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International Journal of Nanomaterials and Biostructures

Universal Research Publications. All rights reserved

ISSN 2277-3851
Original Article
Evaluation of Invitro Antidiabetic Activity of Sphaeranthus
Amaranthoides Silver nanoparticles
Swarnalatha.L*, Christina Rachela, Shruti ranjanb, Payas baradwajc
*Lecturer, Department of Biotechnology, Sathyabama University, Chennai-119.
Corresponding author: Dr.L.Swarnalatha,lecturer,
Department of Biotechnology, Sathyabama University,
Jeppiar nagar, Chennai, India-600119,
E.mail: lokiswarna@gmail.com
Received 20 June 2012; accepted 19 July 2012
Abstract
Diabetes is a syndrome which is characterized clinically as hyperglycemia due to absolute or relative deficiency of insulin
production and progressive resistance of insulin. In recent years there is a fast increase in the occurrence and prevalence of
diabetes mellitus. In the present study sphaeranthus amaranthoides was screened for antidiabetic activity in invitro. In the
present search synthesis of silver phyto nanoparticles and their antidiabetic activity were studied. This is the first attempt of
introducing silver herbal nanoparticles isolation and antidiabetic assessment. This is because silver nanoparticles possess a
very high surface to volume ratio. This can be utilized in areas where high surface areas are critical for success. This could
for example be in the catalytic industry. In biology and biochemistry nanoparticles have attracted much attention. The aim
of the current work is to screen for invitro inhibition of alpha-amylase enzyme activity of extract of sphaeranthus
amaranthoides silver nanoparticles. The silver nanoparticles showed a dose response inhibitory activity on α-amylase.
Acarbose was used a standard drug. The IC50 value for plant extract is 0.28g/ml where as for acarbose is 0.75g/ml.
© 2011 Universal Research Publications. All rights reserved
Key words: Sphaeranthus amaranthoides, antidiabetic activity, α-amylase, hyperglycemia, silver nanoparticles.3
Introduction: side effects. During pregnancy diabetes may cause serious
Diabetes mellitus results from the defects in the insulin problems in both mother and child, however to overcome
secretion and action, this may be characterized by chronic these problems synthetic agents are used vigorously these
hyperglycemia, which is connected with the carbohydrates, are not suitable for continuous use due to side effects[5]
protein and lipid metabolism [1]. Globally mortality rate such as development of hypoglycemia, weight gain,
9% is recorded due to the diabetes. Diabetes mellitus a gastrointestinal disturbances, liver toxicity etc[6]. Based on
well known endocrine disorder and it is most common in the recent studies antioxidants capable of neutralizing free
India now a day. The reason may be life style and genetic radicals are effective in preventing experimentally induced
factors[2]. Due these factors the diabetic monocytes diabetes in animal models [7,8] as well as reducing the
produce increased superoxide anion. (O2-)[3]. In premature severity of diabetic complications9.
atherosclerosis and oxidative stress patient’s diabetes is a Silver nanoparticles are widely used for its unique
major risk factor. Over the centuries, herbal drugs have properties in catalysis, chemical sensing, biosensing,
served as a major source of medicines for the prevention photonics, electronic and pharmaceuticals[10] and in
and treatment of diseases including diabetes mellitus. There biomedicine especially for antibacterial agent[11] and
are more than 200 species of plants exhibit hypoglycemic antiviral agent[12]. These properties can be extended to
properties, including many common plants, such as antidiabetic activity along with the plant extracts. The most
pumpkin, wheat, celery, wax gourd, lotus root and bitter important application of silver and silver nanoparticles is in
melon but the basis of this activity is frequently not medical industry such as tropical ointments to prevent
investigated[4]. infection against burn and open wounds[13]. Biologic
There are many synthetic hypoglycemic drugs to manage synthesis of nanoparticles by plant extracts is at present
post-prandial hyper-glycaemia at digestive level, under exploitation as some researchers worked on it[14, 15]
glucosidase and amylase inhibitors such as acarbose, and testing for antimicrobial activities[16,17].
miglitol and voglibose, but these drugs may cause many In the present study the plant sphaeranthus amaranthoides

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25

Fig.1(a) UV-Vis spectra after 24hrs incubation
Fig.1(b) UV-Vis spectra after 48hrs incubation
extract is made in to silver nanoparticles and investigated
for antidiabetic activity. These silver nanoparticles can
make the plant more effective. This plant is already studied
for the phytochemical analysis and antimicrobial
activity[18].
Materials and methods:
Biogenisis of silver nanoparticles: The whole plant was
used for the experimental purpose. The plant was shade
dried and ground to a fine powder. One mM silver nitrate
was added to the plant extract to make up a final solution of
200 mL and centrifuged at 18,000 rpm for 25 min. The
collected pellets were stored at 4°C. The supernatant was
incubated for 24hrs. A change in the colour of the
solutions was observed during incubation process. The
reduction of pure Ag2+ ions were monitored by measuring
the UV-Vis spectrum of the reaction media after 24hrs by
diluting a small aliquot of the sample in distilled water by
using Systronic 118 UV-Visible Spectrophotometer. Thin
films of the samples were prepared on a carbon coated
cupper gird and SEM analysis of both the samples was
carried out using Hitachi S-4500 SEM Machine. The
EDAX measurements of the silver nanoparticles of the bark
extracts were performed on Hitachi S-3400 NSEM
instrument equipped with thermo EDAX attachments.
Assay for α-Amylase Inhibition:
In monosaccaride glucose can be readily absorbed from
the gastrointestinal tract into the blood stream after the
hydrolysis of glycosidic bonds in digestible carbohydrate
foods containing starch by the enzyme α-amylase.
Inhibition of these enzymes reduced the high post prandial
blood glucose peaks indiabetics. In this study, α-amylase
inhibition in vitro model was used to screen the silver nano
International Journal of Nanomaterials and Biostructures 2012; 2(3) 25-29
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particles of sphaeranthus amaranthoides to evaluate their
potential hypoglycaemic effects. The bioassay method was
adopted from Conforti et al (19). A starch solution (0.5%
w/v) was obtained by stirring 0.1g of potato starch (Sigma)
in 25ml of 20mM sodium phosphate buffer with 6.7mM
sodium chloride, pH 6.9 at 65°C for 15min. The enzyme
solution was prepared by mixing 0.0253g of (α-amylase in
100ml of cold distilled water. Silver nano particles of
Sphaeranthus amaranthoides extract is dissolved in buffer
to give a final concentration from 1mg/ml to 12.5 mg/ml.
The colorimetric reagent was prepared mixing a sodium
potassium tartrate solution (12.0g of sodium potassium
tartrate, tetrahydrate in 8.0ml of 2M NaOH) and 96mM
3,5-dinitrosalicylic acid solution. Both control and plant
extracts were added with starch solution and left to react
with (α-amylase solution under an alkaline condition at
25°C.
The reaction was measured over 3min. The generation of
maltose was quantified by the reduction of 3,5-
dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. This
reaction (corresponding to colour change from orange-
yellow to red) is detectable at 540nm. In the presence of an
(a-amylase inhibitors less maltose would be produced and
the absorbance value would be decreased. Preliminary
experiments were carried out to establish optimal
conditions and these were found necessary: starch 0.25%
w/v; (α-amylase 1 unit/ml; inhibitor concentration 1mg/ml.
Statistics Data were expressed as means ± S.D. Statistical
analysis was performed using Student’s t test. Differences
were considered significant at p≤0.05. The inhibitory
concentration 50% (IC50) was calculated from the Prism
dose response curve (statistical programme) obtained by
plotting the percentage of inhibition versus the
concentrations.
% Inhibition = 100- % reaction ± SD
Results and discussion:
Characterization of synthesized silver nanoparticles:
Silver nanoparticles are formed by reduction of the aqueous
Ag+ during exposure to the ethanol extract of sphaeranthus
amaranthoides were followed by UV–vis spectroscopy. It
is well known that silver nanoparticles exhibit reddish-
brown in water[20]. After 24 h of the incubation process
silver nanoparticles showed reddish-brown color, suggested
the formation of silver nanoparticles in solution. These
colors arise due to excitation of surface plasmon vibrations
in the silver metal nanoparticles [21].
Fig.1a and Fig1b shows the UV–vis spectra recorded from
the ethanol extract of sphaeranthus amaranthoides silver
nanoparticles. It is observed that the silver surface plasmon
resonance band occurs at 340 nm and steadily increases in
intensity as a function of time of reaction without any shift
in the peak wavelength. The frequency and width of the
surface plasmon absorption depends on the size and shape
of the metal nanoparticles as well as on the dielectric
constant of the metal itself and the surrounding metal [22,
23]. Generally the shape of the silver nanoparticles will
be changed when in contact with water or any
biological sample. It is generally recognized that UV–vis
Fig.2 SEM image sphaeranthus amaranthoides silver
nanoparcles
spectroscopy could be used to examine size- and shape-
controlled nanoparticles in aqueous suspensions[24]. Fig. 2
shows representative SEM images recorded from drop-
coated films of the silver nanoparticles synthesized by
treating silver nitrate solution with sphaernathus
amaranthoides ethanol extract. The silver nanoparticles
formed were predominantly tubeular and cubical with
uniform shape the similar phenomenon was reported by
chandran et al[25]. It is known that the shape of metal
nanoparticles considerably change their optical and
electronic properties[26].
Fig.3 EDX plot of sphaeranthus amaranthoides silver
nanoparcles
XRD patterns obtained for silver nanoparticles synthesized
using sphaernathus amaranthoides ethanol extract silver
nanoparticles in figure 4 shows characteristic peaks (at 2θ
= 30.8◦), marked with (111). A number of Bragg reflections
corresponding to the (111) sets of lattice planes are
observed which may be indexed based on the face-centered
cubic and tubular structure of silver. The XRD pattern thus
clearly shows that the silver nanoparticles are crystalline in
nature. The XRD pattern of pure silver ions is known to
display peaks at 2θ= 7.9o, 11.4o, 17.8o, 30o 38o, and 44o
(27). The value of the pure silver lattice constant has been
estimated to be α= 4.081, a value that is consistent with α =
4.0862 A° reported by the JCPDS file no 4-0783. This
estimation confirmed the hypothesis of particle
monocrystallinity. The sharpening of the peaks clearly
indicates that the particles are in the nanoregime. The size
of the silver nanocrystallites as estimated from the FWHM
of the (111) peak of silver using the Scherrer formula is 50
- 150 nm. Analysis through Energy dispersive X-ray (EDX)
spectrometers confirmed the presence of elemental silver
signal of the silver nanoparticles (Fig. 3). The vertical axis
International Journal of Nanomaterials and Biostructures 2012; 2(3) 25-29
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Fig.4 XRD patterns of Ag nanoparticles
Fig.5 FTIR analysis of Sphaernathus amaranthoides silver
nanoparticles
displays the number of x-ray counts whilst the horizontal
axis displays energy in KeV. Identification lines for the
major emission energies for silver (Ag) are displayed and
these correspond with peaks in the spectrum, thus giving
confidence that silver has been correctly identified. The
FTIR spectrum analysis of nanoparticles were shown in
Fig 5 which manifest absorption peaks located at the region
about 3500cm-1 and 650cm-1.The absorption peak at around
638cm-1 can be assigned as Secondary amide N-H wagging,
likewise the peak at 1036cm-1 as phosphorus ester P-OH
stretching,1297cm-1 as Nitrate NO2 symmetric
stretching,1384cm-1 as Aliphatic nitro compound NO2
symmetric,1630cm-1 as Primary amide NH2 bending-amide
II band,1713cm-1 as Aliphatic hydrocarbons C-H
stretching,2922cm-1 as Carboxylic acids O-H
stretching,3429cm-1 as O-H stretching.
The invitro inhibition activity of silver nanoparticles
There are several possible mechanisms through which these
herbs can act to control the blood glucose level[28]. In that
one of the mechanism is that an alteration of the activity of
some enzymes that are involved in glucose metabolism.
The α-amylase inhibitors act as an anti-nutrient that
obstructs the digestion and absorption of
carbohydrates[29]. One of the Synthetic α-amylase
inhibitors is acarbose is a complex oligosaccharides that
delay the digestion of carbohydrates. It inhibits the action
of pancreatic amylase in breakdown of starch. Synthetic
inhibitor causes side effect such as abdominal pain,
diarrhoea and soft faeces in the colon. The reference drug
arcabose was not a potent inhibitor of α-amylase under the
current study assay conditions. This is consistant with other
reports that either described a very weak inhibitory activity
of acarbose (IC50 of about 1 mg/ml) or no inhibition of α-
amylase[30, 31]. The Nanoparticles of Sphaeranthus
amaranthoides inhibited α-amylase with IC50 value of 0.28
µg/ml similar principle to that of acarbose with IC50 value
0.75µg/ml. It should be mentioned here that the calculated
IC50values in the current studies is correlated with earlier
studies[32]. The calculated IC50values of Ag nanoparticles
of Spaeranthus amaranthoides showing lower value

Fig.6 Lineweaver-Burk plot of the activity of α-amylase in
the absence or presence of S.amaranthoides Ag Nano
particles extract
compared the standard suggest that these nanoparticles are
potent than acarbose in inhibiting α-amylase. Furthermore,
fig 6 line weaver burk plot which is constructed against to
the substrate concentration Vs velocity of the enzyme in the
presence or absence of plant silver nanoparticles suggests
that the mode of inhibition of acarbose towards α –amylase
can be mixed non-competitive, this is correlated with
earlier studies[31]. In the current study, the mode of
inhibition of sphaeranthus amaranthoides silver
nanoparticles was found to be competitive. This finding
suggest that some of the α – amylase inhibitory components
in sphaeranthus amaranthoides ethanol extract may be
structural analogs of the substrate of α-amylase
Conclusion:
According to the presnt study we can conclude that plant
medicines are showing a better inhibitory activity when
compare to the synthetic inhibitors. Natural medicines have
lesser or no side effects when compared with chemical
inhibitors. From the present study we can conclude that the
sphaeranthus amaranthoides is showing better antidiabetic
activity when compared with the acarbose which is
synthetic inhibitor for α-amylase.
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Source of support: Nil; Conflict of interest: None declared

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