IET Nanobiotechnology

Research Article

Green synthesis, characterisation and ISSN 1751-8741

Received on 4th July 2015
Revised on 22nd December 2015
biological evaluation of AgNPs using Agave Accepted on 4th January 2016
doi: 10.1049/iet-nbt.2015.0053
americana, Mentha spicata and Mangifera www.ietdl.org

indica aqueous leaves extract

Bashir Ahmad 1, Farah Shireen 1, Shumaila Bashir 2, Ibrar Khan 1, Sadiq Azam 1 ✉
1
Center of Biotechnology and Microbiology, University of Peshawar, KPK, Pakistan
2
Department of Pharmacy, University of Peshawar, Pakistan
✉ E-mail: azam_bbt@yahoo.com

Abstract: The current study was performed to synthesize stable, eco-friendly and bio-compatible silver nano-particles
(AgNPs) of Agave americana, Mentha spicata and Mangifera indica leaves and to screen them for biological activities.
The ultraviolet-visible spectroscopic analysis revealed that l-max for AgNPs range from 350–500 nm. All AgNPs
possessed polycrystalline structure as notified as intense graphical peaks in complete spectrum of 20 values
ranging from 10–80° in X-ray diffraction measurements and supported by scanning electron microscopy data. The
size of the nano-particles was confirmed by transmission electron microscopy (30–150 nm). Mass loss at variable
temperatures was evaluated by simultaneous thermogravimetric and differential thermal analysis revealed
reduction in mass and activity of compounds was notified by temperature increase from 200 to 800 °C, thus
concluding it as thermally sensitive compounds. A. americana AgNPs showed significant (96%) activity against
Methicillin resistant Staphylococcus aureus, Escherichia coli (95%) and Fusarium oxysporum (89%). Good
antioxidant activity was shown by M. spicata AgNPs at 300 µl (79%). M. indica AgNPs showed significant phytotoxic
activity (88%) at highest concentration. No haemagglutination reaction was observed for the test samples. The
above results revealed that AgNPs synthesized from selected plant species possesses significant antimicrobial and
phytotoxic effect.

1 Introduction
Green synthesis of silver nano-particles (AgNPs) is transpiring
domain of nanotechnology as the techniques exploited are
reckoned to be environment-friendly and impregnable than other
alternative conventional strategies [1]. Manipulations of AgNPs
specifically from plant extracts are entailed by the global
commerce due to their substantial pertinence in health and
industrial sphere [2]. AgNPs play a prime part in fields of clinical
therapeutics, predominantly antimicrobial and anti-inflammatory
prospective are highly reputed. They are also accounted to be
utilised in medical devices along bare quandary of nanotoxicity [3].
Agave americana, a monocarpic and succulent plant belonging to
family Agavaceae, having 2 m lingering greyish-green leaves and
huge yellow flowers [4]. It is native drought tolerant Mexican
plant but is dispersedly cultivated across Pakistan, India and other
Mediterranean countries [5]. The long fibres that exist in the leaves
are availed to produce rope, mats and sturdy clothing.
A. americana saccharine nectar is commercialised as natural sugar
comprehending less glycemic index due to exorbitant fructose
contents. The sap are cumulated and brewed from flowering stems
to produce alcoholic drinks such as tequila and pulque [6].
Mentha spicata habitually entitled as spear mint is herbaceous
perennial small plant that belongs to family Lamiaceae [7]. It is
native to Europe and South Asia but widely distributed across the
planet [8]. It is used to manufacture spearmint oil which possesses
carminative and aromatic properties. It is also used in
manufacturing of soaps, shampoos, toothpaste and confectioneries
due to its strong essence [9]. It possesses tremendous antioxidant,
antimicrobial properties and is widely used as seasoning by the
Indians [10, 11].
Mangifera indica conventionally known as mango plant belongs
to family Anacardiaceae. It is cultivated extensively across
temperate regions of the world but wild variety is native to India.
It is used in traditional folk medicine as anti-emetic, anti-diuretic,
anti-diarrheal, acidity, kidney and heart problems [12].
The green synthetic AgNPs have a significant action against
various health problems; the present research activity was
performed by using the aqueous leaves extracts of A. americana,
M. spicata and M. indica for the synthesis, characterisation and in
vitro biological screening of AgNPs.
2 Materials and methods
2.1 Plant materials
Leaves of A. americana, M. spicata and M. indica were collected
from District Peshawar, Khyber Pakhtunkhwa, Pakistan and were
identified by Ghulam Jelani, Department of Botany, University of
Peshawar, Pakistan.
2.2 Extraction
Leaves of selected plants were washed with water. The cleaned
leaves were shade dried and pulverised into fine powder using
electrical grinder. Aqueous plant extracts were prepared by boiling
25 g of pulverised leaves in 500 ml of sterile distilled water for 30
min. The aqueous extracts were cooled and filtered at room
temperature with Whattman No. 1.
2.3 Synthesis of silver nanoparticles
Aqueous extract (10 ml) was added to 90 ml of AgNO3 (1 mM)
solution at room temperature. The concoction was subjected to

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shaking water bath at 75° for 1 h, changing the color from yellow to
dark brownish black indicating reduction of Ag+ ions to Ago
nanoparticles. Finally prepared nanoparticles solution was
concentrated under vacuum at 50° using rotary evaporator.
Concentrated green synthesised AgNPs were dried at room
temperature in sterile petri plates.

2.4 Characterisation of silver nanoparticles

2.4.1 Ultraviolet–visible spectroscopy (UV–vis)

spectroscopy: The optical properties of AgNPs were measured
by UV–vis spectrophotometer (Shimadzu UV-1601), operated at a
resolution of 10 nm between 350–500 nm. Fig. 1 UV–vis spectroscopic analysis of green synthesised AgNP from
selected plants
2.4.2 X-ray diffraction measurements (XRD): Crystalline
metallic properties of AgNPs were examined by XRD (JDX-3532)
equipped with Cu Ê (á) radiation of 1.54187 nm, using Ni as filter blotting paper and then was allowed to dry by putting it under a
at 30 kV/30 mA. mercury lamp for 5 min [13].

2.4.3 Scanning electron microscopy (SEM): Using SEM
(JEOL-JSM-5910), thin films of AgNPs were subjected on carbon
coated copper grid. Extra solution was wiped out with blotting
paper. The thin film was then allowed to dry by placing it under
mercury lamp for 5 min and subjected to electron microscope for
evaluation at 150, 500 and 1000× magnification.
2.4.4 Transmission electron microscopy (TEM): TEM was
conducted utilising a TEM (Techni-G2-300kV). By dropping very
small amount of sample onto the carbon coated copper grid, thin
films of sample were prepared. Extra solution was scraped off with
2.4.5 Energy-dispersive X-ray spectroscopy (EDX):
xElemental composition of AgNPs was determined by EDX
(INCA-200) to eliminate the possibility of presence of other
elements.
2.4.6 Simultaneous thermogravimetric and differential
thermal analysis (TG-DTA): Evaluation of physical and
chemical properties of AgNPs was determined by simultaneous
thermo-gravimetric and differential thermal analysis (Shimadzu
DTG-60/DTG-60A). Mass gain and loss of nanoparticles was
determined at variable increasing temperatures.

Fig. 2 a, b, c: XRD spectra of AgNPs of the selected plants

a Agave americana
b Mentha spicata
c Mangifera indica

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Fig. 3 a, b and c: SEM of AgNPs of the selected plants
a Agave americana
b Mentha spicata
c Mangifera indica
2.5 In vitro biological studies of silver nanoparticles
2.5.1 Antibacterial activity: Antibacterial activity of AgNPs
were determined against Pseudomonas aeruginosa, Methicillin
resistant Staphylococcus aureus (MRSA), Vancomycin resistant
S. aureus (VRSA), Proteus mirabilis, Escherichia coli, Streptomyces
griseus and Bacillus subtilis using the agar well diffusion assay [14].
Autoclaved nutrient agar (Sigma-Aldrich, Germany) was poured
into sterilised Petri-plates. Uniform bacterial lawn was prepared on
Petri-plates using 18–24 h old culture and 6 mm wells were made
using a sterile borer. 100 µl from stock solution (3 mg/ml) of
AgNPs in dimethyl sulphoxide (DMSO, <1%) was poured into
respective wells. Amoxicillin and DMSO were used as positive
and negative control, respectively. After 24 h of incubation at 37°,
zones of inhibition (mm) were measured and by using the given
formula, per cent inhibition was calculated. (see equation at the
bottom of the page)
2.5.2 Antifungal activity: Antifungal activity of AgNPs was
determined against Verticillium dahliae, Aspergillus niger,
Aspergillus parasitica, F. oxysporum and Penicillium notatum
using agar tube dilution assay [14]. Stock solution (24 mg/ml) of
AgNPs was prepared in DMSO. 4 ml Sabouraud dextrose agar
(Sigma-Aldrich Germany) was poured in test tubes and autoclaved
at 121° for 15 min at 21 Psi. After autoclavation the tubes were
allowed to cool (50°), 66.6 µl from stock solution was added and
allowed to solidify in slanting position. With the help of sterilised
inoculating loop, fresh culture of test fungi was inoculated to
labelled slants. Miconazole and DMSO were used as positive and
negative control, respectively. All the test tubes were incubated at
28 ± 1° for 5–7 days. After incubation, the visible linear growth
inhibitions of fungal strains were calculated in comparison with
controls. (see equation at the bottom of the page)
2.5.3 Antioxidant activity: Antioxidant activity of AgNPs were
evaluated by their ability to eliminate the free radicals using 1,
1-diphenyl-2-8 picrylhydrazyl (DPPH) as per reported procedure
[14]. The test samples were prepared at concentration of 100, 200
and 300 µg/ml. Absorbance at 517 nm was determined after half
an hour at room temperature and finally antioxidant activity were
calculated as percentage of free radical reduction. Experiment was
run in triplicate. DPPH was used as a reference compound.
2.5.4 Phytotoxic activity: AgNPs were evaluated for phytotoxic
activity against Lemna minor as per reported procedure [14].
Percent inhibition = Zone of inhibition of sample (mm)/Zone of inhibition of positive control (mm) × 100
% linear growth inhibition = linear growth in sample (mm)/linear growth in negative control (mm) × 100
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20 mg/ml stock solutions of nanoparticles were prepared in
methanol. E-medium was prepared for the growth of L. minor.
Test samples at concentration of 10, 100 and 1000 µg/ml were
poured to sterile flask and methanol was allowed to evaporate.
After evaporation, 20 ml of the E-medium was poured to all
flasks. Finally 16 healthy L. minor were picked and transferred to
each flask and incubated at 27 ± 1° for 7 days. The results were
recorded at the end of incubation period by counting the damaged
plants.
2.5.5 Haemagglutination activity: Haemagglutination activity
of AgNPs was determined as per procedure [14]. Stock solution
(1 mg/ml) was prepared in phosphate buffer (pH 7.0) and different
dilutions (1:2, 1:4, 1:8 and 1:16) were prepared. Fresh blood
samples from healthy volunteers were collected and centrifuged to
isolate red blood cells (RBC). Using phosphate buffer, 2% RBC’s
suspension was prepared. From each dilution, 1 ml of test sample
was transferred to sterilised test tube and 1 ml of the RBC’s
suspension was then added to it. Reaction mixture was incubated
at 37 °C for 30 min. The test tubes were centrifuged to observe
positive and negative results indicated by rough and smooth button
formation, respectively.
3 Results and discussion
3.1 Characterisation of silver nanoparticles
3.1.1 UV–vis spectroscopy: From UV–vis spectroscopic
analysis it was validated that the ƛ-max for the AgNPs lies in
range of 350–500 nm. The ƛ-max for A. americana and M. indica
AgNPs was 430 nm with absorbance of 0.72 and 0.81,
respectively, while for M. spicata, it was 410 nm with 0.68
absorbance. Results of spectroscopic analysis are presented in Fig. 1.
3.1.2 X-ray diffraction measurements: From crystallographic
evaluation of the biosynthesised AgNPs, it was validated that all of
the three AgNPs possessed polycrystalline structure with an intense
graphical peaks in complete spectrum of 2θ values, ranging from 10–
80° as shown in Figs. 2a–c.
3.1.3 Scanning electron microscopy: At 500 and 1000× in
SEM, the structure and shape of synthesised AgNPs were revealed
as polycrystalline as shown in Figs. 3a–c.
3.1.4 Transmission electron microscopy: The TEM analysis
revealed that major particle size falls in the range of 30–150 nm.
However, few particles of 15–20 nm were also spotted.
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Fig. 4 a, b and c: Transmission Electron Microscopic analysis of AgNPs of selected plants
a Agave americana
b Mentha spicata
c Mangifera indica

Conformations of fabricated AgNPs were localised as spherical, nanoparticles occurred as temperature increased from 200–800 °C
semi-spherical, oblong, rods and scarily triangular. The obtained as depicted in Figs. 6a–c of TG-DTA spectra. The analysis
results were supported by the reported work of Jingqi et al. [15] revealed that the stated nanoparticles are temperature sensitive.
Results are displayed in Figs. 4a–c.

3.1.5 Energy-dispersive X-ray spectroscopy: From EDX
analysis (Figs. 5a–c), it was revealed that composition of AgNPs
is precise and consists of 34.91% (by weight) of silver for
A. americana, 9.96% for M. spicata and 9.93% for M. indica,
along with other elements; organic carbon, oxygen, magnesium,
silicon, sulphur, chlorine, potassium and calcium.
3.1.6 Simultaneous thermogravimetric and differential
thermal analysis: The reduction in mass and activity of
3.2 Biological activities of silver nanoparticles
3.2.1 Antibacterial activity: AgNPs of A. americana possess
significant antibacterial potential against MRSA (96%) and E. coli
(95%). Good activity was observed against VRSA (76%),
P. mirabilis (72%) and B. subtilus (64%). Moderate activity was
observed against P. aeruginosa (48%) and S. griseus (45%).
AgNPs of M. spicata showed significant inhibitory potential
against E. coli (83%), moderate activity was observed against

Fig. 5 a, b and c: EDX of AgNPs of selected plants

a Agave americana
b Mentha spicata
c Mangifera indica

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Fig. 6 a, b and c: TG-DTA of AgNPs of selected plants
a Agave americana
b Mentha spicata
c Mangifera indica

Fig. 7 Antibacterial activity of AgNPs of selected plants

Fig. 8 Antifungal activity of AgNPs of the selected plants

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Fig. 9 Antioxidant activity of AgNPs of the selected plants
MRSA (57%), P. merabilis (56%), P. aeruginosa (55%), VRSA
(46%) and S. griseus (45%) and low activity against B. subtilis
(20%). M. indica AgNPs exhibited good antibacterial activity
against; B. subtilis (76%), P. aeruginosa (74%), S. griseus (70%),
MRSA (69%) and P. mirabilis (60%). Moderate activity was
observed against E. coli (52%) and VRSA (42%). Results are
presented in Fig. 7.
From antibacterial studies of biosynthesised AgNPs, it has been
manifested that AgNPs possessed pre-eminent antibacterial
prospective to disrupt microbial membranes, prevent biofilm
formation, obstruct replication by intercalating between bases or
proliferate reactive species formations which act as bacteriostatic
and bactericidal agents, which can assist in industrial, agricultural
and medical sphere to cease and terminate pandemics of both
Gram positive and negative bacterial infections particularly caused
by Klebsiella, E. coli, Proteus, Pseudomonas and Staphylococcus
species [16–18].
3.2.2 Antifungal activity: The AgNPs of A. americana
possessed significant antifungal activity against F. oxysporum
(89%) and V. dahliae (82%), good activity against A. niger (70%)
Fig. 10 Phytotoxic activity of AgNPs of the selected plants
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and moderate activity was observed against P. notatum (55%).
M. indica AgNPs possessed significant inhibitory effect against
A. niger (80%), moderate inhibitory effect was observed against
P. notatum (51%) and F. oxysporum (41%). The test sample was
found inactive against V. dahliae and A. parasiticus. M. spicata
AgNPs showed low activity against P. notatum (39%) and
V. dahliae (12%) while it was inactive against the other test fungi.
Results are given in Fig. 8.
From the antifungal studies of AgNPs conducted earlier, it has
been manifested that green synthesised AgNPs have the capacity
to hinder the growth of fungal strains particularly Alternaria
brassicicola, Trichoderma harizanum and F. oxysporum by
shattering their membranes and increased endocytosis leads to
ROS formation which terminate growth of fungal species by
blocking its genomic machinery and thus can be used in treatment
of fabrics mainly silk and cotton [18].
3.2.3 Antioxidant activity: All the tested AgNPs presented good
antioxidant potential at the dilutions of 200 and 300 µl with values of
73 and 68%, 79 and 73% and 64 and 62% for A. americana,
M. spicata and M. indica, respectively, as shown in Fig. 9. Earlier
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reported studies have displayed that biosynthesised AgNPs of
various plants exhibited good antioxidant activity in comparison
with the plant extract and thus they are helpful to treat many
oxidative stress related diseases [19, 20].
3.2.4 Phytotoxic activity: The synthesised AgNPs were
screened for phytotoxic activity against L. minor at various
concentrations. The results revealed that AgNPs of M. indica
showed significant phytotoxic potential (88%) at concentration of
1000 µl, moderate (50%) at 100 µl and low activity (25%) was
observed at 10 µl. A. americana AgNPs showed good (63%)
phytotoxic activity at higher concentration (1000 µl) while at lower
concentrations (100 and 10 µl) the test sample showed low
activity. The phytotoxic activity of AgNPs of M. spicata was 50,
31 and 13% at 1000, 100 and 10 µl, respectively, as shown in
Fig. 10. Results depict strong herbicidal potentials by blocking its
vegetative pathways particularly vascular bundles.
3.2.5 Haemagglutination activity: The result of
haemagglutination activity of AgNPs at all dilutions; 1:2, 1:4, 1:8
and 1:16 was negative as shown by smooth button formation. The
results revealed that the nano-particles of the selected plants
species lack phytolectins.
4 Conclusion
From the above results it may be concluded that the selected plant
species can be used for the synthesis of AgNPs. AgNPs (30–150 nm)
of A. americana, M. spicata and M. indica leaves extract possessed
remarkable antimicrobial activity and thus can be exploited in
pharmaceutical industry to formulate topical creams and ointments. It
can also be used in food and dairy industry to prevent food borne
infections and spoilage. At various dilutions, good and moderate
antioxidant activity was observed by the biosynthesised AgNPs.
Significant phytotoxic activity of the AgNPs may be a green sign for
its utilisation by industrial and agricultural sectors to manufacture
non-toxic herbicides. Finally no haemagglutination activity was
observed due to absence of phytolectins.
5 Acknowledgment
The authors are thankful to Directorate of Science and Technology
(DOST) Government of Khyber Pakhtunkhwa, Pakistan, for
financial support.
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& The Institution of Engineering and Technology 2016
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