflection pattern of the face centered cubic structure of metallic silver (reference file JCPDS no.

04-0783). As evident from the observed pattern, the most significant peak is centered at 2 θ =

38.175ᵒ, which is originated from the face centered cubic silver 30. This result clearly shows that

the top crystal plane (basal plane) of the synthesized nanoparticles must be the (111) plane.

Similar diffraction patterns have been previously reported for silver and gold nanoparticles using

plant extract as reducing and capping agents 31. When compared the broadness of XRD peaks for

silver and gold nanostructures, it is seen (Fig. 4) that XRD peaks are much broader for silver

nanoparticles as compared to gold NPs, suggesting a smaller size distribution for silver relative

to gold nanoparticles. The silver nanoparticles with smaller size have wider peaks (Fig 4 A-ii) as

compare to their larger counterparts (Fig 4 A-i). This pattern is also supported by the result

obtained from high resolution TEM. Similarly, the XRD pattern of gold nanoparticles with

different particles size has been given in fig. 4 B.

The average particles size of the synthesized nanoparticles was calculated from the Debye-

Scherrer's equation (1)


 =   ! "
(1)
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13

Where, d is particle size, K is the Scherer constant with value from 0.9 to 1 (shape factor), λ is

the wave length of the X-ray source, β is full width half maxima in radians (FWHM) and θ is the

Bragg angle. The sizes of silver and gold nanoparticles (under optimized conditions, 5 mL

extract) were about 7 and 25nm respectively. These results were in agreement with the sizes of

nanoparticles obtained from the HRTEM study. The calculated d spacing (fringe spacing)

between the lattice planes for (111) plane was found to be 0.236 nm, which was in agreement

with the lattice spacing of the reference (JCPDS file No. 04-0784d = 0.2359 nm). Similarly, the

lattice constant "a" of AgNPs was determined from the following equation (2).

# = $%& '(ℎ* + , * + - * ) (2)

Where "a" is the lattice parameter, dhkl is plane spacing for (111) plane and hkl are the

crystallographic dimensions. The lattice parameter for silver nanoparticles (4.08 Å), as calculated

from the above equation (equ. 2), was in full agreement with the reference standard 4.086 Å

(JCPDS 04-0783).

3.3. EDX analysis

Fig. 5 shows the EDX pattern of Ag and Au nanoparticles. Appearance of strong peak around 3

KeV designate the position of elemental silver which arises from the localized surface plasmon
21
resonance in AgNPs . Similarly, the appearance of strong peak around 2 KeV confirmed the

elemental gold as major constituent along with small signals from C, O and Cu atoms in gold

nanoparticles. Overall EDX result exhibited the presence of elemental silver and gold as major

constituents with small amount of carbon and traces of copper signals. These weak signals may

be originated from the surface bound macromolecules and the copper grids used as supporting

filament (Cu signal).

3.4. High-resolution transmission electron microscopy (HRTEM)

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The biological activities associated with silver and gold nanoparticles are size, shape, and degree

of dispersion dependent. These three parameters of the synthesized nanoparticles were

determined by transmission electron microscopy (HRTEM) and the result is shown in fig. 6.

Different concentrations of the plant extract were used to get the narrow size distribution of

nanoparticles. Under the optimized conditions (5 mL extract/30 mL, 2.5 mM gold solution), the

synthesized gold nanoparticles were well dispersed, mostly hexagonal in shape with scattered

pyramids (Fig. 6a). The biogenic synthesis of gold nanoparticles of mixed shapes has also been

previously recorded with plant extracts using 10 mM HAuCl4 [32]. The approximate particles

size was in the range of 20-40 nm. This result is in agreement with particle size calculated from

XRD pattern using Scherer's formula (̴ 25 nm). However, when high concentration of the plant

extract (10 mL/30 mL, 2.5 mM gold solution) was used, the resulted particles were larger in size

(50-80 nm) and were mostly fused together (Fig. 6b). The synthesized silver nanoparticles using

3 mM AgNO3 and 5 mL plant extract were mostly spherical in shapes with approximate grain

size of 5-8 nm (Fig. 6c). The same way, silver nanoparticles synthesized under high plant

concentration were larger in size (5-15 nm) with some degree of aggregation (Fig. 6d). It has

been previously reported that particle size is directly related to the total phenolic content in the

plant extract. Higher concentration of the phenolic content results in larger particle size and our

results are in close agreement with the previous findings 33. The observed increase in particle size

at higher plant concentration may be due to the elevated level of poly-phenolic contents. These

compounds play a critical role in the formation of nanoparticles as previously been reported for
33
other plants . It is also important to note that both Ag and AuNPs are surrounded by a thin

layer, suggesting that biomolecules present in Sargentodoxa cuneata have capped and adhered to

the surface of these particles 31.

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3.5. FTIR analysis

‒1
FTIR spectrum of AgNPs revealed significant peaks at 3432, 2922, 1628 and 1040 cm while
‒1
several weak bands were also observed at 2852, 1511, 1382 and 1129 cm (Fig. 7a). The peak

appeared at 3432 is attributed to the O-H stretching frequency of hydroxyl group of phenolic
‒1 ‒1
compounds, peaks at 2852 cm and 2922 cm could be due C-H stretching vibration of
‒1 ‒1
methyl or methylene groups [35]. The strong peaks at 1628 cm and 1040 cm represent the

carbonyl group stretching vibration of flavonoids and ester bonds in polyphenolic (tannin) [36]

[32, 37]. Other weak peaks around 1500 cm ‒1 and 1129 cm ‒1 correspond to stretching vibration

of aromatic -C-C- groups and C-O functional in tannin/tannic acid 38, 39.

Similar pattern of FTIR spectra was also observed for the gold nanoparticles with the exception

of a strong peak at 1021 cm‒1 (Fig. 7b). This peak corresponds to the ester bonds in phenolic

compounds (tannins), suggesting a strong involvement of tannin in the synthesis of gold

nanoparticles. The possible phytochemicals of the selected plant such as poly-phenolics such as

flavonoids and tannin, as indicated from the FTIR spectra, may be involved in metal reduction

and capping for the synthesis of silver and gold nanoparticles 40, 41.

Polyphenolic compounds (tannins) contain sufficient hydroxyl and other suitable groups (such
42
as carboxyl) to form strong complexes with various metal ions . Tannins are water soluble

polyphenols that constitute a major portion (more than 10 % of the dry weight) of higher

herbaceous and woody plants, where they play an important role in protection from microbial

attack and their colonization 43.

Phytochemical analysis of the aqueous extract of Sargentodoxa cuneata reveals the presence of

flavonoids, tannins, phenolic glycosides, and reducing sugars (Table 1). Previous studies also

verify the presence of phenolic glycosides, flavonoids, anthraquinones, and organic acids in the
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44-46
aqueous extract of Sargentodoxa cuneata . Most of the detected compounds in the aqueous

extract of Sargentodoxa cuneata such as flavonoids, tannins and other phenolic compounds

possess strong antibacterial and anti-parasitic activities 47, 48.

Based on the FTIR spectral analysis of silver and gold nanoparticles for the surface adhered

biomolecules and experimental work, we proposed a mechanism (Fig. 8) for the reduction and

capping of metal ions into metal nanoparticles. It is suggested that flavonoids and other phenolic

compounds in the plant extract such as tannin may be responsible for the reduction and

subsequent stabilization of silver and gold ions. These compounds possess sufficient hydroxyl

and carboxyl groups, which are able to bind to metals. The chelating ability of phenolic

compounds is possibly related to the strong nucleophilic character of the aromatic rings rather

than to specific chelating groups within these molecules 49.

3.6. Synthesis of AgNPs in the absence of phenolics

When the aqueous extract of Sargentodoxa cuneata (after the extraction of polyphenols) was

used for the synthesis of silver nanoparticles, it was observed that the rate of metal reduction was

very slow as evident from the sluggish color change after 10 h of the reaction. This observation

suggests that phenolic compounds in the plant extract are majorly responsible for the reduction

of metal ions into metal nanoparticles. The role of tannin as a reductant and stabilizer was also

investigated in an experiment when AgNPs were prepared with the aqueous extract after tannin

precipitation with polyvinyl polypyrrolidone (PVPP). The LSPR pattern of these nanoparticles

was of low intensity with significant red shift, indicating a possible increase in particles size.

Furthermore, aggregation and fusion among the particles was also observed, revealing the lack of

sufficient capping agent, which could stabilize the metal nanoparticles (Fig. 6 e). These

observations suggest that in the absence of tannin in the aqueous extract, the metal nanoparticles
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were not fully stabilized and hence resulted aggregation and fusion among them. All these

observation suggest that polyphenolic compounds are the main players in the synthesis of metal

nanoparticles. Our results are in agreement with previous findings that phenolic compounds are

mainly responsible for metal reduction and capping 32, 50.

3.7. Antibacterial activity

The emergence of new infectious diseases and increased incidence of bacterial resistance is a

serious health problem worldwide. There is an urgent need for the discovery of new, safer, and

more potent therapeutic agents to treat the emerging microbial resistance and other medical

conditions. The potential of a medicinal plant "Sargentodoxa cuneata" was exploited in the

synthesis of silver and gold nanoparticles to enhance its biological activities. The synthesized

nanoparticles were tested against four bacterial strains and the results showed variation with

respect to antibacterial response (Table 2). The silver nanoparticles exhibited significant activity

against staphylococcus aureus, Pseudomonas araginosis and bacillus subtilis with zone of

inhibition 32 ±2.6, 16 ± 2 and 18 ± 2 mm respectively, even more potent than the standard drug

used (Fig. 9). However, both silver and gold nanoparticles were moderately active against E. coli

with zone of inhibition 12 ± 1 and 11 ± 1 mm respectively. The strong antibacterial activity of

AgNPs (compared to AuNPs) may be attributed to their small size and spherical morphology.

Smaller particles sizes with spherical morphology have large surface area for contact with

microorganisms, and are therefore more active than their larger counterparts. Furthermore, the

surface adhered biomolecules in metal nanoparticles also contribute to the biological activities of

these nanostructures. Sargentodoxa cuneata mediated silver nanoparticles exhibited strong

antibacterial activity against Klebsiella Pneumoneae (17 ± 1.4) and Bacillus subtilis (16 ± 1.5) in

a control experiment as compared to chemically synthesized silver nanoparticles (10 ± 1.2, 12 ±

1.4 respectively). This result clearly suggests that the improved biological activity of
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Sargentodoxa cuneata mediated AgNPs are not simply due to capped silver and gold atoms but

also to their surface macromolecules (Fig. 9).

It has been suggested that antibacterial effect is mainly due to silver release from the AgNPs,
18, 51
followed by the interaction of silver ions with the cellular targets . Silver ions exert their

antimicrobial activity by several mechanisms such as (i). Ag ions interact with the thiol group of

critically important enzymes and inhibit them (ii). Interfere with the respiratory mechanism (iii).

interact with phosphate group in DNA and interfere its ability to replicate (iv) cause K+ depletion

thus disrupt cellular transport system (v) physical contact of silver nanoparticles disrupt cell

membrane and (vi) production of reactive oxygen species from silver ions and silver particles 16-
18
. Previous findings indicate that control release of silver from nanoparticles induces the
17, 52
production of reactive oxygen species in pathogenic bacteria . When the levels of these

reactive species overcome the scavenging capacity of bacterial cell, they cause damage to
17
bacterial protein and DNA . The most important result of the present study is that AgNPs

exhibited significant activity against Pseudomonas araginosis, which was resistant to several

antibiotics including streptomycin. The Pseudomonas araginosis is usually isolated from the

burn wounds, and the AgNPs could be successfully applied to treat the multi-drug resistant

bacteria in such medical conditions. Secondly, the AgNPs also showed significant activity (32 ±

3 mm) against a streptomycin resistant staphylococcus aureus, making these nanoparticles of

significant importance as antibacterial agents.

3.8. Antileishmanial activity

The antileishmanial activity of silver and gold nanoparticles was tested against Leishmania

tropica as a modal parasite. The efficacy of all the tested nanoparticles was studied for 96 h and

the antileishmanial activity was expressed as percent inhibition. To make determinations, the
 RSC Advances Page 20 of 44

19

number of promastigotes was counted in both control and experimental groups at different time

intervals (24, 48, 72 and 96 h). It was observed that the number of cells significantly decreased in

each group, when compared with the control group, after exposure to silver and gold

nanoparticles. Fig. 10 (a, b) shows the time dependent antileishmanial activities of silver and

gold nanoparticles. The observed decrease in the parasite count after 24 h of incubation is shown

in fig. 10 c. It is obvious from the results that the parasite count significantly decreased in the

first 24 h of incubation with the nanoparticles. Ag and Au nanoparticles showed 90 and 62%

inhibition within this time period respectively. The number of cells count was further decreased

in the treated samples when examined at 48 h of incubation. Both the Ag and Au nanoparticles

showed significant activity against the parasite, however, the silver nanoparticles presented an

excellent antileishmanial activity with maximum of 95.45 % inhibition at 48 h of incubation.

From then onward, AgNPs showed a negligible decline in activity after 48 h of incubation, while

an increase in leishmanicidal activity was observed for AuNPs (77.5%).

Several mechanisms have been proposed for the antimicrobial property of metal nanoparticles. A

number of reports suggest that the antimicrobial effect relay mainly on the slow release of silver

ions from nanoparticles surface, followed by interaction with microbial cell surface, penetration

into the cytoplasm and binding with the target sites 17, 53. Furthermore, noble metal nanoparticles

are able to produce reactive oxygen species, which destroy pathogenic microbes by a process

called respiratory burst mechanism. It has been stated earlier that Leishmania is highly sensitive

to these oxygen species and the drug, which could generate ROS, will be efficient

antileishmanial agents. Macrophages (host cells for Leishmania) produce high concentration of

these ROS to disinfect microbial agents 54. However, Leishmania bypasses the oxidative damage

caused by ROS by inhibiting the enzymes that are involved in the process of ROS production 55.
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The use of Ag and Au nanoparticles as leishmanicidal agents will act as a large reservoir of

silver and gold ions, which will provide a non-enzymatic source of ROS and will destroy the

invaded parasite. Electron spin resonance spectroscopy has confirmed the generation of free

radicals from silver ions, and these radicals damage microbial cells by several mechanisms 56.

Previous studies also indicate that metal containing compounds have promising antileishmanial

activities. Gold containing complexes are efficient inhibitors of metabolically important enzymes

in Leishmania. According to researchers, Trypanothione reductase is a critically important

enzyme, which controls the polyamine-dependent redox metabolic activity of Leishmania, and is

therefore an attractive target for designing leishmanicidal drugs. Recent studies have

demonstrated that gold containing compounds exert antileishmanial activity by inhibiting

57
Trypanothione reductase . Trypanothione reductase and Trypanothione synthatase are two

essential enzymes for Leishmanial survival. These enzymes play an important role in protecting

Leishmania form the oxidative damage, and allowing the delivery of the reducing equivalents for

DNA synthesis. The absence of Trypanothione reductase and Trypanothione synthatase in

mammalian host and the well-known sensitivity of Leishmania to ROS make these enzymes as

selective target for antileishmanial drugs. Furthermore, gold-based drugs are also efficient

inhibitors of thioredoxin glutathione reductase, and inhibition of this enzyme upsets the

intracellular redox balance, followed by induction of oxidative stress and subsequent cytotoxic

effects 58. The strong antileishmanial activities of our samples may be due to inhibitory effects on

some of the metabolically important enzymes.

The corresponding concentrations of Maytenus extract were also used to investigate the

antileishmanial activity of the aqueous extract leishmania tropica cells as control experiments.
 RSC Advances Page 22 of 44

21

The maximum mortalities of 42.24 % were obtained after 72 hours incubation in the highest

concentration of aqueous extracts. This indicates the moderate antileishmanial activity of

Maytenus royleanus aqueous extract. Concerning the antileishmanial activity of biogenic silver

and gold nanoparticles, it can be concluded that the coating of gold and silver nanoparticles with

Maytenus extract displays synergistic antimicrobial effects. The significant results of our

findings thus clearly demonstrate that low concentrations of silver nanoparticles can significantly

inhibit Leishmania and may be a promising agent for the treatment of leishmaniasis.

3.9. Cytotoxicity of biogenic silver nanoparticles

The development of more effective and less toxic agents are required to treat a particular

pathological condition. Silver nanoparticles are strong candidates possessing broad-spectrum

antimicrobial activities. However, it is important to evaluate the cytotoxic effects of these

nanostructures on normal cells in order to ensure their safe use. The phyto-synthesized silver

nanoparticles were evaluated for their effect on cell viability against murine macrophages (J774

cell line) in a dose dependent manner. Results indicated that AgNPs did not show any prominent

cytotoxicity at low concentrations and less cytotoxicity was observed at higher concentration

above 80 µg/mL (Fig. 11). The biosynthesized AgNPs exhibited 50% cell inhibition (IC50) at 116

µg/mL which is very high than the inhibitory concentration against leishmania tropica (IC50 =

4.37 µg/mL). Our results also indicate that AgNPs synthesized with Sargentodoxa cuneata are
59-61
less cytotoxic than previously reported biogenic silver and gold nanoparticles . Hence, the

present investigation suggests that at limited dosage, the Sargentodoxa cuneata mediated AgNPs

can be used as an effective antileishmanial agent with minimum toxicity.

4. Conclusion
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The present contribution demonstrated the synthesis of silver and gold nanoparticles employing

an eco-friendly green approach. The aqueous extract of Sargentodoxa cuneata was successfully

used as reducing and capping agent without any aided supportive chemicals. Subsequent

characterization of the synthesized nanoparticles was carried out using UV-visible spectroscopy,

XRD, EDX, HRTEM and FTIR techniques. Silver nanoparticles showed significant activity

against Staphylococcus aureus (32 ± 3), Pseudomonas araginosis (16 ± 2) and Bacillus subtilis

(18 ± 2) while moderate activity was recorded with E. coli (10 nm ± 1.2). Importantly, the Ag

and AuNPs size can be controllably varied by changing the concentration of Sargentodoxa

cuneata stem extract. The absorption spectra of nanoparticles were tuned, by carefully

optimizing various process parameters for the synthesis of small sized nanoparticles.

Promising antileishmanial activity was shown (for promastigotes form) by AgNPs, inhibiting the

growth of parasite by 95 %. Gold nanoparticles also presented a significant activity against

Leishmania tropica at 48 h of incubation (75% ± 14). The green synthesized silver nanoparticles

were shown to have minimum toxicity (IC 50 = 116 µg/mL) against the normal human cells.

Thus, this study provides a platform to synthesize more effective and less toxic antimicrobial

agents by a green method for use in pharmaceutical industry. Furthermore, the use of silver and

gold nanoparticles may represent a future alternative to current antileishmanial drugs. This is the

first report on biogenic synthesis of silver and gold nanoparticles using the aqueous extract of

Sargentodoxa cuneata.

Acknowledgment

The authors are highly grateful to International Science & Technology Cooperation Program of

China (Grant No. 2013DFR90290) for supporting the current project.

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Page 29 of 44 RSC Advances

Figure 1
 RSC Advances Page 30 of 44

Fig. 1. Time dependent evolution of UV-visible spectra of silver and gold nanoparticles under

varying amount (mL) of plant extract at 3 and 2 mM silver and gold ions respectively. (a) 2 mL

extract and 3 mM AgNO3 (b) 5 mL extract (c) 10 mL extract and (d) silver nanoparticles under

the optimized conditions (5 mL extract, 3 mM AgNO3, pH 8 and 60 ºC. (e, f, g) formation of

gold nanoparticles under 2, 5 and 10 mL plant extract and (h) under the optimized conditions

(pH = 8, 50 C and 20 min) at 2 mM gold solution

Page 31 of 44 RSC Advances

Figure 2

Fig, 2. UV-Vis spectra of silver and gold nanoparticles under varying amount of silver and gold
ions keeping the plant concentration constant (5 mL), (a, b) Silver nanoparticles at 2 and 4 mM
AgNO3. (c, d) gold nanoparticles at 1 and 3 mM gold solution
 RSC Advances Page 32 of 44

Figure 3

Fig. 3. Optimization of various parameters for the synthesis of silver and gold nanoparticles (a)
pH (b) temperature and (c) time
Page 33 of 44 RSC Advances

Figure 4

Fig. 4. X-ray diffractogram of silver and gold nanoparticles. (a) XRD pattern of silver

nanoparticles of different sizes as evident from the comparative broadening of the representative

peaks, (i) comparatively narrow than (ii) (b) Represents the comparative XRD pattern of gold

nanoparticles of different sizes, (i) larger particles with narrow peaks and (ii) comparatively

broader peaks indicating small particles size

 RSC Advances Page 34 of 44

Figure 5

Fig. 5. EDX analysis of silver and gold nanoparticles (a) EDX pattern of nanoparticles

confirming the presence of elemental silver as major constituent, (b) EDX pattern of gold

nanoparticles, showing gold signal as major peak

Page 35 of 44 RSC Advances

Figure 6

Fig. 6. (a) HRTEM image of gold nanoparticles synthesized at 5 mL plant extract (3mM

(HAuCl4.4H2O). (b) Gold nanoparticles synthesized at 12 mL plant extract. (c,d) AgNPs

 RSC Advances Page 36 of 44

synthesized at 5 and 12 mL plant extract (3 mM AgNO3). (e) AgNPs synthesized with aqueous

extract after tannin precipitation with polyvinyl polypyrrolidone (PVPP).

Page 37 of 44 RSC Advances

Figure 7

Fig. 7. FTIR spectra of (a) Sargentodoxa cuneata mediated synthesis of silver and (b) gold
nanoparticles
 RSC Advances Page 38 of 44

10

Figure 8

Fig. 8. Possible mechanism involved in the phytosynthesis of gold nanoparticles

Page 39 of 44 RSC Advances

11

Figure 9

Fig. 9. Antibacterial activity (1-4) of Sargentodoxa cuneata mediated silver and gold
nanoparticles against Staphylococcus aureus, E. coli, Pseudomonas araginosis, and Bacillus
subtilis. (5,6) Comparative antibacterial activities of Sargentodoxa cuneata mediated (1) and
 RSC Advances Page 40 of 44

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chemically synthesized (3) silver nanoparticles against Klebsiella pneumoneae and Bacillus
subtilis respectively.
Page 41 of 44 RSC Advances

13

Figures 10
 RSC Advances Page 42 of 44

14

Fig. 10. Antileishmanial activity of silver and gold nanoparticles (a) Number of viable cells

count in control and treated samples at different time intervals. (b) % growth inhibition of

Leishmania tropica after exposure to silver and gold nanoparticles and (c) dose dependent

antileishmanial activities of silver and gold nanoparticles. (d) Microscopic view of parasites (i) in

the control group (not exposed to silver nanoparticles (ii) in the group exposed to silver

nanoparticles in the dark, and (iii) in the group exposed to gold nanoparticles in the dark for 24 h

and (iv) showing the distorted morphology of leishmania.

Page 43 of 44 RSC Advances

15

Figure 11

Fig. 11. Dose dependent cytotoxicity of Sargentodoxa cuneata mediated silver nanoparticles
against murine macrophages. Results are expressed as a mean with bars showing mean ± SD, CC,
control group.
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Table 1. Phytochemical screening of aqueous extract of Sargentodoxa cuneata

Phytochemicals Inference

Alkaloids +
Flavonoids +
Terpenoids +
Tannins +
Saponins +
Glycosides +

Table 2. Antibacterial activity of silver and gold nanoparticles

Bacteria AgNPs AuNPs Streptomycin

Zone of inhibition
(mm)
Staphylococcus aureus 32 ± 2.6 12 ± 0.8 00

Pseudomonas araginosis 16 ± 2 08 ± 0.5 00

Bacillus subtilis 18 ± 2 06 ± 0.5 08 ± 0.6

E. coli 12 ± 1 11 ± 1 21 ± 2.2