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Study of Antioxidant Property of the Rhizome Extract of Roscoea purpurea Sm. (Kakoli) and its Use in Green Synthesis of Gold nanoparticles

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The antioxidant property of the rhizome extract of Roscoea purpurea (commonly known as Kakoli) has been studied against a long lived 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical at room temperature. The radical scavenging activity of the extract was compared with the naturally occuring antioxidant Ascorbic acid (Vitamin C) at the same concentration. The phytochemicals present in the rhizome extract have been utilized for the synthesis of gold nanoparticles under very mild conditions without any extra stabilizing agent. The synthesized gold nanoparticles were characterized by Surface Plasmon Resonance spectroscopy, High resolution transmission electron microscopy, X-Ray diffraction and FTIR studies.
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Bag et al. Int. J. Res. Chem. Environ. Vol.4 Issue 2 April 2014(174-180)
[174]
International Journal of Research in Chemistry and Environment
Vol. 4 Issue 2 April 2014(174-180)
ISSN 2248-9649
Research Paper
Study of Antioxidant Property of the Rhizome Extract of Roscoea purpurea Sm.
(Kakoli) and its Use in Green Synthesis of Gold nanoparticles
*Bag Braja Gopal, Dash Shib Shankar, Roy Avishek
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, INDIA
(Received 10th March 2014, Accepted 24th March 2014)
Available online at: www.ijrce.org
Abstract: The antioxidant property of the rhizome extract of Roscoea purpurea (commonly known as
Kakoli) has been studied against a long lived 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical at room
temperature. The radical scavenging activity of the extract was compared with the naturally occuring
antioxidant Ascorbic acid (Vitamin C) at the same concentration. The phytochemicals present in the
rhizome extract have been utilized for the synthesis of gold nanoparticles under very mild conditions
without any extra stabilizing agent. The synthesized gold nanoparticles were characterized by Surface
Plasmon Resonance spectroscopy, High resolution transmission electron microscopy, X-Ray diffraction
and FTIR studies.
Keywords: Astavarga, Roscoea purpurea (Kakoli), antioxidant, DPPH, rhizome, polyphenols, gold nanoparticle.
Introduction
Ayurveda (a Sanskrit word, Ayusmeans life
and ‘Vedameans science or knowledge) is a science
of long life practiced in India since the prehistoric
period. The aim of Ayurveda is to use the inherent
principles of nature to maintain and prolong the life of
a person by restoring a balance among body, mind and
spirit. In early days, the knowledge of Ayurveda used
to be taught orally in India through the lineages of
sages until it was collated into text thousands of years
ago. The Charak Samhita written by Maharshi Charak
(600 BCE) and the Sushruta Samhita written by
Maharshi Sushruta (600 BCE) are the conveyer of this
eternal tradition[1]. Astavarga, a set of eight medicinal
plants formulated by the reputed Ayurvedic wonder
healers Ashwini Kumars, could rejuvenate the old, frail
and emaciated body of the Rishi Chyavan and
miraculously restored his youth. Since then this
Ayurvedic preparation containing the set of eight
medicinal plants has been known as Chyavanprash
after the name of Rishi Chyavan and has been an
important and demanding Ayurvedic health tonic for
the Kings and the rich people. The eight Astavarga
plants namely Kakoli, Kshrikakoli, Jeevak, Rishvok,
Meda, Mahameda, Riddhi and Vriddhi grow naturally
in small patches in particular ecological environments
in the Himalaya especially in the North-Western
Himalaya at an elevation of 1200 4000 m above the
sea level. However, because of the lack of proper
documentation and written details, identification of the
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plants became difficult and illusory. Recent attempts
by a group scientists and sages have enabled the
proper identification of the eight Astavarga plants[2].
Thorough literature search by us have revealed that
there is no report of the active chemical constituents of
most of the Astavarga plants.
In recent years, the role of active oxygen
species and free radicals has been recognized as the
cause of various of physiological disorders, stress and
age related diseases including cancer, tumor, etc.[3,4].
Antioxidants including polyphenolic compounds
inhibit the cell damage and cell death by quenching the
free radicals generated by uncontrolled oxidation in
the physiology. Moreover, the antioxidants prevent
age related diseases, promote proper functioning of the
immune system, fight and prevent against cancer, etc.
To find out whether the Astavarga plants contain
antioxidants, we have studied the antioxidant
properties against a long lived 2, 2-
diphenylpicrylhydrazyl (DPPH) radical at room
temperature. Herein we report the results of our
investigations on the studies of antioxidant property of
the rhizome extract of Roscoea purpurea (Kakoli).
The radical scavenging activity of the extract was
compared with the naturally occurring antioxidant
Ascorbic acid (Vitamin C) at the same concentration.
The rhizome extract has also been utilized for the
green synthesis of gold nanoparticles under very mild
conditions without any additional stabilizing or
capping agents. The synthesized gold nanoparticles
have been characterized by Surface Plasmon
Resonance (SPR) spectroscopy, High Resolution
Transmission Electron Microscopy (HRTEM) and X-
Ray diffraction studies.
Material and Methods
Chemicals: DPPH was purchased from Sigma-
Aldrich. HAuCl4 was purchased from SRL. Ascorbic
acid (Vitamin C) was purchased from Merck. Ferric
chloride (FeCl3) was procured from Himedia. All the
chemicals were analytical grade and used without
further purification. Double distilled water was used
for the experiment.
Plant Material: The plant Kakoli was collected in the
Himalayan region at an altitude of 3000 m from the
sea level, identified by a group of scientist from
Patanjali and deposited at Patanjali. Medicinally
important Rhizome of this plant was used for the study
of anti-oxidant property and synthesis of AuNPs at
room temperature.
Au (III) stock solution: HAuCl4 (35.4 mg) was
dissolved in distilled water (10 mL) to obtain a 10.42
mM Au (III) stock solution.
Preparation of ethanol extract of rhizome of Kakoli
: Finely dried powdered of rhizome (5.93 g) was
suspended in ethanol (50mL) and refluxed with
continuous magnetic stirring for 3 h, cooled at room
temperature and then filtered using a sintered glass
funnel. Volatiles of the yellowish filtrate were
removed under reduced pressure to afford a light
yellow sticky material (0.34g). The extract (16.8mg)
was suspended in distilled water (10mL) and sonicated
for 15 minutes using an ultra sonicator bath to get a
semi transparent light yellow solution (1680 mgL-1).
Preparation of methanol extract of rhizome of
Kakoli: Rhizome of Kakoli was collected and dried in
air. Then it was finely powered using a grinder. Finely
dried powdered of Rhizome (5.94 g) was suspended in
methanol (50mL) and refluxed with continuous
magnetic stirring for 5 h, cooled at room temperature
and then filtered using a sintered glass funnel.
Volatiles of the greenish filtrate were removed under
reduced pressure to afford a greenish yellow sticky
material(0.350g). The extract (11.5mg) was suspended
in distilled methanol (10 mL) and sonicated for 5
minutes using an ultra sonicator bath to get a clear
greenish yellow solution (11.5 g/L).
DPPH assay: The most extensively used stable DPPH
radical was employed for the study of anti oxidant
property of rhizome extract of Kakoli. Anti oxidants
present in the extract react with DPPH radical and
convert it to 1-1-diphenyl-2-picryl hydrazine. The
color change from violet to yellow within 30 minutes
upon addition of rhizome extract to DPPH solution
indicate the anti oxidant property of the extract.
The reduction in the absorbance intensity at
517 nm in the UV-visible spectrum has also been used
to measure the antioxidant property of rhizome extract.
In this experiment different volume of the stock
solutions (1150 g/mL) was mixed with methanolic
solution of DPPH (0.04 mL each) and diluted to 4 mL
where concentration of plant extract varies from 20-
120 g/mL. The solutions were allowed to keep in
dark at room temperature for 1 hour. The UV-visible
spectrum of the colored solution was measured and the
absorbance at 517 nm was followed. Reduction in
absorption intensity at 517 nm was observed when
compared with a control solution of DPPH in methanol
at the same concentration. % scavenging was
calculated using the following formula.
OD = Optical Density. The radical scavenging
activity was compared with ascorbic acid at the same
concentration.
Synthesis of gold nanoparticles: Aliquots of Au (III)
solution (0.2 mL, 10.42 mM each) were added drop-
wise to the rhizome extract (1680 mgL-1) to prepare a
series of stabilized AuNPs where concentration of the
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rhizome extract varies from was 100, 200, 300, and
400 mgL-1 and the concentration of Au (III) was fixed
at 0.42 mM. UV-visible spectrum of the colloids was
taken after 15 hours of HAuCl4 and rhizome extract
were mixed together.
Characterization:
Figure 1: Photographs of Roscoea purpurea Sm. (Kakoli): (a) the whole plant, (b) flower of Kakoli, (c)
rhizome of Kakoli
Figure 2: Antioxidant activity studies of the rhizome extract of Kakoli: (i) UV-visible spectra of (a) Extract,
(b) DPPH, (c) DPPH + ethanol extract, Inset: photographs of the vials containing the respective solutions;
(ii) reaction scheme showing quenching of DPPH by the antioxidant (A-H); (iii) plot of % DPPH radical
scavenging by the methanol extract of rhizome at 20, 40, 60, 80, 100, 120 g/mL concentration and its
comparison with ascorbic acid (120 g/mL, brown); (iv) and (v) UV-visible spectra of DPPH with increasing
concentration of the methanol extract of rhizome (RE)
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HRTEM images of AuNPs were taken from
JEOL 2100 instrument. X-ray diffraction (XRD)
patterns of the stabilized AuNPs were recorded Rigaku
Miniflex II diffractometer with Cu-Kα radiation (λ=
1.54 Ǻ). Mass spectra were recorded in Shimadzu
GCMS QP 2100 Plus instrument. UV-visible
spectrophotometry was carried out in Shimadzu 1601
spectrophotometer. FTIR spectra of samples were
analyzed using a Perkin Elmer FTIR Spectrum Two
model using KBr pelllet.
Results and Discussion
Kakoli is a perennial rhizomatous herb up to
15-30 cm in height (Figure 1). It is found in alpine
grassland, grassy hillsides and stony slopes of central
to eastern Himalaya from Uttarakhand to Assam and
Sikkim, up to an altitude of 3300m. Its roots
(rhizomes) are light brown in color, thick, fleshy and
bundled (Figure 1a,c). The rhizomes are used for the
treatment of haematemesis, excessive thirst, rheumatic
pain, etc. Immunostimulant properties of the ethanolic
extract of rhizomes have recently been demonstrated.5
Mass spectral studies of the rhizome extract in
our laboratory revealed the presence of several
polyphenolic compounds including flavanoids along
with steroids and other plant secondary metabolites
(supporting information Figure S1). A positive ferric
chloride test also supported the presence of phenolic
compounds along with other easily oxidizable
compounds. Realizing the significance of antioxidants
in the prevention of age related diseases and cell
damage, we investigated the anti-oxidant properties of
the alcoholic extract of rhizome against a stable DPPH
free radical at room temperature.
Determination of Antioxidant activity by DPPH
Assay: The DPPH assay is one of the most popular
and frequently employed methods to test the ability of
compounds to act as free radical scavengers or
hydrogen donors, and to evaluate the antioxidant
property of food and medicine. The radical
scavenging activity of the rhizome extract of Kakoli
was tested against a methanolic solution of DPPH
according to the procedure described in the literature.6
The reducing ability of antioxidants towards DPPH
radical can be evaluated by monitoring the change in
the absorbance intensity at 517 nm in the UV-visible
spectrum. Interestingly, on treatment of the methanol
extract of rhizome of Kakoli with DPPH solution,
decrease in intensity of its deep violet color was
observed visually (Figure 2(i)). With increasing
concentration of the methanol extract, the decrease in
the absorption intensity of DPPH at 517 nm was
observed (Figure 2(iii), (iv), (v)). The % of radical
scavenging activity calculated were 20.2, 40.0, 48.0,
55.2, 63.6 and 70.7 when concentration of the rhizome
extract were 20, 40, 60, 80, 100, and 120 g/mL.
Ascorbic acid (Vitamin C), a naturally occurring anti-
oxidant has been widely used as a standard to compare
the antioxidant efficacy of a sample. Significant
antioxidant activity of the rhizome extract was evident
from the DPPH assay by comparing the % DPPH
scavenging values of the rhizome extract and ascorbic
acid at 120 g/ml (Figure 2(iii)).
Synthesis of AuNPs: Gold nanoparticles (AuNPs)
with its unique optoelectronic and magnetic properties
have found applications in nanobiodiagnostics,
pharmaceuticals, catalysis, etc.7,8,9,10,11 Many of such
applications require the AuNPs to be dispersed in
water and stabilized with non-toxic biomolecules to
avoid any undesired environmental effects[12]. The
green syntheses of AuNPs from the extracts of Acacia
nilotica[13],Punica granatum[14], Saraca indica bark[15],
Ananas comosus (L.)[16], Terminalia arjuna bark[17],
Ocimum sanctum stem[18], Azadirachta indica bark[19],
have recently been reported. The polyphenolic
compounds along with other easily oxidizable
phytochemicals present in the plant extracts were
capable of forming AuNPs from Au(III) and then
stabilize them in aqueous medium. As the rhizome
extract of Kakoli is rich in polyphenolic compounds
along with other easily oxidizable compounds, it
occurred to us that it can also be utilized for the
efficient synthesis of AuNPs. To test this, we treated
the rhizome extract of Kakoli with an aqueous solution
of HAuCl4. Interestingly, the color of the rhizome
extract changed from light yellowish to bluish violet
within 2 h indicating the formation of AuNPs. With
increasing concentration of the plant extract increase
in intensity of color was observed (Figure 3).
Figure 3: UV-visible spectrum of (a) HAuCl4 and
(b-e) colloidal AuNPs at 100, 200, 300, 400 mgL-1
concentration of rhizome extract.
Inset: photograph of vials containing gold
nanoparticles
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UV-visible spectroscopy of the solutions
containing HAuCl4 and varied amounts of the rhizome
extract was carried out to find out the shifts in the
absorption maxima. Two peaks at 220 and 290 nm
were observed in the UV-visible spectrum of HAuCl4
(0.52 mM) solution due to charge transfer interaction
between the metal and chloro ligands. On addition of
the rhizome extract to HAuCl4, decrease in intensity of
these two peaks were observed with concomitant
formation of a new peak around 560 570 nm due to
Surface Plasmon Resonance (SPR) of the AuNPs.
With gradual increase in concentration of the rhizome
extract from 100 mgL-1 to 200, 300 and 400 mg L-1, a
red-shift of the absorption maxima was observed
respectively from 563.5 nm to 564.0, 567 and 571.5
nm. Such a red-shift of the absorption maxima has
been observed by us previously20 and others21
suggesting the formation of assembly of AuNPs.
Formation of flower-like assembly of AuNPs was
indeed observed by HRTEM study (discussed below).
HRTEM, FTIR and XRD studies: The size
distribution, shape and morphology of the AuNPs were
studied by high resolution transmission electron
microscopy (HRTEM) (Figure 4). Mostly spherical
shaped AuNPs of 6 nm average diameter (calculated
from 60 spherical particles) were observed at 400
mgL-1 concentration of rhizome extract. The AuNPs
have a tendency to assemble together to form flower-
like assemblies of 20-200 nm diameters (Figure 4d-g).
This observation is also supported by the red-shift
observed by SPR spectroscopy (discussed earlier). A
thin layer of the colloidal AuNPs coated over a glass
plate were analyzed by X-ray diffraction. The
reflections of the planes (111), (200), (220) and (311)
at 2 = 38.39, 44.37, 64.86 and 76.73 respectively
resembled the characteristic reflections of crystalline
metallic face centered cubic Au (JCPDS file no. 04-
0784).
FTIR spectra of rhizome extract of Kakoli and
the stabilized AuNPs synthesized from it were
compared to investigate the biomolecules present in
the rhizome extract and their roles in the stabilization
of AuNPs. In the FTIR spectrum of the rhizome
extract of Kakoli, a peak around 3422 cm-1 appeared
due to the symmetric stretching vibration of
intermolecularly H-bonded ‘OH’ groups present in the
phenolic compounds. The peak in the vicinity of 2926
cm-1 is due to saturated CH stretching vibration. The
peak at 1643 cm-1 is the characteristic of ‘C=O’
stretching of amide-I. A sharp peak with poor intensity
appeared at 1075 cm-1 might be due to ‘C-O’ bond
stretching vibration[14]. Interestingly, in the FTIR
spectrum of stabilized AuNPs the peak due to ‘OH’
group became narrower and shifted to slightly higher
region possibly due to partial destruction of H-bonding
among the phenolic hydroxyl groups and their
interactions with AuNPs.
Mechanism of the formation of Stabilized AuNPs:
The rhizome extract of Kakoli rich in different types of
plant secondary metabolites such as flavanoids,
tannins, saponins, etc. Evidence for the presence of
polyphenolic compounds was obtained from the ferric
chloride test (supporting information).
Figure 4: (a-c) HRTEM Images Kakoli rhizome stabilized AuNPs at 400 mgL-1 concentration of rhizome
extract; (d-g) flowere-like assembly of AuNPs; (h) histogram of particle size distribution
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Figure 5: Mechanism of formation and stabilization of AuNPs by phytochemicals present in the rhizome
extract
Figure S1: Mass spectrum of methanolic extract of rhizome of Kakoli. Probable structures of the
compounds have shown by arrows
Mass spectral analysis (supporting information figure
S1) of the rhizome extract carried out by us also
supported the presence of the quercetin, kaempferol,
pinocembrin, galangin, quinic acid or their analogues
(supporting information S2). A schematic
representation of the possible mechanism for the
formation of AuNPs and their stabilization by the
phytochemicals is shown in Figure 8. Ortho-dihydroxy
compounds along with other phytochemicals can
reduce Au (III) to Au (0) with concomitant oxidation
of the phytochemicals to a higher oxidation state.
Collision of the neighboring Au (0) atoms with each
other leads to the formation of the AuNPs. The
AuNPs can be stabilized by the polyphenolic
compounds, quinones as well as the other coordinating
phytochemicals present in the rhizome extract.
Supporting Information: To test the presence of
polyphenolic compounds in the rhizome extract of
Kakoli, FeCl3 test was performed. A freshly prepared
aqueous solution of FeCl3 was added to the rhizome
extract and shaken at room temperature. Instant
appearance of greenish color indicated the presence of
phenolic compounds in the rhizome extract of Kakoli
at room temperature.
Conclusion
Evidence for the presence of antioxidants
including polyphenols has been obtained in the
rhizome extract of Roscoea purpurea (Kakoli). The
antioxidant activity of the rhizome extract has been
studied against the long lived 2,2-
diphenylpicrylhydrazyl (DPPH) radical at room
temperature. The radical scavenging activity of the
extract has been compared with the naturally occurring
antioxidant Ascorbic acid (Vitamin C) at the same
concentration. Very efficient radical scavenging
activity of the rhizome extract of Kakoli opens up its
use the prevention of free radical related diseases. The
phytochemicals present in the rhizome extract have
been utilized for the synthesis of gold nanoparticles at
room temperature under very mild conditions without
any additional stabilizing agents. The synthesized
gold nanoparticles have been characterized by Surface
Plasmon Resonance spectroscopy, HRTEM and X-Ray
diffraction studies and a mechanism for the synthesis
of the gold nanoparticles has been proposed.
According to our knowledge, this is the first report of
the study of antioxidant properties of the rhizome
extract of Kakoli and its utilization in the green
synthesis of gold nanoparticles. As the rhizome extract
of Kakoli has tremendous medicinal significance, the
studies described will be useful in biomedical
Bag et al. Int. J. Res. Chem. Environ. Vol.4 Issue 2 April 2014(174-180)
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applications as well as nanoscience and
nanobiotechnology.
Acknowledgement
We gratefully acknowledge the Patanajali
Yogpeeth, Haridwar for the generous gift the rhizome
sample of Kakoli. We thank Acharya Balkrishnaji,
Anupam Srivastavaji, Shambhu Patelji for helpful
discussions, cooperations and help. BGB thanks CSIR
and UGC for funding. SSD thanks CSIR, New Delhi
for senior research fellowship.
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