sRI VENKATESWARA UNIVERSITY TIRUPATI

Accredited by NAAC with A+ Grade

“Production of Silver Nanoparticles using Couroupita

guianensis and their Characterization”

Project submit ed for the Academic year

2020-21 by
Ms. G.Bhavani (Regd. No: 20221062029) Mr. S.Vishnu vardhan (Regd.
No: 20221062030) Ms. P.Shahina (Regd. No: 20221062031) Ms.
S.Mounika (Regd. No: 20221062032)

Under the Guidance of

Prof. _________

DEPARTMENT OF BOTANY
SV UNIVERSITY, TIRUPATI 517 502
PRODUCTION OF SILVER NANO PARTICLES USING COUROUPITA
GUIANENSIS AND THEIR CHARACTERIZATION

ResearchArticle
Aim: Production of silver nano particles from the fruits of medicinal plant Couroupita guianensis.

Objective:To identify the efficient plant pod(fruits) for the synthesis of silver nano
particles.

Review of literature: Couroupita guianensis is known by a variety of common

names including Cannonball tree, is a deciduous tree in the flowering plant family Lecythidaceae. It is native to the
tropical forests of Central and South America.

It has beautiful, large and fragrant flowers as well as interesting fruits. Fruits
are edible and are occasionally eaten,but the smell of the white flesh discourages most
people from trying them. Fresh leaves, flowerpetals and fruitpulp were used to prepare aqueous extracts.

Keywords:Green synthesis ,silver nano particles,Couroupita guianensis, aqueous

extracts,characterization.

INTRODUCTION

Biomolecules present in plant extracts can be used to reduce metal ions to nanoparticles. The
biological reduction of metal ion to base metal is quite simple, rapid and easily conducted at room
temperature. It is an ecofriendly way of synthesis of nonoparticles. The reducing agents involved
include the various water soluble plant metabolites (e.g. alkaloids, phenolic compounds, terpenoids)
and co-enzymes. Extracts of a diverse range of plant species have been successfully used in
synthesizing silver nanoparticles.
Silver is a naturally occurring precious metal, most often as a mineral ore in association with other
elements. It has been used in a wide variety of applications as it has some special properties like high
electrical and thermal conductivity. Ancient civilizations used this precious metal in medicine, eating
utensils, plates, cups, food containers, jewellery, coins, cloths and as a disinfectant for water and
human infection1.
In recent years nanoparticles of silver have been found to exhibit interesting antibacterial activities2,3.
Presently, the investigation of this phenomenon has regained importance due to the increase of
bacterial resistance to antibiotics. Recently, silver nanoparticles exhibiting antimicrobial activity have
been synthesized by many researchers using plant extracts4, 5. Antibacterial activity of the silver
containing materials can be used, for example, in medicine to reduce infections as well as to prevent
bacteria colonization.
Couroupita guianensis Aubl. is a highly medicinal valued plant belonging to the family
Lecythidaceae. It is also known as Cannon ball tree and Ayahuma, Kailaspati, Nagalingam etc. The
flowers have acquired some religious significance in the country and are offered at Buddhist temples
and shrines. This plant is used to treat number of ailments such as, cold, intestinal gas formation and
stomach ache. The leaf has been found to show antioxidant activity, anthelmintic activity, immune
modulator and antinociceptive activity6, 7. The present study is an attempt to synthesize and
characterize the silver nanoparticles (SNPs) produced by using the leaf extract of threatened medicinal
plant Couroupita guianensis, which have been using in traditional medicine without any validation.

MATERIALS AND METHODS

Collection of Plant Materials and Extract Preparation: Couroupita guianensis is also known as
Cannon ball tree or Nagalingam in different regions. It is a highly medicinal tree species. The plant
material was collected from the coastal area of Pondicherry, India. The Couroupita guianensis trees
were identified with help of Gamble Flora7, and selected in the Botanical garden, Puducherry and in
the campus of Tagore Arts College. Fresh, green and mature leaves, flowers and fruits were harvested
during the months of July, 2012 to June, 2013. The leaves and flower petals were thoroughly washed
with distilled water and finely cut in small pieces (Fig. 1).
The cannon ball fruits were broken with help of hammer because outer cover is so hard and could not
be cut by help of anything. The fresh fruit pulp (white in color which converts into blue to brown
within minutes) was collected for the synthesis of nanoparticles. The plant extracts (broth solutions)
were prepared by using 5 gm of washed and cut leaves, flower petals and fruit pulp in a 250 ml
Erlenmeyer flask with 50 ml of sterile distilled water and then boiling the mixture for 5min. The
herbal aqueous extract was collected in separate conical flasks by standard filtration method and
stored at 4oC in a refrigerator.
 F G H
A,B,C. Mature fruits, broken fruit and fruit pulp used for biogenesis of AgNPs.

Preparation of 1mM Aqueous Solution of Silver Nitrate: 0.017gm of Silver Nitrate (AgNO3)
(Himedia, Mumbai) was added to the 100 ml of distilled water and the solution was stirred well
continuously until the silver nitrate is dissolved. This 1mM Silver Nitrate solution is stored in brown
bottle at 4oC for further use for the synthesis of Silver Nanoparticles from Couroupita leaf, flower and
fruit pulp extracts.
Methods of making Nanoparticles using Plant Extracts: Plant mediated nanoparticle synthesis
using whole plant extract or by living plant was reported in literature by several researchers8, 9.
Synthesis of AgNPs: 1mM aqueous solution of Silver nitrate (Himedia, Mumbai) was prepared for
synthesis of silver nanoparticles. For the synthesis of AgNPs, two boiling tubes were taken, one
containing 10 ml of 1mM AgNO3 solution as control and the second containing 9 ml of 1mM Silver
nitrate solution and 1 ml of plant leaf extracts as test solution. These were incubated at room
temperature for 1-2 hours. The color change of the leaf extracts from pale yellow to dark brown was
checked periodically. The brown color formation indicates that the silver nanoparticles were
synthesized from the plant extracts and they were centrifuged at 5000 rpm for 15 minutes in order to
obtain the pellet which is used for further study. Supernatant is discarded and the pellet is dissolved in
deionized water. The silver nanoparticles were confirmed by color changes and qualitatively
characterized by UV-Visible spectrophotometer.
Silver nitrate is used as reducing agent as silver has distinctive properties such as good conductivity,
catalytic and chemical stability. The aqueous silver ions when exposed to herbal extracts were reduced
in solution, there by leading to the formation of silver hydrosol. The time duration of change in color
varies from plant to plant. The pellets were used for characterization or identification of size of the Ag
Nanoparticle with the help of FTIR. The same protocol was repeated for three times with regular time
intervals.
Characterization of Silver Nanoparticles: To establish the formation of silver
nanoparticles, it is necessary to characterize them. The formation of silver nanoparticles
is characterized visually with naked eyes by a change in color to yellowish brown.
UV-Vis Spectrophotometeric analysis: AgNPs can be characterized using a UV-VIS
spectrophotometer (Systronics Double Beam Spectrophotometer, Model 2202,
Systronics Ltd.). UV VIS absorption spectra of the silver colloids were acquired by
using wave length scan between 200nm and 800nm. On an average, a plasmon peak at
410nm implies the formation of approximately 12nm silver nanoparticles. Larger
wavelength points to the formation of larger sized nanoparticles.
FTIR Spectral (Fourier Transform Infrared) analysis: Infrared spectroscopy is an
important technique in organic chemistry. It is an easy way to identify the presence of
certain functional groups in a molecule. Also, one can use the unique collection of
absorption bands to confirm the identity of a pure compound or to detect the presence of
specific impurities. To remove any free biomass residue or compound that is not the
capping ligand of the nanoparticles, the residual solution of 100 ml after reaction was
centrifuged at 5000 rpm for 10 min and the resulting suspension was redispersed in 10ml
sterile distilled water. The centrifuging and redispersing process was repeated three
times. Thereafter, the purified suspension was freeze dried to obtain dried powder.
Finally, the dried nanoparticles were analyzed by FTIR (Shimadzu,Model- IRAffinity-1,
SHIMADZU Corporation, Kyota, Japan).

RESULTS AND DISCUSSION

In the last decade, biosynthesis of metal nanoparticles is a growing need to develop

clean, nontoxic chemicals, environmentally benign solvents and renewable materials10
and hence the focus turned towards “green” chemistry and bioprocesses. Inspiration
from nature comes through yeast, fungi, bacteria and plant extracts for the control
synthesis of biocompatible metal and semiconductor nanoparticles11.
Biosynthesis of Silver Nanoparticles: Extracts from plants may act as reducing and
capping agents in silver nanoparticles synthesis. The reduction of Ag + ions by
combinations of biomolecules found in these extracts (e.g. enzymes/proteins, amino
acids, polysaccharides, vitamins etc.) is environmentally benign, yet chemically
complex12. The extract of lower plants (algae, fungi etc.) was also used to synthesize
AgNPs at room temperature. Proteins in the extract provide dual function of Ag +
reduction and shape control in the nanoparticle synthesis. The carboxyl groups in
aspartic and/or glutamine residues and the hydroxyl groups in tyrosine residues of the
proteins were suggested to be responsible for the Ag + ion reduction13.
The green synthesis of silver nanoparticles using leaf, flower petals and fruit pulp
extracts of Couroupita guianensis was carried out in present investigation. The color
was changed in the cell free leaf extract when challenged with 1mM AgNO 3 from pale
yellow to dark brown (Fig. 2A,B & C) within 15 min and attained maximum intensity
after 12 hrs with intensity increasing during the period of incubation indicative of the
 formation of silver nanoparticle. The reaction was completed early (within 30 min) in
case of flower petals extract when subjected to 1mM AgNO 3 and it did not attain much
color changes with time duration as compared to leaf extract (Fig. 2D,E & F). In case of
cell free extract of fruit pulp the color change was not that much significant as
compared to leaf and flower petal extracts (Fig. 2G, H & I). Control (without silver
ions) showed no change in color of the cell filtrates when incubated under same
conditions. Sadowski et al.,14 exploited the fungus, Penicillium sp. for the extracellular
synthesis of silver nanoparticles the fungal cell filtrate was treated in the dark with Ag +
ions for the biosynthesis process. The reaction mixture showed color change from
colorless to brown which intensified with the increase in incubation period.

GH I

Fig. 2: D, E & . Changes in the color of the mixture with time when fruit pulp extract mixed with
AgNO3 solution.

Characterization of AgNPs: It is well known that silver nanoparticles exhibit dark brown color in
aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles15. The
appearances of yellowish brown color in the reaction vessels suggest the formation of silver
nanoparticles (SNPs)16. When the cell free extracts were mixed with 1mM AgNO3 the color was
changed from pale yellow to dark brown in 15 min and attained maximum intensity after 12 hrs with
intensity increasing during the period of incubation indicative of the formation of silver nanoparticle.
Shekhawat et al.,4 reported biogenesis of silver nanoparticles with help of leaf extract of Turnera
ulmifolia. The color change reaction was completed within 12 hrs in this study.
UV-Vis spectral analysis: The synthesis of AgNPs had been confirmed by measuring the UV-Vis
spectrum of the reaction media. The UV-Vis spectrum of colloidal solutions of AgNPs synthesized
from Couroupita guianensis leaf extract have absorbance peaks at 415-425 nm regions (Fig. 3), which
are identical to the characteristics UV-visible spectrum of metallic silver. The weak absorption peak at
shorter wave lengths was due to the presence of several organic compounds which were known to
interact with silver ions. Again, the time duration of change in color and reaction completion is varies
from plant to plant. Boswellia ovalifoliolata synthesized silver nanopartcles within 10 min whereas
Shorea tumbuggaia and Svensonia hyderobadensis took 15 min to synthesize nanoparticles17.
The UV-visible spectra of the control samples depicted absorption bands at 260-270nm corresponding
to proteins, α-NADPH and hydroxyquinoline. The spectra of reaction mixture showed strong surface
plasmon resonance at 413nm which intensified with time while, the absence of absorption band at
413nm for the reaction mixture in the absence of enzyme clearly depicted that the reduction of silver
involves enzymatic reduction of nitrate to nitrite. The first two spectral graphs did not show any
AgNPs presence but with time duration (After 11 hrs) a very fine peak was observed in this
experiment.

AB

C
D
 Fig. 5: A, B & C. UV-Visible Spectra of AgNPs synthesized using fruit pulp extract after every 1hr;
D. A combined UV-Visible Spectra of AgNPs synthesized using fruit pulp extract after every 1hr.

FTIR analysis: A hypothetical mechanism for the synthesis of silver nanoparticles was corroborated
according to the FTIR study of silver nanoparticles. The FTIR spectra of silver nanoparticles depicted
intense peak at 1119 cm-1 corresponding to stretching vibrations of Ag-N bonds and two broad bands
at 1405 and 1642 cm-1 attributed to symmetric and asymmetric C=O stretching vibration of CO2.
Selective enhancement of these Raman bands indicated that C=O bonds and Ag-N bonds lie
perpendicular to the nanosilver surface and gets associated with the formation of a cap over
nanoparticles. Also, the symmetric and asymmetric stretching bonds of CO2 significantly broaden due
to distortion of the respective bond angles and bond lengths which further support in the encapsulation
of silver nanoparticles. The band at 240 cm-1 confirmed the formation of a chemical bond between
silver nanoparticles and the nitrogen of amino groups19.
FTIR analysis was used for the characterization of the extract and the resulting nanoparticles. FTIR
absorption spectra of water soluble extract after reduction of Ag ions are shown in Fig. 6. Absorbance
bands in the figure are observed in the region of 500–4000 cm-1 are 1119, 1364, 1405, 1642 1734cm-1.
These absorbance bands are known to be associated with the stretching vibrations for –C C–C O, –C
C– [(in-ring) aromatic], –C–C– [(in-ring) aromatic], C–O (esters, ethers) and C–O (polyols),
respectively19. In particular, the 1119 cm-1 band arises most probably from the C–O group of polyols
such as hydroxyflavones and catechins. The nanoparticles synthesized using Couroupita guianensis
leaf extract was only investigated for FTIR study in present investigation. Ramesh et al.,18
investigated on reduction of palladium ions by soybean (Glycine max) leaf extract and which was
examined by UV-visible spectroscopic technique. They concluded that the proteins and some of the
amino acids that are exist in soybean leaf extracts were actively involved in the reduction of
palladium ions. Further it was confirmed by Fourier Transformations Infrared Spectroscopic analysis.
A phytoapthogen F. solani (USM-3799) was harnessed for the extracellular biosynthesis of silver
nanoparticles by Ingle et al.,20. The biosynthesized nanoparticles were characterized using FTIR. The
FTIR spectra of the silver nanoparticles depicted presence of functional groups like C-N, C-O-C,
amide linkages and –COO-. These functional were found to play an important role in the capping of
nanoparticles and their further stability in aqueous solution.

CONCLUSION

In conclusion, the bio-reduction of aqueous Ag+ ions by the leaf, flower petal and fruit pulp extracts of
the Couroupita guianensis plant has been demonstrated. In the present study we found that fruits can
also be a good source for synthesis of silver nanoparticles. This green chemistry approach toward the
synthesis of silver nanoparticles has many advantages such as, ease with which the process can be
scaled up, economic viability, etc. Applications of such eco-friendly nanoparticles in bactericidal,
wound healing and other medical and electronic applications, makes this method potentially exciting
for the large-scale synthesis of other inorganic materials (nanomaterials). There is no report describing
synthesis of silver nanoparticles using Couroupita guianensis plant parts, it is first time reported
during present investigation that to by use of flower petals and fruit extract.
 REFERENCES
1. G. Nordberg, L.S. Gerhardsson; Silver. In: H.G. Seiler, H. Sigel, A. Sigel (Eds); Handbook on
Toxicity of Inorganic compounds, Marcell Dekker; New York, 1988, 619-624.
2. A.R. Shahverdi, A. Fakhimi, H.R. Shahverdi, S. Minaian; Nanomed. Nanotech. Biol. Med; 2007, 3
(2), 168-171.
3. S. Pal, Y. Kyung, J. Myong Song; Appl. Env. Microbiol; 2007, 73 (6), 1712-1720.
4. M.S. Shekhawat, N. Kannan, M. Manokari; J. Ecobiotech; 2012, 4 (1), 54-57
5. A. Panacek, L. Kvítek, R. Prucek, M. Kolar, R. Vecerova, N. Pizúrova, V.K. Sharma, T. Nevecna,
R. Zboril; J. Phys. Chem. B; 2006, 110 (33), 16248-53.
6. D. Pradhan, P.K. Panda, G. Tripathy; Nat. Prod. Rad; 2009, 8 (1), 37-42.
7. J.S. Gamble; Botanical Survey of India; Calcutta, India, 1957.
8. J.L. Gardea-Torresdey, E. Gomez, J. Peralta Videa, J.G. Parsons, H.E. Troiani, Jose Yacaman;
Langmuir; 2003, 13, 1357.
9. Y. Park, Y.N. Hong, A. Weyers, Y.S. Kim, R.J. Linhardt; IET Nanobiotechnol; 2011, 5, 69–78. 10.
P. Raveendran, J. Fu, S.L. Wallen, Completely "green" synthesis and stabilization of metal
nanoparticles. J. Am. Chem. Soc; 2003, 125, 13940-13941.
11. S. Shiv Shankar, A. Ahmad, R. Pasricha, M.J. Sastry; Mater. Chem; 2003, 13, 1822 . 12. O.
Collera-Zuniga, F.J. Jimenez, R.M. Gordillo; Food Chem; 2005, 90, 109-114. 13. J. Xie, J.Y. Lee,
D.I.C. Wang, Y.P. Ting; A. C. S. Nano; 2007, 1, 429-439. 14. Z. Sadowski, G.B. Maliszewska, I.
Polowczyk, T. Kozlecki; Mat. Sc. Poland; 2008, 26 (2), 419- 425.
15. A. Thirumurgan, N.A. Tomy, R. Jai Ganesh, S. Gobikrishnan; De. Phar. Chem; 2010, 2, 279-284.
16. S.S. Shankar, A. Rai, A. Ahmad, M. Sastry; Chem. Mater; 2005, 17, 566-572. 17. N.
Savithramma, M.L. Rao, K. Rukmini, P.S. Devi; Int. J. ChemTech Res; 2011, 3(3), 1394- 1402.
18. P. K. Ramesh, V. Singaravelu, M. Misra, A.K. Mohanty, N. Satyanarayana; J. Biomat.   Nanobiotech;
2012, 3, 14‐19.
19. H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra; Physico. Eng. Aspects; 2009, 339,
134–139.
20. A. Ingle, M.K. Rai, A. Gade, M. Bawaskar; J. Nanopart. Res; 2009, Doi 101007/s11051-008-
9573-y.