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The
plant-mediated synthesis of noble metal nanoparticles is widely used for the
preparation of allopathic nano drugs with enhanced bio-efficacy. However,
little attention has been paid towards the use of such green nanotechnological
approach for the bio-fabrication of noble metal nanoparticles using homeopathic
mother tincture (highly dilute plant extracts) for the enhancement of
pharmacological activities. The enhancement in pharmacological bio-efficacy
chiefly depends on the shape and size of the nanoparticles. Among the various
methods used for size segregation of nanoparticles, post-synthesis segregation
of nanoparticles using sucrose density gradient centrifugation is considered a
fast, simple and green method for improving solution-phase heterogenous metal
nanoparticles to homogenous nature with desirable size.
The
present communication warrants the synthesis of gold nanoparticles using Calendula officinalis homeopathic mother
tincture in a single-pot green process, accompanied by thorough characterization
for their optical property, surface morphology, crystalline lattice, particle
size and zeta potential. Post synthesis size segregation has been carried out
using sucrose density gradient centrifugation. The fractionated nanoparticles
sized in the narrow range (Z-average 28.13 to 13.24 nm) have been monitored to
study the effect of nano-sizing on the enhancement in the free radical
scavenging activity (DPPH assay). The
nanosized fractionated gold nanoparticles (Z-average 13.24 nm) exhibited elevated
free radical scavenging activity (28.17%) compared to native Calendula officinalis homeopathic mother
tincture (Z-average 8217 nm). A tentative mechanism has been put forward
explaining the observed enhancement.
Keywords: Density gradient centrifugation, Calendula
officinalis, Homeopathic mother
tincture, Gold nanoparticles, DPPH assay
Abbreviations: Cm@AuNp: Gold Nanoparticles Embedded with Calendula officinalis Homeopathic Mother Tincture; UV-Vis Spectroscopy: Ultra-Violet-Visible Spectroscopy;
SEM: Scanning Electron Microscope; XRD: X-Ray
Diffraction; DLS: Dynamic Light Scattering; RPM: Revolutions per Minute;
DPPH: 2,2-Diphenyl-1-Picrylhydrazyl Radicals; JCPDS: Joint Committee on Powder Diffraction Standards
The growing popularity of complementary
medicine has been accompanied by a call for controlled clinical studies to
examine the efficacy and cogency of its mode of action [1]. Homeopathy is one
of the important complementary medicine and a time-tested two-century-old
empirical system of healing. Homeopathic medicines are prepared through a
characteristic process known as potentization, where serial dilutions are
performed with strong strokes at each step of dilution [2]. Homeopathy is
controversial because its most medicines do not contain one single molecule of
the corresponding starting substance and earlier has been labeled as the
placebo effect. However, homeopathy elicits much more than placebo responses.
It is an empirical puzzle and a challenge to its
orthodox scientific models. In recent perspectives, with the advent of new
tools supplied by nanoscience and nanotechnology, novel research pathways of
homeopathic medicines are to be opened [3].
Size and shape are important parameters for
nanoparticle-based drugs [4] that control the kinetics of internalization [5],
biodistribution [6], cellular membrane deformability [7] and cargo loading
efficiency [8]. It has been shown that smaller nanoparticles escape natural
body clearance mechanisms more efficiently and hence circulate longer in the
blood [9]. In addition, the shape plays an important role in demonstrating that
cylindrical nanoparticles interact with cells very differently to spherical
ones, resulting in dramatic changes in the bio-availability [10]. The
preparation of nanoparticles with the well-defined size is essential for
accurate control of chemical and physical properties [11]. Size-controlled
nanoparticles find its application in medical science, biotechnology
self-assembly, electronics, diagnostics, and sensing [12,13]. The various
influencing factors and different synthetic methods make it challenging to
synthesize nanoparticles with uniform size and morphology. Therefore,
post-synthesis separation methods remain a more feasible way to procure
monodisperse nanoparticles [14]. Several techniques have been employed to
separate nanoparticles of desired size and shape. Techniques such as magnetic
field flow fractionation filtration [15], size exclusion chromatography [16],
size selective precipitation [17], density gradient centrifugation [18] and
cross-flow filtration [19] have been utilized to produce certain nanoparticle
fractions with narrow shape and size distributions. Recently, centrifugation has
been proven to be a more effective and easy method to segregate nanoparticles
based on their size due to its high efficiency, capability of scalable
production, and free of nanoparticle aggregation [20,21]. Density gradient
centrifugation is cost- effective, fast, easy and highly versatile method used
for separation of nanoparticles according to their size [22].
In continuation of our work pertaining to the green
synthesis, characterization and enhancement of various pharmacological
bio-efficacy [23-27], the present piece of work demonstrates the single pot
green synthesis of gold nanoparticles (Cm@AuNp)
using homeopathic mother tincture Calendula
officinalis, thorough characterization and post-synthesis size segregation
of bio-fabricated gold nanoparticles using sucrose density gradient
centrifugation. Further, the effect of size of segregated gold nanoparticles on
the antioxidant bio-efficacy has been studied.
MATERIALS
AND METHODS
Calendula
officinalis homeopathic mother tincture was purchased from the homeopathic
store of SBL India. The synthesis of
gold nanoparticles was carried out using different dilutions of Calendula officinalis homeopathic mother
tincture, keeping the concentration of NaAuCl4.2H2O
solution constant as a function of pH.
Characterization
of Cm@AuNp
The Cm@AuNp was
characterized for optical properties using UV-Vis spectrophotometer (Lab India,
India). The morphology of Cm@AuNp was
studied using SEM (Table tops
SNE-3200M, USA). The X-ray diffraction pattern of Cm@AuNp was recorded using XRD
(Bruker AXS D8 Advance, Germany) over 35°-75° with scan run 40/min, step size
of 0.02° and Cu Kα radiation of λ=1.54Å. The hydrodynamic size distribution
with poly-dispersity index (PDI) and
zeta potential was analyzed using zeta sizer (Nano ZS90 model Malvern,
Germany).
Density
gradient centrifugation (DGC)
Sucrose density
gradient method was lucratively used to fractionate anisotropic Cm@AuNp having different sedimentation rates. A
discontinuous density gradient of sucrose solutions (5%, 10%, 15% and 20%) was developed upon one another. Colloidal solution of Cm@AuNp (5 mL) was added to the capacity
vial and centrifuged at 5000 RPM for
15 min. Nanoparticles depending upon the size were confined to different layers
of the sucrose density gradient. Each layer was separated using a sterile syringe and was further
monitored for the determination of the size of separated nanoparticles using
zeta sizer. Sedimentation coefficient S was calculated for each layer using the
equation: 𝑆 = (𝜌𝑝 − 𝜌𝑚)d2 / 18𝜂
DPPH
radical scavenging activity
The anti-oxidative effect of sucrose gradient concentrations (5%, 10%, 15%, 20%) containing fractionated Cm@AuNp and native homeopathic mother tincture was determined using DPPH assay. Ascorbic acid (1 mM) was used as a positive control. The test samples (2 ml) were incubated with DPPH ethanolic solution (1 ml; 1 mM). The contents after vigorous mixing were allowed to stand for 30 min at room temperature. The absorbance was measured at 517 nm and the free radical scavenging activity was calculated using the formula: (C-T)/C × 100
Where, C is absorbance of control and T is the
absorbance of the test sample.
RESULTS
AND DISCUSSION
Optimized experimental conditions
of bio-fabricated gold nanoparticles were as follows: Calendula officinalis homeopathic mother tincture with 40% dilution
(1 ml), sodium tetrachloroaurate dehydrates solution (1 ml; 1 mM) and
sonication (15 min; 20 KHz) at pH 8. The conversion of Au (III) to its elemental
form (Au°) may be ascribed to the presence of flavonoidal moieties present in Calendula officinalis homeopathic mother
tincture which is nothing but an ethanolic extract of the plant Calendula officinalis. The visual change
in color from pale yellow to ruby red indicated the bio-fabrication of target
nanoparticles. Calendula officinalis
extract has been reported to contain a series of polyphenolics flavonoidal
compounds namely quercetin, isorhamnetin, isoquercetin, narcissin,
isorhamnetin-3-O-β-D-glycoside, calendoflaside, calendoflavoside,
calendoflavobioside, rutin, isoquercitrin, neohesperidoside,
isorhamnetin-3-O-neohesperidoside, isorhamnetin-3-O-2G-rhamnosyl rutinoside,
isorhamnetin-3-O-rutinoside, quercetin-3-O-glucoside and
quercetin-3-O-rutinoside [28-30]. The bio-fabrication of Cm@AuNp using polyphenolics (flavonoids) present in the homeopathic
mother tincture can be explained by the following reaction:
Flavonoidal content not only reduce Au3+ to Au0 but also get coated (capping) on the freshly generated Cm@AuNp and thus adding their medicinal properties [24].
Characterization of gold nanoparticle UV-Vis spectrophotometer
Bragg diffraction peaks 2θ
appeared at 38.17°, 44.23° and 64.29° in the bio-fabricated Cm@AuNp (Figure 2). It could be indexed to (111), (200) and (220) having lattice planes of
face-center cubic compared with (JCPDS file 04-0784). The intensity of the
diffraction peaks (200) and (220) were found lower than the corresponding
crystallographic plane (111). The fact established that the lattice plane (111)
is the transcendent crystallographic plane and is more reactive because
of its high atom density [31].
SEM
SEM images (Figure 3) acquired from the drop-coated film of nanoparticles
indicated their polydisperse spherical shaped morphology.
Dynamic
light scattering
The
dynamic light scattering (DLS)
spectrum highlighted the asymmetric distribution of nanoparticles mainly in the
range (21 to 1281 nm) with poly-dispersive index 0.664 and intercept 0.922 (Figure 4a). However, a little
population was extended in the range of 6000 nm. The average hydrodynamic size
(Z-Average) of Cm@AuNp was found to
be 265.6 nm. Zeta potential of Cm@AuNp determined
in water medium as a dispersant was -20.8 mV (Figure 4b). The magnitude of observed high negative charge on the
bio-fabricated nanoparticles might be acting as a repulsive barrier, avoiding
aggregation of nanoparticles.
Density
gradient centrifugation (DGC)
The size fractionation of Cm@AuNp centrifuged at 5000 RPM for a
fixed time (15 min) are shown in (Figure
5). Different bands obtained after the run indicated successful segregation
of Cm@AuNp as per their sedimentation
rates.
Sedimentation coefficients for Cm@AuNp at 5%, 10%, 15% and 20% sucrose
concentration gradient (Figure 7)
were 95.1 × 10-3 s, 261.02 × 10-3 s, 1121.47 × 10-3
s and 1717.22 × 10-3 s, respectively indicating faster sedimentation
rate of heavier particles. Overall results obtained demonstrate the utility of
density gradient technique to separate mixtures of different size distributions
of nanoparticles to near monodispersity.
DPPH
radical scavenging activity
DPPH is a stable
nitrogen-centered free radical, the color of which changes from violet to
yellow upon the reduction by either the process of hydrogen or electron
donation. Substances that perform this reaction can be considered as
antioxidants and, therefore, radical scavengers [32]. Natural antioxidants
(secondary metabolites of plants) are limited in their radical scavenging
action; therefore, recent efforts are focused on the enhancement of the free
radical scavenging activity of the natural antioxidants by synthesis of noble
metal nanoparticles prepared from different plant extracts. In the present
study Calendula officinalis homeopathic
mother tincture was used to bio-fabricate gold nanoparticles. The free radical
scavenging activity of different sucrose gradient fractions (20%, 15%, 10%, 5%)
containing Cm@AuNp 5 ml each and
native mother tincture was tested. As the size of Cm@AuNp decreases (20% to 5% sucrose gradient fraction) free
radical scavenging activity increases which can be visualized by the change in
color from dark brown to yellow exhibiting size dependent free radical
scavenging activity (Figure 8). The
fact finds support (Table 1) from
the higher free radical scavenging activity of fractionated Cm@AuNp (Z-average (28.13 to 13.24 nm)
compared to native Calendula officinalis
homeopathic mother tincture (Z-average 8217 nm). A perusal of the Figure 9 further highlights that with
the decrease of the size of Cm@AuNp
(Z-average to 13.24 nm), the free radical scavenging activity is increased from
(67.07% to 79.3%). However, sharp increase in the free radical scavenging
activity has been observed with the change in the size of the nanoparticle
(Z-average 26.25 to 15.51 nm). Free radical scavenging activity of segregated Cm@AuNp (5% sucrose gradient fraction),
prepared from native Calendula
officinalis homeopathic mother tincture overall reports 28.17% enhancement (Table 1). The observed enhancement in
free radical scavenging activity may be assigned to the biocompatibility,
astonishing optical properties related to surface plasmon resonance and high
surface area to volume ratio (nanosizing) overall improving permeability in the
cell membrane and higher accumulation of the Cm@AuNp.
The bio-fabricated gold
nanoparticles embedded with flavonoids (Cm@AuNp)
possibly might release 3 electrons for scavenging of 3 free radicals
(enhancement) as depicted in the following reaction [33].
The
simultaneous coating of polyphenolic flavonoids of Calendula officinalis extract on freshly prepared nanoparticles
also contributes in the enhancement of free radical scavenging activity [24,26].
CONCLUSION
The present
communication reports the bio-fabrication of Cm@AuNp using homeopathic mother tincture Calendula officinalis in a single step green method. After thorough
characterization of bio-fabricated gold nanoparticles embedded with biomolecules of Calendula
officinalis mother tincture
(Cm@AuNp) have been
fractionated into smaller
sized gold nanoparticles using sucrose density
gradient centrifugation method.
The fractionated nanoparticles ranging
in the size (Z-average 28.13
to 13.24 nm) are monitored for the effect
of size of nanoparticles on the enhancement in the free radical
scavenging activity (DPPH assay). The enhancement in free radical
scavenging activity (28.17%)
of minimum sized
fractionated (Cm@AuNp: 5%
sucrose gradient fraction) having Z-average 13.24 nm compared to native Calendula officinalis homeopathic mother
tincture (Z-average 8217 nm) have been recorded.
ACKNOWLEDGEMENT
The authors gratefully acknowledge Prof. P. K.
Kalra, Director, and Prof. Sahab Dass, Head Department of Chemistry, Dayalbagh
Educational Institute for providing necessary facilities. Authors also thank
Dr. Vijai Kumar, Advisor, Medical and Health Practices, Dayalbagh Educational
Institute for discussions.
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