Synthesis and characterisation of zinc oxide nanoparticles using terpenoid fractions of Andrographis paniculata leaves
Synthesis and characterisation of zinc oxide nanoparticles using terpenoid fractions of Andrographis paniculata leaves
S. Kavitha 0 1 2 3
M. Dhamodaran 0 1 2 3
Rajendra Prasad 0 1 2 3
M. Ganesan 0 1 2 3
0 Department of Chemistry (R&DC), Bharathiar University , Coimbatore, Tamil Nadu , India
1 A.V.C. Polytechnic College , Mannampandal, Mayiladuthurai, Nagai (DT), Tamil Nadu 609305 , India
2 Department of Biochemistry and Biotechnology, Annamalai University , Chidambaram, Tamil Nadu , India
3 Department of Chemistry, Perunthalaivar Kamarajar Institute of Engineering and Technology (PKIET) (Government of Puducherry Institution) , Karaikal, Puducherry U.T. , India
Zinc oxide (ZnO) nanoparticles have been widely employed for various pharmacological applications. Several approaches were tried to synthesize ZnO nanoparticles. In this study, ZnO nanoparticles were biosynthesized using terpenoid (TAP) fractions isolated from Andrographis paniculata leaves. Subsequently, the ZnNO3 (0.1 N) is treated with the isolated TAP fractions to biosynthesize zinc oxide nanoparticles (Zn-TAP NPs). This nanoparticle preparation has been confirmed by the colour change from green to cloudy-white and the peak at 300 nm by UV-Visible spectra. FTIR analysis of Zn-TAP NPs showed the presence of functional group (i.e.) C=O which has further been confirmed by H1-NMR studies. From SEM and XRD analysis, it has been found that the hexagonal nanorod particle is 20.23 nm in size and ?17.6 mV of zeta potential. Hence, it can be easily absorbed by negatively charged cellular membrane to contribute for efficient intracellular distribution. Therefore, it is suggested that the synthesised Zn-TAP NPs are more suitable in drug delivery processes.
Terpenoids paniculata; ZnNO3; Nanoparticles; Andrographis; Zinc oxide nanoparticles
Nanotechnology is an upcoming field of science which has
its impact in various fields such as energy, environment,
electronics, etc. The widespread practical applications of
metal nanoparticles (particles less than 100 nm) are
attributed to their unique properties . Different physical
and chemical processes are widely used to synthesize metal
nanoparticles . However, these production methods are
usually expensive, labor-intensive, and are potentially
hazardous to the environment and living organisms .
Thus, there is an obvious need for an alternative,
costeffective and at the same time, safe and environmentally
sound method of nanoparticles production . During the
past decades, it has been demonstrated that many
biological systems, including plants and bacteria  and fungi 
can transform inorganic metal ions into metal nanoparticles
via the reductive capacities of the proteins and metabolites
present in these organisms.
The ability of plant extracts to reduce metal ions has
been known since the early 1900s, although the nature of
the reducing agents involved was not well understood.
Because of its simplicity, the use of live plants or whole
plant extracts and plant tissue for reducing metal salts to
nanoparticles has attracted considerable attention within
the last 30-years . Plant extracts act both as reducing and
stabilizing agents in the synthesis of nanoparticles . The
source of the plant extract is known to influence the
characteristics of the nanoparticles . This is because
different extracts contain different concentrations and
combinations of organic reducing agents . Typically, a
plant extract-mediated bioreduction involves mixing
aqueous extract with an aqueous solution of the relevant
metal salt. The reaction occurs at room temperature and is
generally completed within a few minutes. Due to the
number of different chemicals involved, the bioreduction
process is relatively complex.
Nanoparticles have gained importance due to the
awareness in biological processes. The chemicals present
in the plant with anti-oxidant property are the basis for the
preparation of zinc oxide nanoparticle. It has become a
necessity to develop nanoparticles which in turn can be
targeted on different applications . Nanoparticles of
zinc oxide are under intensive study for their applications
in the field of optical devices, catalysis, biotechnology,
DNA labeling, drugs and medical and chemical sensors.
Nanosized zinc oxide has found various applications
(sunscreen coatings and paints) due to its high absorption
in UV [12, 13].
Currently, researchers are focusing on the synthesis of
nanoparticles using green methods. Synthesis of
nanoparticles using green methods increases the biological
effectiveness. Bio-nanoparticles have greater catalytic activity
due to the increase in the surface area. The possibility of
using plant materials as nano-precursors has also been
studied. The plant species A. paniculata, commonly known
as Nilavembu in India, belongs to the Acanthaceae family. It
is found in a large extent throughout South China, Asian
countries and Sri Lanka. It is also known as ‘‘King of bitters’’
[14, 15]. Despite its bitter taste, this species possesses
pharmacological properties, i.e. antimicrobial, antioxidant,
antiinflammatory, antiparasitic, antihyperglycemic,
hypoglycemic and antiallergic . Andrographis paniculata
reduces oxidation level due to its steroidal characteristics and
destroys infected somatic cells. It contains diterpenes,
lactones and flavonoids. The leaf and stem extracts have
glycosides, flavonoids, gums, steroids, terpenoids, tannins,
saponins and phenolic compounds . Compared to its
other parts, the leaf, which has multiple clinical applications,
has huge amounts of terpenoid (TAP) (2.39%) which
accounts for the bitter taste in leaves. This reason has been an
impetus to isolate the medically most active and major
compound TAP from the leaf. Therefore, in this study TAPs
were isolated from A. paniculata and used to prepare ZnO
nanoparticles for possible pharmacological applications.
Materials and methods
The plant A. paniculata was collected from the campus of
A.V.C. Arts and Science College, Mayiladuthurai. The AR
grade chemicals and solvents like zinc nitrate tetrahydrate,
sodium hydroxide, ethanol, chloroform, silica gel and
CDCl3 were procured from Merck Chemicals, Pune, India.
The collected A. paniculata leaves were cleaned with tap
water then rinsed with distilled water, dried, cut into small
parts and ground into fine powder. It was further stored at
The terpenoid fractions were separated by column
chromatography. In this method, 25 g silica gel powder was
filled in column apparatus and mixed with ethanol up to
slurry formation. Then the solvent was completely eluted,
and added 50% mixture (65 mL CHCl3 and 1 mL
methanol). It was followed by adding 10 mL ethanolic sample.
Finally, the remaining mixture was added. The solvent was
eluted. Within 12 h terpenoid was collected in test tubes.
Phytochemical test for terpenoid (TAP) fractions
from A. paniculata
Confirmative test for terpenoids
Salkowskis test TAP was mixed with a few drops of
chloroform and concentrated sulphuric acid. The formation
of yellow colour indicates the presence of terpenoids.
The extract was treated with 1 mL of CHCl3, 1 mL
CH3COOH and few drops of concentrated sulphuric acid.
Appearance of brown ring indicates the presence of terpenoids.
Synthesis of zinc oxide nanoparticles (Zn-NPS)
Zinc oxide nanoparticles were prepared by green synthesis
(co-precipitation) method. 0.1 N aqueous solution of zinc
Fig. 1 TAP mediated Zn-TAP NPs synthesis
Fig. 2 Schematic diagram of
Zn-TAP NPs synthesis
Vigorous stirring for 10 mts and
At 60o C for 3 hrs by adding 0.1 N NaOH in time
Cloudy- White precipitate
(Zn- nanopar cles)
nitrate tetrahydrate (Zn (NO3)2 4H2O) was added to 50 mL
distilled water by continuous shaking. Later 0.1 N NaOH
was added in 10 m gap for an hour. Following that, the
time gap was increased for adding the NaOH. The
procedure was repeated for 2 h. The obtained white solution was
stirred for 2 h at pH 12. The product was washed with
distilled water and ethanol to get the final product. It is then
dried overnight. The whole mode of proposed method for
the synthesis of Zn-NPs mediated by the aqueous extract
Characterisation of terpenoid zinc oxide
nanoparticles (Zn-TAP NPs) from A. paniculata
Particle size and zeta potential measurements
DLS and zeta potential were based on the direction and
velocity of particles under the influence of known electric
field. Malvern Zetasizer ZS (Malvern Instruments,
Malvern, UK) instrument was used to measure particle size,
size distribution and zeta potential of Zn-TAP NPs. To suit
the above situation a homogenous suspension was created
using the lyophilized nanoparticles in double distilled
water and repeated thrice.
Scanning electron microscopy (SEM)
The samples were placed on a carbon plated platinum strip.
Splash drops were wiped off. It was dried in mercury lamp
for 5 m and examined under SEM (using JEOL JSM-6610
LV SEM machine).
UV spectroscopic analysis
The Zn-TAP NPs were dissolved in distilled water (1 mg/
mL) and scanned in a Perkin Elmer Lambda 25 UV–Vis
spectrometer at 25 C in the range of 250–650 nm. The UV
spectrum was repeated three times.
Fourier transform infrared spectroscopy (FTIR) analysis
Zn-TAP NPs and potassium bromide 10 and 100 mg were
respectively mixed to form a salt plate. Spectra between
4000 and 400 cm-1 were noted in a Bio-Rad FTIR–40
Proton nuclear magnetic resonance spectroscopy
An approximately 30 mg of Zn-TAP NPs was made
soluble in 0.5 mL CDCl3 (99.9%). At 27 C 1H-NMR spectra
were recorded by Luo and Fan method (2011).
Green synthesis of zinc oxide nanoparticles
The biosynthesised Zn-TAP NPs were isolated (Fig. 1)
using 0.1 N Zinc nitrate with NaOH of co-precipitation
method at pH 12 (Fig. 2).
Characterisation of Zn-TAP NPs from A. paniculata
X-ray diffraction (XRD) analysis
Particle size and zeta potential of Zn-TAP NPs
At k = 0.1546 nm, running at 40 kV and 30 mA in X-ray
diffractometer (X’Pert PRO-PANalytical Philips). Zinc
oxide nanoparticles were recorded in the region from 10 to
80 at a scan speed of 2h per minute.
Figure 3a shows the size distribution of the Zn-TAP NPs in
aqueous medium. It was measured by DLS. The average
particle size was 20.23 nm. Figure 3b reveals that the zeta
potential of synthesized ZnO nanoparticles was 17.6 mV.
Fig. 3 a Particle size for Zn-TAP NPs from A. paniculata leaves. b Zeta potential of Zn-TAP NPs. c XRD images of Zn-TAP NPs. d SEM for
XRD analysis of Zn-TAP NPs
X-ray diffraction was used to confirm the crystalline nature
of the particles by no discernible peak in the low range
(2h = 1 –10 ). Figure 3c shows a representative XRD
pattern of the ZnO nanoparticles synthesized by the A.
paniculata extract after the complete reduction of Zn2? to
Zn0. A number of Bragg reflections were present which can
be indexed on the basis of the hexagonal Wurtzite structure
of ZnO. The diffraction peaks at (100), (002), (101), (102)
and (110) were obtained with those reported values of
standard card (JCPDS no: 36–1451) .
Fig. 4 a UV absorption of TAP isolated from A. paniculata leaves.
b UV absorption of Zn-TAP NPs from A. paniculata leaves
From the XRD peaks, to estimate the average
particle size was calculated by using Scherrer’s equation
as 22.23 nm. These values are merely similar to DLS
SEM analysis of Zn-TAP NPs
The synthesised Zn-TAP NPs morphology was examined
by SEM in JEOL JSM-6610 LV instrument. When the
nanoparticle was placed on carbon coated platinum grid,
after completion of reaction, it showed a hexagonal shape
as indicated in Fig. 3d.
Fig. 5 a and b FTIR of TAP
and Zn-TAP NPs from A.
UV spectroscopy studies
The formation of zinc oxide nanoparticles might be due to
reduction of Zn2? to Zn0 by terpenoids present in the leaf
of A. paniculata. The colour transformation of A.
paniculata extract treated with zinc nitrate might be due to
vibrations in surface plasmon resonance of zinc .
Because it has free }-electrons (C=O). The broad band at
300 nm indicates the reduction of Zn2? ions which further
confirmed the formation of zinc oxide nanoparticles in
Fig. 4b. Whereas, the isolated active compound TAP has
no absorbance change in the range of 280–350 nm
Fourier transform infrared spectroscopy (FTIR) studies
The FTIR spectroscopy measurements were studied to
identify the possible bio-molecules responsible for
reducing the ZNO-NPs synthesised through TAP. The IR spectra
of TAP (Fig. 5a) exhibit strong absorption band at
3334.71 cm-1 for O–H stretching vibration. The C–O axial
stretching band appears at 1656.36 cm-1. The peak at
2852.85 cm-1 corresponds to C–H stretching vibration.
Another report was assigned that the peak at 1656.36 cm-1
is due to –C=C– aromatic stretching.
The peak was observed for Zn-TAP NPs between 513
and 466 cm-1 (Fig. 5b). The bands appeared between 600
and 400 cm-1 which may be assigned to the metal oxide or
metal chloride [20, 21], which confirm the formation of
ZnO NPs at 466.77 cm-1. The absorbance at
3479.58 cm-1 indicates the O–H stretching vibration in
hydroxyl functional group of alcohols and phenol
compounds of ZnO NPs. The absorption peak at 2929.87 cm-1
corresponds to –CH stretching and 2767.85 cm-1 for
aldehydic –CH vibration mode. 1764.87 cm-1 is assigned
Fig. 6 H1NMR for Zn-TAP
NPs from A. paniculata leaves
to aldehydic carbonyl (–CH=O) group. Some other bands
at 1637.56, 1382.96, 1004.91 and 640.37 cm-1 were also
seen which correspond to aromatic –C=C–, C–H group, –
CH2 group and mono substituted ring which indicate that
the TAP of A. paniculata reduced by ZnO NPs might be
surrounded by aromatic ring polyphenol . The band at
1637.56 cm-1 remained in ZnO NPs and this is due to
groups with a benzene ring. Another report has assigned
that the peak at 1637.56 cm-1 is due to –C=C– aromatic
In CDCl3 solvent, H1-NMR spectrum (Fig. 6) showed the
3H, s, [C=CH2 group at d 1.603. The peak at d 2.178
assigned to phenyl group stretching. The absorption peak at
d 4.481 corresponds to H3C–O–CO–CH3 stretching
vibration. The band appears at d 7.265 for aromatic
compound. Another report was assigned that the peak at d
2.178 and d 4.481 corresponds to aliphatic –OH and
aromatic –OH group respectively.
TAP could be adsorbed on the surface of nanoparticles
possibly by interaction through carbonyl groups or
}electrons. The formation of ZnO NPs was confirmed by
colour changes and was characterised by UV–Visible
spectrophotometer. The broad band was observed at
300 nm. It was proved by IR spectra band at 466.77 cm-1.
The presence of functional groups in ZnO NPs was also
confirmed by IR and H1-NMR studies. The SEM analysis
shows hexagonal shape particles with 20.23 nm size which
was merely close to Scherrer’s value (i.e.) 22.23 nm.
ZnTAP NPs has positive zeta potential value 17.6 mV.
Hence, it was proposed that it can be easily absorbed by
negatively charged cellular membrane and then contributes
to efficient intracellular distribution of drug.
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1. Daniel , M.C. , Astruc , D. : Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology . Chem. Rev . 104 ( 1 ), 293 - 346 ( 2004 )
2. Kundu , S. , Maheshwari , V. , Niu , S. , Saraf , R.F. : Polyelectrolyte mediated scalable synthesis of highly stable silver nanocubes in less than a minute using microwave irradiation . Nanotechnology 19 ( 6 ), 065604 ( 2008 )
3. Gan , P.P. , Ng , S.H. , Huang , Y. , Li , S.F. : Green synthesis of gold nanoparticles using palm oil mill effluent (POME): a low-cost and eco-friendly viable approach . Bioresour. Technol. 113 , 132 - 135 ( 2012 )
4. Raveendran , P. , Fu , J. , Wallen , S.L. : Completely ''green'' synthesis and stabilization of metal nanoparticles . J. Am. Chem. Soc. 125(46) , 13940 - 13941 ( 2003 )
5. Kalishwaralal , K. , Deepak , V. , Pandian , S.R.K. , Kottaisamy , M. , BarathmaniKanth , S. , Kartikeyan , B. , Gurunathan , S. : Biosynthesis of silver and gold nanoparticles using Brevibacterium casei . Colloids Surf. B Biointerfaces 77 ( 2 ), 257 - 262 ( 2010 )
6. Kitching , M. , Ramani , M. , Marsili , E. : Fungal biosynthesis of gold nanoparticles: mechanism and scale up . Microb. Biotechnol . 8 ( 6 ), 904 - 917 ( 2015 ). doi:10.1111/ 1751 - 7915 . 12151
7. Ankamwar , B. : Biosynthesis of gold nanoparticles (green-gold) using leaf extract of Terminalia catappa . Eur. J. Chem . 7 , 1334 - 1339 ( 2010 )
8. Kumar , V. , Yadav , S.C. , Yadav , S.K. : Syzygium cumini leaf and seed extract mediated biosynthesis of silver nanoparticles and their characterization . J. Chem. Technol. Biotechnol . 85 , 1301 - 1309 ( 2010 )
9. Kumar , V. , Yadav , S.K. : Plant-mediated synthesis of silver and gold nanoparticles and their applications . J. Chem. Technol. Biotechnol . 84 , 151 - 157 ( 2009 )
10. Mukunthan , K. , Balaji , S. : Cashew apple juice (Anacardium occidentale L.) speeds up the synthesis of silver nanoparticles . Int. J. Green. Nanotechnol . 4 , 71 - 79 ( 2012 )
11. Gnanasangeetha , D. , Sarala Thambavani , D. , et al.: One pot synthesis of zinc oxide nanoparticles via chemical and green method . Res. J. Mater. Sci. Int. Sci. Congr. Assoc . 1 ( 7 ), 1 - 8 ( 2013 )
12. Gnanasangeetha , D. , Sarala Thambavani , D. , et al.: Facile and eco-friendly method for the synthesis of zinc oxide nanoparticles using Azadirachta and Emblica. Int. J. Pharm. Sci. Res . 5 ( 7 ), 2866 - 2873 ( 2014 )
13. Fan , Z. , Lu , J.G. , et al.: Zinc oxide nanostructures: synthesis and properties . J. Nanosci. Nanotechnol . 5 ( 10 ): 1561 - 1573 ( 2005 )
14. Hosamani , P.A. , Lakshman , H.C. , Sandeepkumar , K. , et al.: Antimicrobial activity of leaf extract of Andrographis paniculata wall . Sci. Res. Rep . 1 ( 2 ), 92 - 95 ( 2011 )
15. Sivarajan , V.V. , Balachandran , I. , et al.: Ayurvedic drugs and their plant sources . pp. 570 - 95 . Oxford and IBH Publishing Co . Pvt. Ltd., New Delhi ( 1994 ) http://jsrr.in Hosamani et al. ISSN: 2249- int)
16. Sule , A. , Ahmed , Q.U., Samah , O.A. , et al.: Screening for antibacterial activity of the treatment of skin infections . Ethnobot. Leafl . 14 , 445 - 456 ( 2010 )
17. Goodman , S.L. , Gilman , A. : The pharmacological basis of therapeutics , 9th edn, pp. 959 - 975 . Macmillan Publishing Co Inc, New York ( 2000 )
18. Kohler , G. : An continuous cultures of fused cells secreting antibody of predefined specificity . Nature 256 , 495 - 497 ( 1975 )
19. Gao , Q.Q. , Yu , Q.X. , Yuan , K. , et al.: Influence of annealing atmosphere on room temperature ferromagnetism of Mn-doped ZnO nanoparticles . Appl. Surf. Sci . 264 , 7 - 10 ( 2013 )
20. Nakamoto , K. : Infrared and Raman spectra of inorganic and coordination compound, 4th edn . Willey, New York ( 1986 )
21. Vinod , K. , Pandey , O.P. , Soumitra , K. : Synthesis and physicochemical and biological studies on ruthenium (III) complexes with Schiff bases derived from aminocarboxylic acids . Transit. Met. Chem . 12 , 509 - 515 ( 1987 )
22. Rajasekar , R. , Priyadharshini , S. , Rajarajeshwari, T. , et al.: Bioinspired synthesis of silver nanoparticles using Andrographis paniculata whole plant extract and their anti-microbial activity over pathogenic microbes . Int. J. Res. Biomed. Biotechnol . 3 ( 3 ), 47 - 52 ( 2013 )
23. Raghunandan , D. , Bedre , M.D. , Basavaraja , S. , et al.: Rapid biosynthesis of irregular shaped gold nanoparticles from macerated aqueous extracellular dried clove buds (Syzygium aromaticum) solution . Colloids Surf. B Biointerfaces 79 , 235 - 240 ( 2011 )