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Materials Today: Proceedings 3 (2016) 2394–2398 www.materialstoday.com/proceedings

Recent Advances In Nano Science And Technology 2015 (RAINSAT2015)

Synthesis and Spectral Characterization of Novel nano-

Hydroxyapatite from Moringaoleifera Leaves
Dharman Govindaraj, Mariappan Rajan*
*a
Biomaterialsin Medicinal Chemistry Lab, Department of Natural Products Chemistry,
School of Chemistry, Madurai Kamaraj University, Madurai - 625021, India.

Abstract

The conversion of green food materials into biocompatible materials (biomaterials) used in medical surgery and therapeutics is a
strategy. Nano- hydroxyapatite (n-HAP, Ca10 (PO4) 6 (OH) 2) was produced from Moringaoleifera leaves using a rapid
microwave irradiation method. Moringaoleifera leaves was converted to flakes like n-HAP particles using EDTA as a chelating
agent after an appropriate pre-treatment and an irradiation step in a microwave. The as-synthesized n-HAP was characterized
using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray
spectroscopy (EDS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR). The process of
transforming Moringaoleifera leaves into nano-hydroxyapatite is an environmentally friendly process. Moringaoleifera leaves
based nano-hydroxyapatite stand as good chance of reducing the cost of treatment in bone repair.

© 2015Elsevier Ltd.All rights reserved.

Selection and Peer-review under responsibility of [Conference Committee Members of Recent Advances In Nano Science and
Technology 2015.].

Keywords: Biomaterials; Bone; Hydroxyapatite; Moringa oleifera Leaves;

1. Introduction
Natural biomaterials like bone substitutes have been successfully used, such as allograft (human), xenografts and
wound dressings made of biologically derived calcium alginate, collagen, chitin etc., have recently started to surpass
the synthetic biomaterials by providing an abundant source for novel biomedical applications [1-5]. Bone is a living

* Corresponding author. E-mail address:rajanm153@gmail.com

2214-7853© 2015 Elsevier Ltd.All rights reserved.

Selection and Peer-review under responsibility of [Conference Committee Members of Recent Advances In Nano Science and Technology 2015.
].
 Dharman Govindaraj, Mariappan Rajan/ Materials Today: Proceedings 3 (2016) 2394–2398 2395

tissue that serves for structural support and calcium metabolism. Bone matrix is organic and consists of a network of
collagen protein fibers impregnated with mineral salts (85% of calcium phosphate, 10% of calcium carbonate, and
5% of calcium fluoride and magnesium fluoride)[6]. The mineral compartment of bone is predominantly present in
the form of calcium hydroxyapatites (HAP, Ca10 (PO4) 6 (OH) 2). Commercially available HAP is expensive due to
the use of high purity reagents [7]. However, problems do arise due to the variability of physical and chemical
properties of the raw material [7]. The Moringaoleifera leaves represent 1g of the total weight of the
Moringaoleifera leaves and is composed predominantly of calcium carbonate (95%), calcium phosphate (1%),
organic matter (3%) and magnesium carbonate (1%). Though these are frequently used as a food due to their high
content of calcium and phosphate [8], most of these are discarded as waste. By utilizing these organic food
materials, namely Moringaoleifera leaves, the cost of high quality calcium source for the preparation of HAP can be
avoided.
There are several reports on the synthesis of HAP from natural sources using direct or indirect conversion
methods [9-12]. Direct conversion process is done by time consuming hydrothermal method whereas the indirect
process involves the preparation of calcium precursor from Moringaoleifera leaves as a first step and then the HAP
preparation by the conventional methods like wet precipitation, sol-gel, microwave irradiation, etc., as a second step
[8]. Microwave method has become a very popular branch of nano-biomaterials science recently thanks to its
advantages. The primary impact of a microwave-assisted hydroxyapatite synthesis approach on the reaction rate is
favorable in many reactions, particularly in conventional methods requiring heating the reaction vessel for prolonged
times. In addition to the outcomes such as thermal effects along with shortened reaction times, the minimization of side
reactions and acquiring better quality in terms of purity makes microwave-assisted nano-hydroxyapatite synthesis
studies interesting [13]. On the other hand, HAP nanostructures with different morphologies have been synthesized
using the chelating agent such as citric acid, ethylene diamine tetra acetic acid (EDTA), etc., [14–15].. The aim of the
present work is to propose a simple and rapid microwave irradiation method for conversion of the green
Moringaoleifera leaves to obtain flake-like HAP nanostructure using EDTA as a chelating agent.
2. Experimental
All the chemicals used for the experiments were analytical grade obtained from sigma Aldrich. Moringaoleifera
leaves were collected and the leaves were washed with Millipore water then rinsed with acetone to remove the
surface contaminants. After drying, they were ground into fine powder by ball milling and immersed in 5 ml of
conc. HNO3 and then put into microwave irradiation for 2 min at 50W to remove organic components. Then, they
were extensively washed with Millipore water and dried in vacuum oven for 2 h at 150 °C. 1 g of Moringaoleifera
leaves powder was then mixed with 0.1 M of EDTA solution to form Ca–EDTA complex. Then, 0.06 M of
(NH4)2HPO4 solution was slowly added with obtained Ca–EDTA complex and sonicated for 30 min. The pH of the
reaction mixture was adjusted to 10 by the addition of NaOH solution. Subsequently, the prepared reaction mixture
was put in a microwave oven (A CEM Discover microwave synthesizer 2455 MHz, 300 W) and irradiated with
microwave 100 W for 5 min. The white precipitate obtained was washed and dried in hot air oven at 150 °C for 3 h.

2.1. Characterization

The chemical and phase composition of the nano-HAP was determined by Fourier transform infrared
spectroscopy (FTIR-Perkin Elmer, USA), X-ray beam photoelectron spectroscopy (KRATOS AXIS-165) and X-ray
diffraction analysis (XRD- Bruker D8). The structural and morphological features of the as-synthesized
nanocomposites were investigated with a field emission scanning electron microscope (FESEM-Curl Jdiss Supra 40-
2007, Germany) and transmission electron microscope (TEM, JEM-2010, JEOL).
3. Results and Discussion
Figure 1 show, which the spectrum of as-synthesized HAP using microwave irradiation, the wide band at 3455
and 1630 cm−1 corresponds to adsorbed water, while the sharp peak at 3572 cm−1 is analogous to the stretching
vibration of OH− ions in the n-HAP lattice. The strong band (ν3) at 1032 and 1092 cm−1 was assigned to the P–O
stretching vibration of PO43− [16]. The P-O symmetric stretching (ν1) and bending (ν4) vibration bands of phosphate
2396 Dharman Govindaraj, Mariappan Rajan/ Materials Today: Proceedings 3 (2016) 2394–2398

groups was appeared at 602 and 570 cm-1 respectively. The 469 cm−1 band resulted from the ν2 phosphate mode.
The above results revealed that the as-synthesized sample had high pure [17].

Fig.1. FT-IR spectrum of synthesized n-HAP Fig.2. XRD spectrum of as-synthesized n-HAP

Fig. 3 XPS spectrum of n-HAP

Fig.4. FESEM images of a) Moringaoleifera leaves powder, b) obtained n-HAP

 Dharman Govindaraj, Mariappan Rajan/ Materials Today: Proceedings 3 (2016) 2394–2398 2397

Fig.5. TEM image of as-synthesized n-HAP

Table. 1. Elemental composition of Moringaoleifera leaves powder and the final product n-HAP obtained from EDX.

Samples Elemental Composition (Wt.%)

Ca P C O
Moringaoleifera Leaves 34.38 2.56 14.75 1.63
n-HAP 24.62 14.34 6.30 1.20

The XRD patterns of HAP nano-flakes shown in Fig. 2. The pattern exhibited the entire characteristic peak for
HAP nano-flakes and no other impure phases were found, which is in good agreement with the ICDD (File No. 09-
0432, International Center for Diffraction Data, ICDD). The XRD pattern showed HAP crystalline phase, at the
corresponding planes (002), (210), (211), (310), (222), (213) and (004), respectively.
To determine further crystallinity form of n-HAP, XPS on the n- HAP was carried out. The n-HAP result of XPS
curve is shown in Fig. 3. The binding energy of calcium (Ca), oxygen (O) and phosphor (P) has observed from HAP
(O: 531.5, Ca: 351.8 & 347.3, and P: 134.5 ev) [16]. This indicates that hydroxyapatite successfully synthesized
from Moringaoleifera leaves.
SEM image (Fig. 4(a)) demonstrates the surface morphology of the Moringaoleifera leaves powder. EDX
analysis indicates the presence of Ca, P, C, and O in Moringaoleifera leaves and the obtained elemental composition
is shown inTable.1. In Fig. 1(b) shows the SEM image of the product obtained after microwave irradiation. It
displays typical flakes-like nano HAP. The related EDX taken from individual nano-flake indicates the presence of
Ca, P, O, and C in the obtained product and its elemental composition is shown in Table1. The formation of nano-
flakes is beneficial because the soft and hard tissue may grow easily inside the pores which will display a good
application in future tissue repair engineering. The morphology of the as synthesized n-HAP is further analyzed
using TEM analysis (Fig. 5). It is observed that the particle shows flakes like morphology with nano size [18, 19].

4. Conclusions

To conclude, we have used a green low-cost, safe, extremely available green food and waste for the consistent
and rapid synthesis of n-HAP nano-flakes. The n-HAP nano-flakes were green synthesized using rich calcium
source of Moringaoleifera leaves by calcium extraction. As revealed by FT-IR, XPS and XRD analyses, the product
is pure & high crystalline nature. The particles are mostly flakes with nano-size and the obtained n-HAP seems to be
a promising material for biomedical applications.
2398 Dharman Govindaraj, Mariappan Rajan/ Materials Today: Proceedings 3 (2016) 2394–2398

Acknowledgements

One of the authors, M. Rajan, is grateful to the University Grant Commission (UGC), Government of India, for
providing financial assistance under the scheme of “UGC-BSR Research Start Up Grants” (Ref. no. F.30-21/20014
(BSR)).

References

[1] B. Ben-Nissan, Curr. Opinion Solid State Mater. Sci. 2003; 7 : 283.
[2] M. S. Block and J. N. Kent, Journal of Oral and Maxillofacial Surgery. 1997; 55: 1281–1286.
[3] J. M. Rueger, W. Linhart, and D. Sommerfeldt, Orthopade. 1998; 27: 89–95.
[4] M. Bohner, Injury. 2000; 31: D37–D47.
[5] M. Bohner, European Spine Journal. 2001; 10 : S114–S121.
[6] A. Berberi, A. Samarani, N. Nader, Z.Noujeim, M. Dagher, W. Kanj, R. Mearawi, Z. Salemeh, and B. Badran. BioMed Research
International. 2014; 2014:1-9.
[7] M.D. Adak, and K. M. Purohit, Trends Biomater. Artif. Organs. 2011; 25: 101-106.
[8] S. Toba, Anjorin, P. Ikokoh, S. Okolo. Asian Journal of Plant Sciences. 2005; 4: 417-421.
[9] Sanosh KP, Chu M, Balakrishnan A, Kim TN, Cho S.Mater Lett. 2009; 63 :2100–2.
[10] Meski S, Ziani S, Khireddine H. J Chem Eng Data. 2010; 55: 3923–8.
[11] Liu J, Li K, Wang H, Zhu M, Yan H. Chem Phys Lett. 2004; 396 : 429–32.
[12] Martins MA, Santos C, Almeida MM, Costa MEV. J Colloid Interface Sci. 2008; 3: 210–6.
[13] J. Han , H. Song , F. Saito, B. Lee. Materials Chemistry and Physics. 2006; 99: 235–239.
[14] E. Bashir, F. Lagerlöf. Caries Res. 1996; 30:213-7.
[15] S. P. Tinling, R. T. Giberson and R. S. Kullar. Journal of Microscopy, 2004; 215: 230–235.
[16] D. Govindaraj, M. Rajan, M.A. Munusamy, M. D. Balakumaranc and P. T. Kalaichelvan. RSC Adv, 2015; 5: 44705-44713
[17] A. Jillavenkatesa, R.A. Condrate Sr., J. Mater. Sci. 1998; 33: 4111.
[18] D. Govindaraj, M. Rajan, M .A. Munusamy, Akon Higuchi, RSC Adv., 2015, 5, 58980-58988.
[19] C. Chen, Z. Huang, W. Yuan, J. Li, X. Cheng , R. Chi, CrystEngComm, 2011, 13, 1632–1637.