ISSN 0974-3618
(Print) www.rjptonline.org
0974-360X (Online)
RESEARCH ARTICLE
Synthesis, Characterization and
Antimicrobial Activities of Turmeric Curcumin and Curcumin Stabilized Zinc
Nanoparticles - A Green Approach
Jayandran. M1,
Muhamed Haneefa. M2*, Balasubramanian.V2
1Faculty of Chemistry, Mahendra Engineering
College, Namakkal, India
2Faculty of Chemistry, AMET University,
Chennai, India
*Corresponding Author E-mail: honey79101@gmail.com
ABSTRACT:
Metal
nanoparticles are versatile platforms for biomedical applications and
therapeutic intervention. But there is a need to develop new method in the
preparation of nanoparticles which should not be harmful to environment. Recent
studies demonstrated that several metal nanoparticles synthesized by green
methodologies have shown potential antibacterial, antifungal activities. This
paper investigates the synthesis of turmeric curcumin as well as zinc
nanoparticles in the greener route by using natural lemon extract as a reducing
agent and curcumin as a stabilizing agent. The obtained curcumin was confirmed
by UV-Vis, IR analysis and Zn nanoparticles were characterized by UV-Vis, IR,
XRD, SEM and TEM techniques. The experimental results displayed that the Zn
nanoparticles with an average diameter of about 23-46 nm. The antimicrobial
activity of curcumin stabilized Zn nanoparticles exhibited significant effects against
all the bacterial and fungal species. But sensitivity to nanoparticles was
found to vary depending on the microbial species.
KEYWORDS: Metal
nanoparticles, Green method, Curcumin, Soxhlet extract, Antimicrobial,
Therapeutic agent.
INTRODUCTION:
New technologies
often create new challenges to science in addition to their benefits, raise
concerns about health and various environmental problems. Recently,
nanotechnology holds a promise and a broad aspect towards wide applications of
nanoparticles in a multiple way of emerging fields of science and technology which
referring at the nanoscale, i.e. 1 to 100 nm. Metal nanoparticles are being
given considerable attention in nanoscale science and engineering technology
over the last decades due to their interesting properties and potential
applications in many areas of industry. Nanoparticles possess high surface to
volume ratio due to its small size, which gives very distinctive features to
nanoparticles1-3.
Received on 03.03.2015 Modified on 19.03.2015
Accepted on 25.03.2015 © RJPT All right reserved
Research J. Pharm. and Tech.
8(4): April, 2015; Page 445-451
DOI: 10.5958/0974-360X.2015.00075.X
Metal
nanoparticles have been widely used for various applications like catalysis,
optoelectronics, magnetic, thermal, sensors, fine chemical synthesis, solar
energy conversion and medicine etc. The vast applications of nanoparticles in
medical sciences are drug delivery, imaging, tissue repair, immune assay and
diagnosis4-5. Mostly nanoparticles have been synthesized using a
variety of techniques typically characterized as either a physical or a
chemical method. Physical methods are capable of producing a wide range of
metallic nanoparticles; however the quality of the material is not as high as
chemically synthesized materials. In chemical synthesis techniques, the growth
and assembly of metallic nanoparticles is controlled by optimizing reaction
parameters6-9. Although chemical and physical methods may
successfully produce pure, well-defined nanoparticles, these are quite
expensive and possibly dangerous to the environment. At present, there is an emergent
need to develop environmentally benevolent nanoparticles synthesis routes,
which can be proceeded by biological method instead of using toxic chemicals10-11. Nanomaterials are
the leading in the field of nanomedicine and in that respect nanotoxicology
research is gaining great importance. Accordingly, the researchers in the field
of nanoparticles synthesis and assembly have turned to biological systems for
inspiration. The green synthesis method plays a significant role on the
effective consumptions of nanoparticles12-13. Proper utilization of
environmentally benevolent solvents and nontoxic chemicals are some of the key
issues in green in synthesis approach deliberations14.In the
green-nanotechnology, various metal nanoparticle synthesis have been reported
using microorganisms, plant extracts and other biological natural materials.
Use of natural plant extracts in the preparation of nanoparticles by greener
route provides advancement over chemical and physical method as it is cost
effective, environment friendly15-16.
Curcumin is an active component of turmeric
plant; it is responsible for its characteristic yellow color and therapeutic
potential17. Curcumin is of considerable interest because of its
antioxidant18-19, anti-inflammatory20, antimicrobial21
and anticancer activities22 etc. But its poor bioavailability
remains a major challenge due to the presence of olefinic groups in its
structure this β-diketone of poor aqueous solubility rendering it of
relatively low bioavailability23. In order to improve the
bioavailability of curcumin, various approaches have been used. One of the
possible approaches to increase the bioavailability of curcumin is its
conjugation on the surface of metal nanoparticles24-25.
Zinc
nanoparticles (ZNPs) have wide applications in optoelectronics, field emission,
vacuum fluorescent, luminescent light, catalysis and also photo degradation
etc.26-27.Various researches have shown that antimicrobial
formulations in the form of nanoparticles could be used as potential
bactericidal materials. The emergence of nanoscience and nanotechnology in the
last decade presents opportunities for exploring the antimicrobial effect of Zn
nanoparticles. The antimicrobial effect of Zn nanoparticles has been attributed
to their small size and high surface to volume ratio, which allows them to
interact closely with microbial membranes and is not merely due to the release
of metal ions in solution. Furthermore, Zn nanoparticle appears to effectively
resist microorganisms and much more stable and has a longer life than organic
based disinfectants and antimicrobial agents. Zinc is an essential constituent
for cell growth and in inhibiting bacterial enzymes like dehydrogenase and
certain protective enzymes such as thiolperoxidase and glutathione reductase28-30.
But the surface functionalization of ZNPs with curcumin may give a new way of
using the curcuminoids with Zn nanoparticles towards highly improved
antimicrobial activity.
Hence, in this
work we report the synthesis of curcumin from turmeric (Scheme 1) as well as
the synthesis of the zinc nanoparticles by using lemon extract as reducer and
curcumin as stabilizing agent (Scheme 2) to enhance the solubility of curcumin
in water and its biological activities. The Zn nanoparticles functionalized
with curcumin showed excellent inhibition activity against the microbial
strains tested over all.
MATERIAL AND
METHODS:
All the chemicals and solvents used were of analytical
reagent grade and obtained from Merck (Indi) Ltd and all samples were prepared
by using fresh double-distilled water throughout the experiment. Curcumin was
isolated from turmeric (BSR-01) which was purchased from Agricultural College
and Research Institute, Madurai.
Collection
of extracts:
Lemon fruits were collected from the local markets. They
were washed in double distilled water, cut into pieces and squeezed well to
make 5 to 10 ml pure extract. The extract was then filtered using Whatmans No.
1 filter paper. The filtrate was collected in a clean and dried container and
it was stored for further uses.
Isolation
of curcumin (CR) from turmeric:
Curcumin was quantitatively extracted from turmeric in soxhlet apparatus
by using 95% ethanol as per our previous work31 and the
curcumin content was estimated by
Manjunath et al., 199132. The high curcumin yield turmeric variety,
BSR-01 was incorporated in this method for better result. The brief process is
described as follows. Turmeric dried powder weighed (5 g) and taken in soxhlet
apparatus and 250 ml of ethanol was poured into the apparatus. The extraction
process was carried out for 2-3 hour and the final curcumin extract absorbance
was measured at 425 nm against alcohol blank and the curcumin percentage
was calculated. The above ethanol residual extract was evaporated and dried
then recrystallized by 95% ethanol for further uses.
CHARACTERIZATION:
The UV-Visible absorption spectra of the samples were
measured on a Shimadzu UV-Vis V-530A spectrophotometer
in the range of 425nm. The nanoparticles were examined for FT-IR spectra
analysis and recorded on a Jasco FT-IR/4100 spectrophotometer with 4 cm-1
resolution in the range of 4000 to 400 cm-1. X-ray measurement of
the prepared solids was carried out using a Panalytical XPert Powder
XCelerator Diffractometer (Netherland) in the range of 10ο to
80ο 2θ of 2ο min-1. Scanning
electron microscopy (SEM) images were recorded by using a JEOL Model JSM - 6390LV scanning electron microscope.
High resolution transmission electron microscopy (HRTEM) was carried out using
a 300 kV JEOL-3011 instrument to determine the morphological changes.
Synthesis of Zinc nanoparticles (ZNPs):
1mM aqueous
solution of zinc acetate was prepared and used for the synthesis of ZNPs.
Double distilled water has been used throughout the synthesis process. The
filtered stored pure lemon extract (10 ml) was taken in a beaker and freshly
prepared zinc solution (10 ml) was mixed with the extract with constant
stirring for the reduction of zinc ions. The reaction mixture was kept in the
magnetic hot stirrer at 50-
Biological assay:
The antibacterial activity of the synthesized ZNPs were
tested by disc diffusion method against two gram positive bacteria (Staphylococcus
aureus and Bacillus subtilis), two gram negative bacteria (Escherichia
coli and Staphylococcus bacillus) and antifungal activity was
carried out by agar well diffusion method againstfour funguses (Candida
albicans, Curvularia lunata, Aspergillus niger and Trichophyton
simii). For disc diffusion method, stock cultures incubated in nutrient
agar were transferred to test tube of Muller-Hinton broth (MHB) for bacteria
that were incubated for 24 hour at 37oC. The cultures were diluted
with fresh Muller-Hinton broth to get 2.0Χ106 CFU/ml for bacteria.
The Muller Hinton Agar (MHA) plates were prepared by pouring 15 ml of molten
media into sterile petri plates. The sample was loaded placed on the surface of
the cultured agar plates and incubated at 37oC for 24 hours then
inhibition zones formed around the disc were measured and the results were
compared with standard antibiotic, Chloramphenicol. For agar well diffusion
method, the fungal strains were suspended in sabourauds dextrose broth for 6
hour to give concentration 105 CFU/ml and then inoculated with the
culture medium. A total of 8 mm diameter wells were punched into the agar and
filled with the sample and solvent blanks (hydro alcohol and hexane). Standard
antibiotic, Fluconazole (concentration 1 mg/ml) was used as positive control
and fungal plates were incubated at 37oC for 72 h. The diameters of zone of
inhibition observed were measured.
RESULTS AND
DISCUSSION:
Synthesized ZNPs are known in the solution by the color
changing from pale green to colorless due to Zn metal ion reduction and from
colorless to dark brown color due to capping of stabilizing agent, curcumin.
The color change can be easily identified by the naked eye. It was clearly
indicates that the formation of well reduced and stabilized ZNPs.
UV-Vis spectra
studies:
The most
convenient and advance technique for characterization of curcumin is UV-Vis
spectroscopy. The synthesized turmeric curcumin (CR) was confirmed by the strong
broad absorption band observed at around 425 nm. This can be due to either an n→π*
transition or a combination of π→π* and n→π*
transitions which is shown in Figure 1.
Fig. 1UV-Vis spectrum of
Curcumin
UV-Visible
spectroscopy can be used as a simple and reliable method for monitoring the
stability of nanoparticle solutions. The
absorption spectrum of Zn nanoparticle is shown in Figure 2. It exhibits a strong absorption
band at around 315nm which meant the formation of Zn nanoparticles. An excitonic
absorption peak is found at about 225nm might be due to the formation of
aggregation of Zn nanoparticles which lie much below the band gap wavelength of
315nm. It is also evident that significant sharp absorption of ZNPs indicates
the monodispersed nature of the nanoparticle distribution.
FT-IR analysis:
FT-IR spectroscopy was used to investigate the interactions between
different species and changes in chemical compositions of the mixtures. Figure
3 shows the FT-IR spectrum of curcumin. The important peaks observed from
curcumin are 3502 cm-1(Phenolic OH), 1625 cm-1
(C=O), 1520 1350 cm-1 (Aromatic C=C, 3 bands), 1272 cm-1
and 1024cm-1(C-O, 2 bands). Figure
4 shows the FT-IR spectrum of curcumin stabilized zinc nanoparticles. From
the data obtained, phenolic OH showed its weak broad band in the range of 3389
cm-1 which is assigned to (Ph-OH) group of curcumin moiety. The peak observed at 2926 cm-1
which can be assigned to the -OH stretching of water or ethanol present in the
system. The C=O stretching of curcumin at 1625 cm-1 was
shifted to a higher wave number at 1704 cm-1 due to interaction with
zinc nanoparticles. Three characteristic peaks in the range of 1520 1350 cm-1
conforms the aromatic unsaturation (C=C) of stabilized curcumin system. The
(C-O) band presence was assigned by the peaks found at 1000-1250 cm-1.
The small broad peak obtained at 1030 cm-1 might be due to some oxidation of
zinc nanoparticles, i.e., (Zn-O) stretching vibrations.
SEM studies
Morphology of synthesized zinc
nanoparticles was characterized by SEM analysis. The samples were placed in an
evacuated chamber and scanned in a controlled pattern by an electron beam.
Interaction of the electron beam with the specimen produces a variety of
physical phenomenon that detected, were used to form images and provide
information about the specimens. The SEM images of curcumin stabilized zinc
nanoparticles are shown in Figure 5. It can be view that the ZNPs formed are
well dispersed and evenly distributed in all direction. SEM images of those
compounds had shown very clear that most of the particles are cubic shaped
morphology of material. The incorporation of low concentrate HCl in the
synthesis process helped to avoid agglomeration of nanoparticles.
Fig. 2 UV-Vis spectrum of
Zn nanoparticles
Fig. 3FT-IR spectra of curcumin
Fig. 4FT-IR spectra of zinc nanoparticles
Fig. 5 SEM monographs of ZNPs
TEM studies
Figure 6 shows
the TEM image of the zinc nanoparticles. These images shows that the particles
formed are of nearly spherical morphology. The nanoparticles are moderately
dispersed and the average crystallite size of particles in the range of around
23-46 nm.
Fig. 6TEM monographs of ZNPs
From the TEM
pictures the aggregations of Zn nanoparticles were observed which confirms the
complex formation between curcumin and Zn nanoparticles which reveals the
medicinal property of synthesized nanoparticles.
Antibacterial activity:
The antibacterial
activity of curcumin and curcumin stabilized zinc nanoparticles was evaluated
against two gram positive bacteria (S.aureus, B.subtilis), two
gram negative bacteria (E.coli, S.bacillus). The compared
antibacterial activity resultswith Chloramphenicol are tabulated (Table 1).
|
|
|
|
Fig. 7Antibacterial
activity data of complexes
Table 1Effect of curcumin and zinc nanoparticles on antibacterial
activity
Bacterial Species |
Zone of inhibition diameter (mm sample-1) |
||
Standard drug (C) |
Curcumin (CR) |
Zinc nanoparticles (ZNPs) |
|
S. aureus |
16 |
13 |
18 |
B.subtilis |
18 |
16 |
19 |
E. coli |
20 |
17 |
16 |
S.bacillus |
21 |
15 |
18 |
Figure 7 exhibits the typical antibacterial test results of curcumin and
curcumin functionalized zinc nanoparticles obtained by disc diffusion method.
It is found that the curcumin stabilized ZNPs have exhibited an
appreciable inhibition activity whereas synthesized pure curcumin exhibited
moderate inhibition activity only. Zone of inhibition tests showed that ZNPs
have a very good antibacterial activity against S. aureus, B. subtilis
and S. bacillus strains.
Substantially, the zone of inhibition observed by ZNPs towards S. aureus and B. subtilis species has been revealed higher bactericidal activity
than standard drug, Chloramphenicol. Therefore, a significant difference is
observed from the screening tests that curcumin has shown low antibacterial activity due to its
poor bioavailability and it showed an excellent activity when it is
functionalized with ZNPs.
Antifungal
activity:
Curcumin and curcumin stabilized zinc
nanoparticles were determined for their antifungal activity against antifungal
activity was carried out by agar well diffusion method against four funguses (C.
albicans, C. lunata, A. niger, T. simii) and their comparable
activity results are tabulated (Table 2).
Table 2 Effect of Curcumin and zinc nanoparticles on antifungal
activity
Fungal Species |
Zone of inhibition diameter (mm sample-1) |
||
Standard drug (C) |
Curcumin (CR) |
Zinc nanoparticles (ZNPs) |
|
C.albicans |
19 |
16 |
17 |
C.lunata |
17 |
14 |
16 |
A.niger |
20 |
15 |
23 |
T.simii |
17 |
16 |
17 |
|
|
|
|
Fig. 8Antifungal activity
data of complexes
Figure 8 displays
the typical antifungal test results of curcumin and curcumin functionalized
zinc nanoparticles obtained by agar well diffusion method. The zone of
inhibition tests proved that the curcumin stabilized ZNPs has shown considerable
antifungal activity against all fungal species tested when compared with
synthesized pure curcumin which exhibited a lower inhibition activity only. The
obtained results indicated that curcumin stabilized ZNPs have significant
inhibition activity against A. niger
and T. simii species which was
greater than the activity observed by standard drug, Fluconazole. Moreover,
ZNPs has shown moderate inhibition activity against C. albicans and C. lunata
fungal species. Therefore, curcumin stabilized zinc nanoparticles reveals an
attractive antifungal activity over all.
CONCLUSION:
The present work demonstrated a simple and
easy synthesis method of curcumin from turmeric plant and preparation of zinc
nanoparticles through green route by using lemon extract as a reducer and
synthesized curcumin as a stabilizing agent. The experimental results indicated
that Zn nanoparticles obtained are in the range of 23-46 nm which are highly
stable and appreciable size. The synthesized ZNPs have exhibited an excellent
inhibition activity towards S. aureus, B. subtilis bacterial
strains and A. niger, T. simii fungal species. Interestingly,
the observed inhibition activity was greater than the standard drug tested
here. Therefore, the current study clearly provides a promising method to
synthesize ZNPs with a natural compound curcumin as a stabilizing agent and
prepared Zn nanoparticles are very improved in therapeutic efficacy as
antimicrobial agents.
ACKNOWLEDGEMENTS:
We gratefully
acknowledge Advanced
Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi for
TEM analysis and Nanotechnology
Research Centre, SRM University, Chennai for SEM analysis facility. We thank
PG and Research Department of Chemistry, V.O. Chidambaram College, Tuticorin
for providing IR spectral analysis facility and Department of Chemistry, SFR
College for women, Sivakasi for providing UV analysis facility. We also thank
AMET University, Chennai, India for their support to do this work.
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