Rautela et al.

Journal of Analytical Science and Technology (2019) 10:5

https://doi.org/10.1186/s40543-018-0163-z
Journal of Analytical Science
and Technology

RESEARCH ARTICLE Open Access

Green synthesis of silver nanoparticles from

Tectona grandis seeds extract:
characterization and mechanism of
antimicrobial action on different
microorganisms
Akhil Rautela*, Jyoti Rani and Mira Debnath (Das)

Abstract
Green synthesis of silver nanoparticles makes use of plant constituents, like carbohydrates, fats, enzymes, flavonoids,
terpenoids, polyphenols, and alkaloids, as reducing agents to synthesize silver nanoparticles. The present study for
the first time utilized seed extract of Tectona grandis (teak) for reduction of 1 mM silver nitrate solution to silver
nanoparticles. The method proved to be very simple, cost-efficient, and convenient. Synthesis of nanoparticles was
confirmed by visual detection in which the colorless solution gets changed to a brown-colored solution. Further
characterization was done by UV-visible spectroscopy, XRD, FTIR analysis, SEM/EDS, FESEM, and TEM. Size of silver
nanoparticles was found to be 10–30 nm approximately as determined by transmission electron microscopy (TEM).
Energy-dispersive spectra (EDS) revealed that nanoparticles contain silver in its pure form. Well diffusion method
showed the antimicrobial effect of AgNPs on different microorganisms with the zone of inhibition of 16 mm for
Staphylococcus aureus, 12 mm for Bacillus cereus, and 17 mm for E. coli when 50 μg of AgNPs was used. Minimum
inhibitory concentration was found to be 5.2, 2.6, and 2.0 μg/ml for Bacillus cereus, Staphylococcus aureus, and E.
coli respectively. Mode of action of antimicrobial activity of nanoparticles was investigated by determining leakage
of reducing sugars and proteins, suggesting that AgNPs were able to destroy membrane permeability.
Keywords: Silver nanoparticles, Green synthesis, Characterization, Antimicrobial activity

Introduction and pesticides (Asthana et al. 2016; Das et al. 2012), sens-
Nanotechnology is the process of synthesizing particles ing of food adulterants (Ping et al. 2012), detection of
which are in the nano range, ranging from approxi- DNA (Thompson et al. 2008), etc.
mately 1 to 100 nm. They have large surface area to vol- Chemical reduction which is the most commonly used
ume ratio due to which they possess optical properties method for synthesis of nanoparticles uses an organic
as they are small enough to confine their electrons and solvent, like ethylene glycol (Wiley et al. 2004), and
produce quantum effects by which their detection be- reducing agents, like hydrazine (Guzmán et al. 2009),
comes easy. Intensive research is being done on silver sodium borohydride (Song et al. 2009), trisodium citrate
nanoparticles (AgNPs) owing to their wide range of ap- (Rashid et al. 2013), and ascorbate (Qin et al. 2010). There
plications in medical devices (He et al. 2013), pharmaceu- was an earnest need for the development of cleaner and
ticals (Kumar et al. 2011), clothing (Vigneshwaran et al. safer methods as chemical reduction gives low yield, re-
2007a), and water purification (Lin et al. 2013) due to their quires complicated purification and high energy. This gave
antimicrobial properties and also in adsorption of metals rise to green synthesis which utilizes microorganisms
(bacteria (Natarajan et al. 2010), fungi (Vigneshwaran
* Correspondence: akhilr.bce15@iitbhu.ac.in et al. 2007b), yeast (Kowshik et al. 2002), actinomycetes
School of Biochemical Engineering, Indian Institute of Technology (BHU),
Varanasi, India (Sastry et al. 2003)) and plant extracts for the reduction

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 2 of 10

of silver to silver nanoparticles. Synthesis of nanoparticles anemia and shows anti-hemolytic anemia activity (Diallo
from microbes can be extracellular (Kowshik et al. 2002) et al. 2008). Its seeds are acclaimed to be hair tonic, which
or intracellular (Mukherjee et al. 2001). The major disad- is reported to increase the number of hair follicles in the
vantage of the use of microbial source is the maintenance anagenic phase (Jaybhaye et al. 2010). Its leaves, bark, and
of aseptic conditions, high cost of isolation, and their wood show antioxidant properties, with wood showing
maintenance in culture media due to which plants the highest (98.6%) inhibition against DPPH (Krishna
promise to be excellent sources for reducing agents for and Jayakumaran 2010).
the synthesis of nanoparticles. Parts of plants used for In the present study, Tectona grandis seed extract
synthesis of AgNPs are leaf (Prakash et al. 2013), bark was utilized for the reduction of silver nitrate to silver
(Sathishkumar et al. 2009), seeds (Bar et al. 2009), roots nanoparticles, and characterization of the synthesized
(Suman et al. 2013), etc. There are numerous examples of nanoparticles was carried out by UV-visible spectros-
AgNPs synthesis from diverse plant sources, like Chrysan- copy, scanning electron microscope (SEM), energy-
themum morifolium (He et al. 2013), Cassia auriculata dispersive X-ray spectroscopy (EDX), field emission
(Kumar et al. 2011), Mimusops elengi (Prakash et al. scanning electron microscope (FESEM), transmission
2013), Cinnamon zeylanicum (Sathishkumar et al. electron microscope (TEM), X-ray diffraction (XRD), and
2009), Jatropha curcas (Bar et al. 2009), and Morinda Fourier transform infrared spectroscopy (FT-IR) analysis.
citrifolia (Suman et al. 2013). Constituents of plants, Antimicrobial activity of the synthesized nanoparticles
like carbohydrates, fats, enzymes, flavonoids, terpe- was investigated against representative human patho-
noids, polyphenols, and alkaloids, are capable of redu- genic microorganisms (Bacillus cereus, Staphylococcus
cing silver to nanoparticles. aureus, and Escherichia coli). Minimum inhibitory con-
Antimicrobial activity of silver nanoparticles has been centration of silver nanoparticles against these microor-
reported in many research papers. The mechanism of ganisms was determined. Besides, the mechanism of
antibacterial action of silver nanoparticles is a topic of action of antimicrobial activity was understood by de-
debate and is not well understood. But many assump- tecting leakage of reducing sugars and proteins through
tions and theories are there. In one of the study on E. coli DNS and Bradford’s method, which indicated that kill-
and Staphylococcus aureus, it was seen that silver ions get ing of microorganisms was through the destruction of
released by nanoparticles and accumulate around the cell membranous structure and permeability.
wall or inside the cell and affect DNA replication and
interact with thiol groups of protein, inducing protein in- Materials and methods
activation (Feng et al. 2000). This also leads to the forma- Materials
tion of reactive oxygen species (Matsumura et al. 2003). Tectona grandis seeds were collected from BHU campus.
Tectona grandis, commonly called as timber, teak, Sagun, Before washing, the outer covering of the harvested seeds
has potential medicinal value. It is widely used for making was removed. Seeds were dried at room temperature for
furniture, cabinets, and musical instruments (Indira and 3–4 days so that moisture gets removed completely. Dried
Mohanadas 2002). Its bark has an antibacterial compound seeds were crushed to fine powder and stored in dry and
5-hydroxy-1,4-naphthalenedione (Juglone) which shows airtight container for further use. Silver nitrate (AgNO3)
antibacterial activity against Listeria monocytogenes and was purchased from SRL, India. All other reagents were of
methicillin-resistant Staphylococcus aureus (MRSA) (Nea- analytical grade and used as received. All the solutions
matallah et al. 2005). It is also used for the treatment of were freshly prepared with double distilled water for the

Fig. 1 Visual detection [(a) 0 min. (b) 30 min. (c) 1 h]

Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 3 of 10

Fig. 4 FESEM image of AgNPs

Fig. 2 Absorption spectra of silver nanoparticles obtained at different of double distilled water. The mixture in the flask was
time intervals heated in a water bath for 15–20 min at 80 °C. After
boiling, the extract was filtered using a muslin cloth to
remove any coarse material. Volume was made up to
experimental procedure and were kept in the dark to 100 ml using double distilled water. The prepared ex-
avoid any photochemical reaction. tract was stored at 4 °C and used within 1 week.

Microorganisms
Antimicrobial activity of silver nanoparticles was investi- Synthesis of silver nanoparticles using seed extract
gated against Gram-positive bacteria (Bacillus cereus; 1 mM silver nitrate solution in double distilled water
MTCC 9817 and Staphylococcus aureus; MTCC 7443) was the source of silver. Silver nitrate and seed extract
and Gram-negative bacteria (Escherichia coli; MTCC 443). were mixed together in a ratio of 1:9. The reaction mixture
Nutrient agar containing peptone, beef extract, and NaCl was heated below the boiling point and continuously
was used to maintain bacterial strains. Prior to the experi- stirred at 800 rpm using magnetic stirrer. The mixture
ment, bacterial strains were inoculated in nutrient broth turned reddish brown in color within 1 h. The whole
to encourage the growth of microorganisms in the expo- reaction was carried out in the dark. The obtained sus-
nential phase. pension of Ag/T. grandis was centrifuged at 15,000 rpm
for 45 min. The pellet containing silver nanoparticles was
Tectona grandis seed extract preparation washed 3–4 times with deionized water to remove silver
Five grams of seed powder was carefully weighed and ions and seed extract residue. The precipitated nano-
added in an Erlenmeyer flask of 250 ml containing 50 ml particles were lyophilized. Lyophilized nanoparticles were
stored in a cool, dry, and dark place and further their
characterization was carried out.

Fig. 3 SEM image of AgNPs Fig. 5 FESEM mapping

Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 4 of 10

Fig. 6 EDX analysis

Characterization of synthesized nanoparticles sonicated for a sufficient amount of time, the smear
UV-1800 Shimadzu spectrophotometer was used to ob- was made on a platinum grid, and allowed to dry over-
tain UV-visible spectra of AgNPs at different time in- night under vacuum. The grid was then coated with a
tervals using seed extract diluted with water (1:9) as a thin film of palladium and finally subjected to FESEM.
blank. Further shape and size of formed nanoparticles Transmission electron microscopy was done to exactly
were determined by SEM, FESEM equipped with EDS, determine the size of nanoparticles. Sonicated sample
and TEM. A lyophilized sample of AgNPs was sub- was loaded on a carbon-coated copper grid and was
jected to Zeiss EVO-18 scanning electron microscope allowed to dry overnight in a vacuum and subjected to
at 20 kV to study the morphological features of silver transmission electron microscopy (FEI-TECNAI G220
nanoparticles. Further, the shape, morphology, and TWIN). The crystalline nature of AgNPs was confirmed
elemental mapping of AgNPs were studied using field by XRD pattern obtained from Rigaku-MiniFlex 600 X-ray
emission scanning electron microscopy (NOVA Nano- diffractometer at 2θ range from 0 to 100°. The sample for
SEM 450). For this purpose, the lyophilized sample was XRD measurement was prepared by casting the powder of
silver nanoparticles on a glass slide and subsequently air-
drying it under ambient conditions. The pattern was re-
corded by CuKα radiation with λ of 1.5406 Å at a voltage
of 40 kV and current of 15 mA with a scan rate of 10°/
min. Presence of functional groups present in AgNPs was
determined by using FTIR (ALPHA BRUKER Eco-ATR).

Fig. 7 TEM image of AgNPs (inset shows the SAED pattern of

nanocrystalline silver) Fig. 8 X-ray diffractogram of AgNPs
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 5 of 10

Determination of minimum inhibitory concentration (MIC)

Minimum inhibitory concentration is defined as the
minimum concentration of the material that inhibits the
growth of the particular microorganisms. The method is
based on growing microorganisms at varying concentra-
tions of AgNPs in suspension. Sterile test tubes, contain-
ing 5 ml of Luria-Bertani broth were taken. In the test
tubes, varying concentrations of AgNPs were added and
one test tube was taken as control. Then, the test tubes
including the control were inoculated with an equal
volume (200 μl) of freshly prepared bacterial suspension
diluted to an optical density of 0.5. The inoculated test
tubes were incubated in a shaker incubator at 250 rpm and
37 °C for 24 h. The next day, absorbance was taken using
UV-visible spectrophotometer at 600 nm, and the graph
Fig. 9 FTIR spectra of AgNPs was plotted against optical density and the concentration
of AgNPs. The concentration giving the least optical dens-
ity corresponds to MIC of silver nanoparticles for that par-
Four per centimeter resolution was taken in all spectra ticular microorganism. The above method is repeated with
with 500–4000 cm−1 IR range. all the three microorganisms, namely E. coli, Bacillus ce-
reus, and Staphylococcus aureus.

Antibacterial assay Determination of the effect of AgNPs on leakage of

Well diffusion method is commonly used to check the membrane
antimicrobial activity of the nanoparticles. The anti- In this leakage of reducing sugars and proteins through
microbial activity of synthesized AgNPs was checked the membrane was determined. For this, overnight grown
using this method. Three microorganisms, namely E. cultures of E. coli, Bacillus cereus, and Staphylococcus aur-
coli, Bacillus cereus, and Staphylococcus aureus were eus were taken, and 2 ml sample was withdrawn from each
used for this purpose. 1 mg/ml solution of AgNPs was culture and was marked as 0 h sample. One milliliter of
made in Milli Q water and was sonicated properly. AgNPs solution (1 mg/ml) was added to each culture and
Overnight grown culture in Luria-Bertani broth of the was incubated at 200 rpm and 37 °C. Now, the sample was
mentioned microorganisms was taken and diluted to withdrawn after 2 h, 4 h, and 6 h from each culture. All
an optical density of 1. Diluted bacterial suspension the samples were centrifuged at 10,000 rpm for 5 min. Pel-
(500 μl) was spread uniformly on three different let was discarded and the supernatant was preserved at −
Luria-Bertani agar plates for three different microorgan- 30 °C immediately, and the concentration of reducing
isms. After spreading, two wells were cut on each plate sugar and proteins were determined by DNS and Brad-
using well borer of approximately 10 mm diameter. One ford’s method, respectively, as soon as possible.
well was filled with 50 μg and the other with 100 μg of
AgNPs solution in all the three plates. The plates were Results and discussion
incubated at 37 °C for 24 h, and the zone of inhibition Nanoparticles synthesis initiates once the T. grandis seeds
was measured. extract was introduced into 1 mM AgNO3 solution. The

Fig. 10 Zone of inhibition of AgNPs against [(a) B. cereus, (b) S. aureus, and (c) E. coli]
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 6 of 10

Table 1 Antimicrobial activity of AgNPs

Diameter of zone of inhibition (mm)
Concentration of AgNPs B. cereus S. aureus E. coli
50 μg 12 16 17
100 μg 14 17 20

gradual color change of AgNO3/T. grandis solution from FESEM (Fig. 4) clearly shows the presence of synthe-
colorless to yellow and finally to reddish brown indicates sized nanoparticles. The nanoparticles were oval, spher-
the formation of silver nanoparticles as shown in Fig. 1. ical in shape. Most of the nanoparticles were aggregated,
This color change is due to the surface plasmon vibration, and few individual particles were also observed (Suman
an optical property which is unique to the noble metals et al. 2013).
(Ibrahim 2015). The formation of AgNPs was further Elemental mapping of AgNPs by FESEM-EDX shows
confirmed by using UV-visible spectroscopy, scanning the presence of 94% of Ag and 6% of oxides as shown in
electron microscopy, field emission scanning electron Fig. 5. Elemental analysis of AgNPs was confirmed by
microscopy, energy-dispersive X-ray spectroscopy, trans- EDX as shown in Fig. 6. A strong signal of the peak was
mission electron microscopy, X-ray diffraction, and Fou- observed at 3 KeV which is typical for absorption of me-
rier transform infrared spectroscopy. tallic silver nanoparticles. The absence of other elements
Formation of the nanoparticles in the aqueous solution confirms the purity of prepared nanoparticles.
was further confirmed by the UV-visible spectroscopy. Further, an insight into the morphology and size de-
The wavelength scale was fixed between 300 and 600 nm, tails of AgNPs was provided by transmission electron
and the solution was scanned in this range. Maximum ab- microscopy. TEM image as shown in Fig. 7 clearly demon-
sorbance at 440 nm was observed, which is characteristic strates that the AgNPs were spherical in shape. The image
of silver nanoparticles (Bahuguna et al. 2016). The curve shows agglomerates of small grains and some dispersed
(Fig. 2) shows an increase in absorbance with the increase nanoparticles, confirming the results obtained by SEM
in incubation time (30 min, 45 min, and 1 h) of silver ni- and FESEM (Guzmán et al. 2009). The synthesized AgNPs
trate and seed extract. were in the range of 10–30 nm. The selected area diffrac-
Scanning electron microscopy images of the lyophi- tion pattern (Fig. 7, inset) confirms the face-centered
lized silver nanoparticles showed mostly spherical parti- cubic (fcc) crystalline structure of metallic silver.
cles of a size below 100 nm as shown in Fig. 3. The image Figure 8 shows the XRD pattern of silver nanoparticles
was a blurred one as the SEM present at the institute was which confirmed the crystalline nature of AgNPs. The
not able to take images of particles below 100 nm. There- four distinct diffraction peaks at 2θ values of 38.05 θ,
fore, FESEM of the nanoparticles was done. 44.23 θ, 64.41 θ, and 76.66 θ can be indexed to the (1 1

a b

Fig. 11 Broth dilution assay to detect MIC against [(a) B. cereus, (b) S. aureus, and (c) E. coli]
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 7 of 10

a b

Fig. 12 Average bacterial growth after 24 h with varying concentrations of AgNPs. [(a) B. cereus, (b) S. aureus, and (c) E. coli]

a b

Fig. 13 Leakage of reducing sugar from (a) B. cereus, (b) S. aureus, and (c) E. coli cells treated with AgNPs
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 8 of 10

a b

Fig. 14 Leakage of proteins from (a) B. cereus, (b) S. aureus, and (c) E. coli cells treated with AgNPs

1), (2 0 0), (2 2 0), and (3 1 1) reflection planes of face- around the well was due to the release of diffusible inhibi-
centered cubic structure of silver. In addition to the Bragg tory compounds, i.e., silver nanoparticles.
peaks representative of silver nanocrystals, additional peaks From Table 1, it is evident that Gram-negative bacteria
were also observed at 27.77 θ, 32.18 θ, 46.17 θ, and (E. coli) show a higher zone of inhibition, in comparison
54.752 θ. Presence of these peaks was due to seed ex- to Gram-positive bacteria (B. cereus and S. aureus) at
tract which contains organic compounds and is respon- the same concentration of AgNPs. This difference could
sible for the reduction of silver ions and stabilization of be explained by variation in the composition of the cell
resultant nanoparticles (Ibrahim 2015). wall of Gram-positive and Gram-negative bacteria. In
FTIR measurements were carried out to identify the Gram-positive bacteria, the cell wall is constituted of a
major functional groups in the seed extract and their thick peptidoglycan layer, consisting of short peptide
possible involvement in the synthesis and stabilization of cross-linked linear polysaccharide chains. This leads to a
silver nanoparticles. The spectrum of seed powder and more rigid structure, increasing difficulties in penetra-
synthesized AgNPs is represented in Fig. 9. Seed extract tion of the silver nanoparticles. On the other hand, the
showed several peaks indicating the complex nature of Gram-negative bacteria’s cell wall is composed of a thin-
the biological material. The bands appearing at 1745, ner peptidoglycan layer (Shrivastava et al. 2007).
1643, 1508, and 1038 cm−1 were assigned to stretching After confirmation of antimicrobial activity of synthe-
vibration of C=O bond of carboxylic acid or ester, N– sized AgNPs through well diffusion assay, minimum inhibi-
C=O amide bond of proteins, nitro compounds, C–N tory concentration (MIC) of AgNPs against B. cereus, S.
amine bond, respectively. There was a shift in the peaks aureus, and E. coli was determined. Broth dilution method
in synthesized silver nanoparticles which suggests that is the most commonly used technique to determine the
functional groups of seed extract participate in the for- MIC of antimicrobial agents against different microor-
mation of AgNPs. ganisms (Fig. 11). After 24 h of incubation, no growth
The synthesized AgNPs were investigated for antimicro- of B. cereus, S. aureus, and E. coli was seen in the test
bial activity by using well diffusion method. Growth inhib- tubes supplemented with 5.2, 2.6, and 2.0 μg/ml of silver
ition was observed after 24 h on plates loaded with 50 and nanoparticles, and the optical density was 0.016, 0.016,
100 μg of AgNPs (Fig. 10). Bacterial growth suppression and 0.02, respectively (Fig. 12).
Rautela et al. Journal of Analytical Science and Technology (2019) 10:5 Page 9 of 10

MIC results are in relation to the fact that a larger Availability of data and materials
zone of inhibition corresponds to smaller minimum in- Data sharing is not applicable. It will be shared if needed.

hibitory concentration (Mohanty et al. 2010). Declaration

The action of the mechanism of silver nanoparticles on This submitted work has not been published previously (except in the form
microorganisms is still a topic of debate. Figures 13 and of an abstract, and an academic thesis), and is not under consideration for
publication elsewhere, it’s publication is approved by all authors and tacitly
14 reveal that AgNPs could enhance the permeability of or explicitly by the responsible authorities where the work was carried out,
the membrane, and thus leakage of reducing sugars and and if accepted, it will not be published elsewhere in the same form, in
proteins. There was an increase in the release of sugar and English or in any other language, including electronically without the
written consent of the copyright holder. The authors declare that they
proteins in all the microorganisms as the incubation time have no competing interests.
increases. At 0 h, in control and treated samples, amount
of sugar and proteins were almost the same, as there was Authors’ contributions
AR carried out all the experiments mentioned in the manuscript as this was
no time of contact between AgNPs and microorganisms. his M.Tech dissertation work. JR helped to draft the manuscript. MD is the
But at 2, 4, and 6 h amount of sugar and proteins were Senior Professor and was the supervisor of the dissertation work. All authors
higher as compared with control in all the cases, which read and approved the final manuscript.

suggests that AgNPs may have expedited leakage of sugar Competing interests
and proteins from the cytoplasm of microorganisms. The authors declare that they have no competing interests.
The maximum amount of sugars and proteins is re-
leased by E. coli (112.86 and 12.9 μg/ml, respectively) Publisher’s Note
when treated with AgNPs. Whereas, the least amount is Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
released by B. cereus.
Received: 13 August 2018 Accepted: 28 December 2018
Conclusion
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