MRS Communications (2017), 1 of 6

© Materials Research Society, 2017

doi:10.1557/mrc.2017.18

Research Letter

Green synthesis and antibacterial activity of hydroxyapatite nanorods for

orthopedic applications

Govindan Suresh Kumar, and Senkotuvel Rajendran, Department of Physics, K.S. Rangasamy College of Arts and Science (Autonomous),
Tiruchengode 637 215, Tamil Nadu, India
Sekar Karthi, Raji Govindan, and Easwaradas Kreedapathy Girija, Department of Physics, Periyar University, Salem 636 011, Tamil Nadu,
India
Gopalu Karunakaran, Department of Functional Nanosystems and High-Temperature Materials, National University of Science and Technology “MISiS,”
Leninskiy Pr. 4, Moscow 119049, Russia; Department of Biotechnology, K.S. Rangasamy College of Arts and Science (Autonomous), Tiruchengode 637 215,
Tamil Nadu, India
Denis Kuznetsov, Department of Functional Nanosystems and High-Temperature Materials, National University of Science and Technology “MISiS,”
Leninskiy Pr. 4, Moscow 119049, Russia
Address all correspondence to Govindan Suresh Kumar at g.sureshkumar@ksrcas.edu

(Received 16 November 2016; accepted 15 March 2017)

Abstract
Biomaterials with antibacterial activity are widely developed for the treatment of bone infection. In the present study, hydroxyapatite (HAp)
nanorods were prepared by green synthesis using Azadirachta indica and Coccinia grandis leaf extract. The prepared samples were charac-
terized by various characterization techniques and the results indicate that the prepared samples are constituted of phase pure polycrystalline
HAp having hexagonal crystal structure. Moreover, antibacterial activity test confirm that the HAp prepared using leaf extract as a solvent hav-
ing significant antibacterial activity against Escherichia coli and Staphylococcus aureus. Hence, green synthesis can be a prospective way to
develop orthopedic biomaterials with antibacterial properties.

Introduction Azadirachta indica is commonly available plant in India and

The recent trend in biomaterials research is focused towards the
nanotechnology, which offers a distinctive approach to over-
come the shortcomings of bulk materials due to their high sur-
face area and quantum confinement effects.[1,2] Nanocrystalline
hydroxyapatite (HAp) is well-known biomaterial owing to its
excellent bioactivity, biocompatibility, osteoconductivity, and
similarity to the inorganic constituent of calcified tissues.
Hence, it is widely used for several biomedical applications
such as fillers for bone defects, scaffold for tissue engineering
and coating on metallic implants to improve the biocompatibil-
ity, and carrier for drug/protein delivery.[1,2] One of the most
important problems in orthopedics is a bone infection caused
by infective micro-organisms.[3] Hence, HAp in combination
with antibiotics is very useful for the development of biomate-
rial for filling bone defects.[4–6] Moreover, HAp substituted
with silver ions has been developed to address this problem.[7]
On the other hand, green synthesis of nanoparticles using
plant leaf extracts has opened a new era in research and many
researchers have reported the green synthesis of metal nanopar-
ticles using plant leaf extracts and its antibacterial activity.[8–11]
However, green synthesis of HAp nanoparticles using plant leaf
extract is rarely reported.[12] Consequently synthesis of HAp
nanoparticles with antibacterial properties using different plant
extracts is important from the viewpoint of materials processing.
each part of this tree has been used from ancient times as a
homemade remedy against various viral, bacterial, and fungal
infections.[9,10] Similarly, Coccinia grandis is abundantly
available plant in India and it is also well known for antibacte-
rial, antidiabetic, anti-inflammatory, antipyretic, analgesic, and
expectorant activities.[11,13] To the best of our knowledge, there
are no reports in the scientific literature on the green synthesis
of HAp nanorods using A. indica and C. grandis leaf extracts.
Hence, the current study is aimed to synthesize HAp nanorods
using A. indica and C. grandis leaf extract as a solvent and to
evaluate its antibacterial activity.
Experimental procedure
Green synthesis of HAp nanorods
Chemicals such as calcium chloride (CaCl2), di-sodium hydro-
gen phosphate (Na2HPO4), and sodium hydroxide (NaOH)
were obtained from Merck, Mumbai, India. A. indica and
C. grandis leafs (Fig. 1) were collected and they were cleaned
with double distilled water and air dried at room temperature.
About 50 g of finely cut leaves was kept in two beakers each
containing 250 mL double distilled water and was boiled for
2 h separately. After 2 h the extracts were cooled down, filtered,
and stored at 4 °C for further use. For the synthesis of HAp
nanorods using leaf extract, 1 M CaCl2 and 0.6 M Na2HPO4

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 Figure 1. (a) Azadirachta indica and (b) Coccinia grandis leafs.
were prepared using leaf extract as the solvent and separately
brought to pH above 10 using 0.8 M NaOH solution. Then
CaCl2 solution was stirred vigorously using a magnetic stirrer
at room temperature, and then Na2HPO4 solution was added
drop wise into it to produce a gelatinous precipitate. The precip-
itation of HAp can be described as follows:
10CaCl2 + 6Na2 HPO4 + 8NaOH Ca10 (PO4 )6 (OH)2
+ 20NaCl + 6H2 O.
The reaction mixture was then stirred for 1 h and kept at room
temperature for 24 h. The obtained precipitate was centrifuged
to remove byproduct and then dried at 150 °C for 6 h in hot air
oven. Finally, the dried cakes were crushed to obtain powders.
HAp synthesized using A. indica and C. grandis leaf extract
were named as NHA and GHA, respectively. Moreover, HAp
without leaf extract was prepared and named as control (pure
HAp) for comparison.
Characterization
X-ray diffraction (XRD) patterns were obtained (Rigaku
MiniFlex II powder X-ray diffractometer) using Cu Kα mono-
chromatic radiation (1.5406 Å) as a source in the range between
20° ≤ 2θ ≤ 60°. The lattice parameters (a and c) of HAp were
calculated by the method of least squares using the following
equation:
1 4(h2 + hk + k 2 ) l 2
= + 2,
d 2 3a2 c standard disk diffusion method.[4,6] For antibacterial activity
where d is the spacing between the planes in the atomic lattice.
The volume V of the hexagonal unit cell was determined by
using the following equation:
√


3
V = × a2 × c.
2
The average crystallite size of HAp along the c-axis was
calculated from the XRD pattern using the Debye–Scherrer
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Figure 2. XRD pattern of synthesized samples (a) NHA, (b) GHA, and (c)
control (pure HAp).
formula.[14]
Kl
Dhkl = ,
b1/2 cos u
where Dhkl is the average crystallite size, K is the broadening
constant, λ is the wavelength of Cu Kα radiation (1.5406 Å),
β1/2 is the full-width at half-maximum of (002) peak (in radian),
and θ is the diffraction angle (in degree). The diffraction peak
at 2θ = 25.8° corresponding to (002) Miller’s plane of HAp,
was selected for calculation of the crystallite size since it
was sharper and isolated from others. Fourier transform
infrared (FTIR) spectra were obtained using Perkin Elmer
RX1 FTIR spectrometer in the range of 400–4000 cm−1.
Thermogravimetric (TG) analysis was performed using SDT
Q600 thermal analyzer (TA Instruments, New Castle, DE,
USA) under an argon atmosphere. The morphological feature
was analyzed using JEM JEOL-2100 transmission electron
microscope (TEM).
Antibacterial activity test
Antibacterial activity of prepared samples was analyzed by
test, 100 mg of synthesized samples were pressed at ∼24 MPa
to form circular disk, each nominally 14 mm diameter and
1 mm thick. Then, micro-organisms such as Escherichia coli
and Staphylococcus aureus were spread on the surface of nutri-
ent agar plates. After the spreading of individual bacteria in
each plate, disks of each sample was placed on the surface of
the culture plate and incubated for 24 h at 37 ± 0.5 °C. The
microbial inhibition zone including disk was measured after
the incubation period and its images were documented.

Table I. The calculated lattice parameter, unit cell volume, and average crystallite size of prepared samples.
Sample code Lattice constant (Å)
a=b c
NHA 9.5008 6.8626
GHA 9.5182 6.8113
Control 9.4120 6.8650
Results and discussion
XRD pattern of NHA, GHA and control samples are shown in
Fig. 2. XRD patterns of all the samples are well matched with
JCPDS data for HAp, which confirmed that prepared samples
are pure HAp having hexagonal crystal structure. The broad
peak in the region of 30°–34° can be ascribed to (211),
(112), and (300) Miller’s planes of HAp. The resolution of
these peaks depends upon crystallinity and crystallite size.
NHA and GHA shows broad diffraction peaks when compared
with control sample (HAp prepared without leaf extract), which
indicates that they are poorly crystalline in nature. The calcu-
lated lattice parameter, unit cell volume and average crystallite
size for all the samples are given in Table I. Significant varia-
tion in the lattice parameters were observed between the sam-
ples. Moreover, the average crystallite size of the control
sample is high when compared with the NHA and GHA.
This illustrates that the biomolecules present in leaf extracts
play an important role on the crystallization process and inhib-
iting the crystallization of HAp.
Different vibrational modes of the phosphate PO3− 4 and
hydroxyl OH− groups can be used to identify the HAp. FTIR spec-
trum (Fig. 3) of pure HAp (Control) shows vibrational modes of
Figure 3. FTIR spectra of (a) control, (b) GHA, (c) NHA, (d) Coccinia grandis,
and (e) Azadirachta indica leaf extract.
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Research Letter
Unit cell volume, V (Å3) Average crystallite size, D (nm)
536.47 53
534.41 64
526.66 84
phosphate at 462, 565, 605, 961, 1032, and 1108 cm−1, and
hydroxyl group at 3569 and 632 cm−1.[2,4] FTIR spectrum of
NHA and GHA shows vibrational modes of phosphate at
460, 565, 605, 961, 1041, and 1102 cm−1.[2,4] However, vibra-
tional modes of the hydroxyl group are not evidently seen in the
FTIR spectrum of NHA and GHA samples due to poor crystal-
line nature of the samples. The band at 1642 cm−1 and band
extending from 3000 to 3600 cm−1 are attributed to the O–H
vibration modes of water molecule and N–H stretching vibra-
tions of amides and amines.[2,4] The peak at 2925 cm−1 is
attributed to the C–H stretching of polyols.[9–11] The presence
of above functional groups shows that biomolecules such as fla-
vonoids, terpenoids, and protein compounds may be present in
the NHA and GHA, which are incorporated during the synthe-
sis process. Moreover, FTIR spectra of NHA show additional
peaks at 1475 and 1294 cm−1, which are due to C = O stretch-
ing of carboxylic acid. It reveals that the carboxyl group
strongly interacts with HAp crystallites. FTIR spectra of A. ind-
ica and C. grandis leaf extracts were also included in Fig. 3 for
comparison, which showed the vibrations of biomolecules such
as flavanoids and terpenoids. TG analysis of the synthesized
samples is shown in Fig. 4. The weight loss observed up to
200 °C in all samples is due to the desorption of adsorbed
water molecules.[4] With the increase in temperature, there is
significant weight loss that was observed between 200 and
Figure 4. TG analysis of synthesized samples.
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 Figure 5. TEM image and SAED patterns of (a) NHA, (b) GHA, and (c) control (pure HAp).

600 °C for NHA and GHA due to the burning of biomolecules
such as flavonoids, terpenoids, and protein compounds. From
TG analysis, we have determined the amount of biomolecules
in NHA and GHA as 4.90 and 4.79%, respectively.
TEM image and selected area electron diffraction (SAED)
pattern of synthesized samples are shown in Fig. 5. NHA
consist of nanorods having 10–20 nm width and 40–80 nm
length, whereas GHA consist of nanorods of 30–50 nm wide
and 80–150 nm length. On the other hand, control sample
(pure HAp) consist of aggregated elongated spherical particles
having 30–40 nm width and 70–80 nm length. The SAED pat-
tern of all the samples exhibits continuous rings around the
sharp spot, which shows the polycrystalline nature of prepared
samples. When A. indica leaf extract was used as a solvent, the
formation of HAp with the reduced particle size is due to the
strong interaction between the carboxyl groups and HAp,
which effectively controlled the growth of HAp particles.[4,12]
The strong carboxyl band appearing in the FTIR spectrum of
NHA confirmed that there is a strong interaction between car-
boxyl group and HAp crystallites, which support the results

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Figure 6. Antibacterial activity of the prepared samples against E. coli and S. aureus.
obtained from TEM results. It is important to mention that the
particle size of NHA and GHA obtained from the TEM obser-
vation is slightly higher than that of crystallite size of HAp
obtained from XRD data, which may be due to chemical het-
erogeneity of NHA and GHA. FTIR spectrum of NHA and
GHA shows that biomolecules such as flavonoids, terpenoids,
and protein compounds are present along with HAp, which
confirms its chemical heterogeneity.
E. coli and S. aureus are the most common bacteria found in
bone infection such as osteomyelitis.[3] Antibacterial activity of
the prepared samples against E. coli and S. aureus are shown in
Fig. 6. The diameters of inhibition zone of synthesized samples
against E. coli and S. aureus are given in Table II. NHA showed
significant inhibition zone against both the tested bacteria.
However, the GHA showed higher inhibition zone against E.
coli when compared with S. aureus. The observed antimicro-
bial activity may be due to the presence of biomolecules such
as flavonoids, terpenoids, and protein compounds in the sam-
ples, which inhibit the enzymes required for bacterial growth
and replication. It is reported that tetratriterpenoids, including
azadirachtin compound from A. indica leaf extract may be
able to inhibit the production of polysaccharide intercellular
adhesion for the growth of S. aureus.[10,11] Hence, NHA
showed higher antibacterial activity against S. aureus when
Table II. Zone of inhibition of as-synthesized samples against E. coli and
S. aureus.
Sample code Diameters of zone of inhibition (mm)
E. coli
NHA 26 ± 03
GHA 25 ± 02
Control Nil
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Research Letter
compared with GHA. Gopi et al. reported green synthesis of
HAp nanorods using banana, grapes, and tamarind extracts
with excellent antibacterial properties.[12] The difference in
the antibacterial activity depends on the particles size, nature
of the particles, and types of bacteria used for the antibacterial
test.
Conclusion
The phase pure HAp nanorods with different particle sizes
were prepared by green synthesis using A. indica and C. gran-
dis leaf extracts as a solvent. The obtained HAp nanorods
exhibit excellent antibacterial activity against E. coli and S.
aureus. Hence, green synthesis can be a potential method to
develop HAp nanorods with antibacterial properties for filling
bone defects.
Acknowledgments
Dr. Govindan Suresh Kumar would like to express his sincere
thanks to the University Grant Commission, India for financial
support through minor research project scheme [File No: 4-4/
2015-16 (MRP/UGC SERO)]. The work was carried out with
financial support from the Ministry of Education and Science
of the Russian Federation in the framework of Increase
Competitiveness Program of NUST “MISiS”, implemented
by a governmental decree dated 16th of March 2013, N 211.
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