Results in Physics 9 (2018) 400–408

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Results in Physics
journal homepage: www.journals.elsevier.com/results-in-physics

Characterization, antibacterial, antioxidant, antidiabetic,

anti-inflammatory and antityrosinase activity of green synthesized
silver nanoparticles using Calophyllum tomentosum leaves extract
M. Govindappa a,⇑, B. Hemashekhar b, Manoj-Kumar Arthikala c, V. Ravishankar Rai d, Y.L. Ramachandra e
a
Department of Biotechnology, Dayananda Sagar College of Engineering, Shavige Malleshwara Hills, Kumaraswamy Layout, Bengaluru 560 078, Karnataka, India
b
Department of Biotechnology, Shridevi Institute of Engineering & Technology, Sira Road, Tumakauru 572106, Karnataka, India
c
Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México, León C.P. 37684, Mexico
d
Department of Studies in Microbiology, University of Mysore, Manasa Gangothri, Mysore 570006, Karnataka, India
e
Department of P.G. Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Jnana Sahyadri, Shankaraghatta, 577 451 Shivamogga, Karnataka, India

a r t i c l e i n f o a b s t r a c t

Article history: The current research study is to develop an easy and eco-friendly method for the synthesis of AgNPs
Received 23 December 2017 using aqueous leaf extract of Calophyllum tomentosum (CtAgNPs) and evaluated the extract to know
Received in revised form 19 February 2018 the effects of anti-bacterial, antioxidant, anti-diabetic, anti-inflammatory and anti-tyrosinase activity.
Accepted 22 February 2018
Using UV–vis spectrophotometer, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction
Available online 27 February 2018
(XRD), energy dispersive X-ray spectroscopy (EDX) characterized the Calophyllum tomentosum mediated
silver nanoparticles. The leaf extract of C. tomentosum yielded flavonoids, saponins, tannins, alkaloids,
Keywords:
glycosides, phenols, terpenoids and coumarins. AgNPs formation was confirmed by UV–vis spectra at
Calophyllum tomentosum
AgNPs
438 nm. Crystalline structure with a face centered cubic (fcc) of AgNPs was observed in XRD. FTIR had
Characterization shown that the phytochemicals were responsible for the reduction and capping material of silver
Antibacterial nanoparticles. The size and shape of the AgNPs were determined using SEM. From EDX study analysed
Antioxidant the strong absorption property of AgNPs. The CtAgNPs have showed significant antibacterial activity
Antidiabetic on multi drug resistance bacteria. The CtAgNPs had shown strong antioxidant (DPPH, H2O2 scavenging,
nitric oxide scavenging power, reducing power) activities. The CtAgNPs had strongly inhibited the a-
glucosidase and DPPIV compared to a-amylase. The CtAgNPs exhibited strong anti-inflammatory activity
(albumin denaturation, membrane stabilization, heat haemolytic, protein inhibitory, lipoxygenase, xan-
thine oxidase) and tyrosinase inhibitory activity. To our best knowledge, this is the first attempt on
the synthesis of silver nanoparticles using Calophyllum tomentosum leaves extract. Hence, to validate
our results the in vivo studies at molecular level are needed to develop an antioxidant, anti-diabetic
and anti-inflammatory agent.
Ó 2018 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction There are many techniques have been used in preparation of

AgNPs, among them chemical and physical methods but usage of
Particles sizes of 1–100 nm in at least one dimension are con- huge amount of toxic chemicals and also required high tempera-
sidered as nanoparticles [1,2]. The surface area to volume ratio ture [8]. Synthesis of nanoparticles by biological methods using
increases as size of the nanoparticles decreases and leads to trivial micro-organisms, enzymes, plants or plant extracts are suggested
changes in their physiochemical and biological properties. From as eco-friendly method to alternative to chemical and physical
recent years, the nanoparticles have many biomedical applications methods [9,10]. Biological agents (plants, fungi, bacteria and virus)
such as antimicrobial, antioxidant, anti-inflammatory, antiviral, are able to absorb and accumulate metals can be used as reducing
cytotoxic, anticancer, anti-HIV and so on [3–7]. The AgNPs are agent and controls the nanostructural topography of the metal
extensively using for diagnosis and treatment of diseases, drug ions. Green synthesis of AgNPs using natural organisms is simple,
delivery and other purposes because they are eco-friendly. reliable, nontoxic and eco-friendly [11,12].
Calophyllum tomentosum (Calophyllaceae) is an endemic plant
⇑ Corresponding author. commonly known as bintangur grows in Sri Lanka and Western
E-mail address: dravidateja07@gmail.com (M. Govindappa). Ghats regions of Karnataka, India. In Ayurveda, the extracts are

https://doi.org/10.1016/j.rinp.2018.02.049
2211-3797/Ó 2018 Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 M. Govindappa et al. / Results in Physics 9 (2018) 400–408 401

being practiced to treat ulcers, snake bites and eye diseases. Xan- vigorously and left to stand for 30 min in the dark before measur-
thones and triterpenes were identified from bark of C. tomentosum ing the absorbance at 517 nm against a blank [18]. Then the
[13] and flavonoids, saponins and terpenoids were noticed from scavenging ability was calculated using the following equation:
leaf part [14] and they exhibited strong a-glucosidase inhibitory

Per cent inhibition ¼ Ablank  Asample =Ablank  100; ð1Þ
activity. Calophyllum tomentosum also shows alkaloids, flavonoids,
terpenoids, tannins, glycosides, saponins presence [15] is responsi- where Ablank is the absorbance of the control reaction (containing all
ble for inhibition of a-glucosidase activity. reagents except the test compound) and Asample is the absorbance of
In the present work, we investigated the simple, effective, low- the test compound.
cost biosynthesis of stable AgNPs by the bioreduction method
using aqueous extract of Calophyllum tomentosum leaves. The syn-
Hydrogen peroxide scavenging assay
thesized AgNPs was characterized and evaluated for antioxidant,
The H2O2 scavenging activity was assayed, in brief; different
antibacterial, anti-diabetes, anti-inflammatory activity.
concentrations (10, 20, 30, 40, 50, 75 and 100 lg/ml) of AgNPs
and ascorbic acid (control) were mixed with 50 ll of 5 mM H2O2
Experimental details solution (SD Fine Chem, Mumbai) and incubated at room temper-
ature (26 ± 2 °C) for 20 min. The absorbance was measured at
Collection of plant, extract preparation and phytochemical analysis 610 nm. The percentage of H2O2 scavenging was calculated using
Eq. (1).
Collected the plant, Calophyllum tomentosum from Agumbe for-
est region of Western Ghats of Karnataka, India during February Nitric oxide radical scavenging assay of CtAgNPs
2016 and was identified with the help of taxonomist, Dr. Gopal Nitric oxide radicals generated from sodium nitroprusside in
Krishna Bhat, Udupi, Karanataka, India. Water was used for extrac- aqueous at physiological pH interacts with oxygen to produce
tion with leaves of C. tomentosum in microwave method two cycles nitrite ions, which were measured by using the Griess reaction
of 10 min at 100 °C. Preliminary qualitative phytochemical analysis reagent was evaluated by modified method. In brief, nitric oxide
was carried out to identify the secondary metabolites present in radicals, which were generated from 100 ll of 20 mM sodium
the extract [16]. nitroprusside, were incubated with 100 ll (10, 20, 30, 40, 50, 75
and 100 lg/mL) of AgNPs for 60 min, at room temperature. BHT
Total phenol content determination in the extract and NO scavenger were used as a positive control. Nitric oxide rad-
ical scavenging assay was calculated by Eq. (1).
Total phenolic content was measured according to the proce-
dure of Singleton and Rossi [17]. Standard gallic acid and plant Reducing power activity of CtAgNPs
extract (100 lg/ml) were mixed with 0.5 ml of Folin–Ciocalteu In brief, different concentrations (10, 20, 30, 40, 50, 75 and 100
reagent (1:1), 2.5 ml of sodium carbonate (20%) and 6.0 ml of dis- lg/mL) of AgNPs solution were mixed with 2.5 ml of phosphate buf-
tilled. The absorbance of the reaction mixture was measured at fer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The
760 nm. mixture was incubated at 50 °C for 20 min and then cooled rapidly.
Subsequently, 2.5 ml of 10% TCA was added with the above-
Synthesis and characterization (UV, XRD, FTIR, SEM, EDX) of silver mentioned solution and centrifuged at 3000 rpm for 8 min. The
nanoparticles from C. tomentosum leaves extract collected supernatant was mixed with equal amount of Millipore
Milli-Q water. Finally, 1 ml of 0.1% ferric chloride was added with
Five gram of dried leaves of C. tomentosum was milled and the upper layer and the absorbance was measured spectrophoto-
transferred to a 50 ml conical flask with 20 ml of sterilized distilled metrically at 700 nm. The obtained results were compared with
water. The C. tomentosum solution was heated to 100 °C for 10 min. BHT which was used as a positive control. The percentage of reduc-
The extract obtained was mixed with 10 ml of AgNO3 (5 mM) solu- ing power was calculated by Eq. (1).
tion at room temperature (26 ± 2 °C). After 1 h, observed the colour
change of the post-mixtures from light green into dark brown. Antibacterial activity of CtAgNPs
Formation of AgNPs was confirmed by Ultraviolet–visible spec- The AgNPs synthesized from leaves extract of C. tomentosum
tral analysis. The absorbance spectra were recorded using Ultravi- was used to evaluate antibacterial activity using disc diffusion
olet–visible spectroscopy (Agilent Cary 60) at the wavelength method [19]. Four multidrug resistant bacteria such as Escherichia
between 300 and 700 nm. Fourier Transform Infrared Spectroscopy coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella
(FTIR) was performed on Thermo scientificTM Nicolet iSTM50 FTIR species were collected from the Department of Microbiology, Shri-
Spectrometer to detect the possible functional groups in biomole- devi Institute of Medical Sciences and Research Hospital, Tuma-
cules present in the plant extract. The X-ray diffraction (XRD) spec- kuru, Karnataka, India. Standard antibiotic Taxim was used to
trum of AgNPs was recorded by X-ray diffractometer (Panalytical compare the study. Sterilized 5 mm Whatmann paper discs were
Xpert-PRO 3050/60) operated at 30 kV and 100 mA and spectrum impregnated with AgNPs and Taxim (100 lg/discs) and placed on
was recorded by Cu-Ka radiation. The size and surface morphology Petri plates media containing bacteria separately. The zone of inhi-
of the synthesized AgNPs were studied by scanning electron micro- bition was measured after 24 h at 37 °C incubation.
scope (SEM) and Energy dispersive X-ray (EDX) on NOVA-450
instrument. In vitro anti-diabetic activity of CtAgNPs

Determination of antioxidant activity a-Amylase inhibition assay

A 250 ml of 500 mg/ml CtAgNPs extract, 250 ml of starch 2.0% (w.
Free radical scavenging ability on 2, 2-diphenyl-2-picrylhydrazyl v) and 250 ml of 1 U/mL a-amylase solution was homogenously
(DPPH) mixed into a test tube. After incubated at 20 °C for 3 min, 500 ml
To assess the scavenging ability on DPPH, each extract of colour reagent (dinitrosalicylic acid) was added to stop the enzy-
(5–20 mg/ml) in water was mixed with 1 ml of methanol solution matic reaction. The mixture was kept into boiled water and 250 ml
containing DPPH radicals (0.2 mM). The mixture was shaken a-amylase 1 U/mL was added immediately. The mixture was
402 M. Govindappa et al. / Results in Physics 9 (2018) 400–408

heated up to 15 min. Further, the solution was removed from the samples. Per cent membrane stabilization activity was calculated
heating process and cooked at room temperature (26 ± 2 °C) for by the formula mentioned above [22,24].
3 min. A 4500 aL aqua dest was added to obtain a total volume
of 6000 ml. The solution was homogenized using a vortex. The Protein inhibitory action of CtAgNPs
a-amylase activity was determined at 540 nm using spectropho- The test was performed according to the modified method of
tometry to measure product absorbance (maltose) which reduces Oyedepo et al. [25] and Sakat et al. [22]. The reaction mixture (2
DNS. The produced absorbance was compared with a blank. Per ml) was containing 0.06 mg trypsin, 1 ml of 20 mM Tris HCl buffer
cent inhibition was calculated using the equation of Elya et al. [20]. (pH 7.4) and 1 ml test sample of different concentrations. The reac-
tion mixture was incubated at 37 °C for 5 min and then 1 ml of
a-Glucosidase inhibition activity of CtAgNPs 0.8% (W/V) casein was added. The mixture was inhibited for an
A 36 ml of phosphate buffer solution, 30 ml of CtAgNPs solution additional 20 min, 2 ml of 70% perchloric acid was added to termi-
with various concentrations (10, 25, 50, 100 and 150 mg/ml) and nate the reaction. Cloudy suspension was centrifuged and the
17 ml of 4-nitrophenyl-a-D glycopyranoside (PNPG) substrate as absorbance of the supernatant was read at 210 nm against buffer
the concentration of 5 mM were put in 37 °C for 5 min. After 5 as blank. The experiment was performed in triplicate. The percent-
min, 17 ml of a-glucosidase solution 0.15 U/mL was added to each age of inhibition of proteinase inhibitory activity was calculated.
well to obtain a total volume of 100 ml. The mixture was incubated
for 15 min the reaction was spotted by adding 100 ml of sodium Anti-lipoxygenase activity
carbonate 200 mM. Absorbance was measured at 405 nm using a Using linoleic acid as substrate and lipoxidase as enzyme, the
microplate reader. Each test was repeated thrice and the calcula- activity of antilipoxygenase was studied [24]. 2 M (0.25 ml) borate
tion was done [20–21]. buffer with pH 9.0 was mixed with 0.25 ml (20 000 U/mL) lipoxi-
dase enzyme solution and incubated at 25 °C for 5 min 1 ml (0.6
Dipeptidyl peptidase IV inhibition assay of CtAgNPs mM) linoleic acid solution was added to solutions and well mixed.
A 25 ml of CtAgNPs extract was added to 50 ml dipeptidyl pepti- Absorbance was read at 234 nm. Indomethacin was used as refer-
dase (DPP-IV) (500 mg/ml). The mixture was incubated at 37 °C for ence standard. The percentage of inhibition was calculated from
5 min. A 100 ml Gly-Pro-P-Nitroanilide (GPPN) (2 mM) was added Eq. (1). A dose response curve was plotted to determine the IC50
to the wells containing extract and enzyme incubated for 15 min. values. IC50 is defined as the concentration sufficient to obtain
The reaction was terminated by adding 25 ml acetic acid glacial 50% of a maximum scavenging capacity. All tests and analyses
(25%). The absorbance was measured at k = 405 nm [20]. were run in triplicate and averaged [27].

Xanthine oxidase assay

In vitro anti-inflammatory activity of CtAgNPs
The XO inhibitory activity of test compounds was determined
by measuring the rate of hydroxylation of the substrate (xanthine)
Inhibition of albumin denaturation
and subsequent formation of uric acid, which is a colourless end
The reaction mixture was consisting of test extracts and 1%
product of the reaction and showed absorption at 295 nm. Briefly,
aqueous solution of bovine albumin fraction, pH of the reaction
the reaction mixture containing 10 ll of 1 mg/L pure compound or
mixture was adjusted using small amount at 37 °C HCl. The sample
0.2 mg/ml of nanoparticles was dissolved in DMSO, 150 ll of phos-
extracts were incubated at 37 °C for 20 min and then heated to 51
phate buffer (0.05 mol/L, pH 7.4), 0.003 units of Xanthine Oxidase
°C for 20 min after cooling the samples the turbidity was measured
dissolved in buffer (20 ll), and 20 ll of 0.1 mmol/L xanthine as
spectrophotometrically at 660 nm. The experiment was performed
substrate for enzyme. After addition of xanthine oxidase, the mix-
in triplicate [22]. Per cent inhibition of protein denaturation was
ture was incubated for 10 min at room temperature and pre-read
calculated as follows:
in the UV region (Akmax 295 nm). The substrate was added to reaction

%inhibition ¼ Acontrol  Asample =Acontrol   100 mixture, and continuous reading for 15 min at an interval of 1 min
was observed [26]. The percentage inhibitory activity induced by
where Acontrol is the absorbance of solution without extract, Asample the samples were determined against a DMSO blank and calculated
is the absorbance of solution with sample extract/standard. using the following formula.

Membrane stabilization test for CtAgNPs Determination of tyrosinase inhibitory activities

Enzyme tyrosinase transformed the L-tyrosine into L-DOPA by
Preparation of red blood cells (RBCs) suspension hydroxylation and oxidation. Dopaquinone rapidly transformed
Fresh whole human blood (10 ml) was collected and transferred to melanins measured at 492 nm. The PsAgNps was used for tyrosi-
to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for nase inhibition assay and its effects were measured in a 96-well
10 min and were washed three times with equal volume of normal reader. The reaction was carried out in a 50 mM potassium phos-
saline. The volume of blood was measured and re constituted as phate buffer (pH 6.8) containing 20 mM L-tyrosine and 125 U/mL
10% v/v suspension with normal saline [22,23]. mushroom tyrosinase at 30 °C. The reaction mixture was pre-
incubated for 10 min and the enzyme was added. Mixture without
Heat induced hemolytic assay of CtAgNPs the enzyme served as blank. The absorbance was measured at 492
The reaction mixture (2 ml) consisted of 1 ml of test sample nm [28]. The percentage inhibition of tyrosinase was calculated as
solution and 1 ml of 10% RBCs suspension, instead of test sample Eq. (1).where Acontrol and Asample are the absorbance values in the
only saline was added to the control test tube. Aspirin was taken presence and absence of inhibitor.
as a standard drug. All the centrifuge tubes containing reaction
mixture were incubated in water bath at 56 °C for 30 min. At the Results and discussion
end of the incubation the tubes were cooled under running tap
water. The reaction mixture was centrifuged at 2500 rpm for 5 The leaf extract of C. tomentosum yielded flavonoids, saponins,
min and the absorbance of the supernatants was taken at 560 tannins, alkaloids, glycosides, phenols, terpenoids and coumarins
nm. The experiment was performed in triplicates for all the test (Table 1). Triterpenes, flavonoids, saponins, alkaloids, flavonoids,
 M. Govindappa et al. / Results in Physics 9 (2018) 400–408 403

Table 1 Identify and to determine different functional groups present in

Presence of phytochemicals in methanol extract of Calophyllum tomentosum. synthesized AgNPs by C. tomentosum extract. Fourier transform
Phytochemicals Methanol extract infrared spectroscopy measurement was used to find out the inter-
Anthraquinones  action of silver ions with the bioactive components present in the
Flavonoids + fungal extracts that are responsible for stabilizing of AgNPs. The
Saponins  CtAgNPs FTIR showed absorption bands at 3401, 2916, 2844,
Tannins + 1618, 1381, 1042 cm1. The FTIR a spectrum also helps in identify
Alkaloids +
Glycosides +
different functional groups in the CtAgNPs extract. The peak 3401
Phenols + cm1 is strong alcohol O–H stretch with intermolecular bonded.
Terpenoids + The peak obtained 2916 cm1 is strong NAH stretch, may be amine
Coumarins + salt, 2844 cm1 the peak is medium CAH stretch it is aldehyde
*
Repeated the each experiment thrice, + = Presence and  = Absence. doublet, 1618 cm-1 is strong, stretch C-O may be conjugated
ketone, 1381 cm1 is medium CAH bending may be aldehyde
and it was due to presence of aromatic group, 1041 cm1 is strong,
broad stretching (Fig. 2C). Similar results are reported by Vinay
tannin, glycosides, saponins were recorded from leaves of C. tomen- et al. [31]. The peak at 1042 cm1 was strong are due to ether link-
tosum [14,15]. The methanol extract of C. tomentosum was sub- age and it indicates the presence of flavanones adsorbed on the
jected to qualitative analysis of total phenols and it showed 0.83 surface of metal nanoparticles [32]. The C. tomentosum extract have
± 0.06. showed many biologically important active compounds might be
The extract colour changed to brown due to addition of 5 mM actively responsible and involved for reduction of Ag+ to Ag0 [33].
AgNO3 within 30 min and no further colour change was observed The CsAgNPs morphology was examined using SEM and
after 24 h (Fig. 1). Due to presence of active phytochemicals in observed the reduction of AgNO3 with Cladosporium species extract
the extract of C. tomentosum are responsible in reducing silver (Fig. 2D). The SEM result also depicted the morphology and size
metal to AgNPs due to excitation of surface Plasmon resonance of details of AgNPs with high density Ag nanoparticles synthesized
synthesized AgNPs. Bioreduction of Ag+ ions were observed in by Cladosporium species. The assembling, spherical and uniform
the solution of AgNO3 into silver nanoparticles from C. tomentosum AgNPs were observed.
phytochemicals was using UV–visible spectroscopy. The highest Fig. 2E depicts the energy dispersive a spectrum of the synthe-
absorbance peak was observed at 428 nm for C. tomentosum [28], sized nanoparticles suggests the presence of silver as the main
it indicates the formation of AgNPs (Fig. 2A). ingredient element. Metallic silver nanoparticles generally show
The XRD analysis was carried to know the crystalline nature of a typically strong signal peak at 3 keV, due to surface Plasmon res-
the CtAgNPs. The XRD pattern exhibited diffraction peaks corre- onance [34,35]. The Fig. 2E provides information on qualitative
sponding to [1 1 1], [2 0 0], [2 2 0], [3 1 1] and [2 2 2] appearing information of biosynthesized AgNPs. Observed the presence of
at 2h (Fig. 2B). These peaks indicate the crystalline (fcc) nature of elements such as C, N, O, Na, Ag at different percentage (Fig. 5).
the AgNPs and are in agreement with the database of the Joint The synthesized silver nanoparticles show strong absorption in
Committee on Powder Diffraction Standards (JCPDS No. 04-0783). the range 2.5–4 keV [35–37].
Additional intense peaks at 2h angles of CtAgNPs are because of Antibacterial activity was performed using CtAgNPs and
the involvement of the AgNO3 which was used for the synthesis showed significant zone of inhibition against all the bacteria tried
of silver nanoparticles [29,30]. The broadening of Bragg’s peaks (Table 2) (Fig. 3). The CtAgNPs had inhibited the growth of
indicates the formation of nanoparticles and Debye-Scherrer’s Klebsiella aerogenes, S. aureus followed by P. aeruginosa and E. coli
equation calculates the mean size of the silver nanoparticles was at 100 lg/ml. AgNPs synthesized from Lycopersicon esculentum
24 nm. The XRD patterns suggest that crystallization of the bioor- [38], Ochrobactrum species [39], Argemone mexicana and Turnera
ganic phase occurs on the surface of AgNPs. Our results are confir- ulmifolia [28], Azadirachta indica [40], Simarouba glauca [41]. Our
matory with the findings of Rajakumar et al. [29] and Li et al. [30]. results confirm that the C. tomentosum mediated synthesis of

Fig. 1. Preparation of silver nanoparticles A. Calophyllum tomentosum leaves extract and B. Colour changes was observed due to bioreduction of AgNO3 after 24 h of
incubation.
404 M. Govindappa et al. / Results in Physics 9 (2018) 400–408

Fig. 2. C. haracterization of silver nanoparticles synthesized from C. tomentosum, A. UV–vis spectra of AgNPs, B. XRD spectra of AgNPs, C. FTIR spectra of AgNPs, D. SEM image
of AgNPs synthesized by 25 K X magnification, E. EDX pattern of AgNPs.

AgNPs have shown potent antibacterial activity against clinically respectively. Interestingly, H2O2 free radical was consistently
important bacteria. lower than those obtained for DPPH scavenging activity. Surpris-
DPPH is a stable free radical well-known for playing role in ingly, the AgNPs exhibited comparatively better reducing power
reduction of accepting hydrogen or electron from donors. The than ascorbic acid due to the structure and characterization of
DPPH reducing activity of the AgNPs was assessed based on colour the AgNPs. In the presence of hydrogen peroxide, the dispersed
change. The DPPH scavenging activity exhibited potent inhibition AgNPs can induce reactive oxygen species such as hydroxyl radi-
of AgNPs activity when compared with standard, BHT (Fig. 4A). cals. Hydrogen peroxide inside a cell at a low dose can accelerate
The AgNPs DPPH activity was high and it was dose dependent. the dissolution of AgNPs and produce much stronger oxidative
Hence, the CtAgNPs had shown more inhibition with 90% scaveng- stress [44]. AgNPs can produce greater accounts of hydrogen per-
ing DPPH activity. The colour change was noticed due to scaveng- oxide and induce greater inflammasome formation because they
ing DPPH due to donation of hydrogen atom to stable the DPPH can cause stronger leakage of cathepsins from impaired lysosomes
molecule is responsible after adding CtAgNPs into DPPH solution and efflux of K+ ions may contribute to the production of superox-
[42,43]. The antioxidant potential of CtAgNPs could be attributed ide and hydrogen peroxide in the membranes of mitochondria
to functional groups adhered to them which were originated from [45]. Our results are in good accordance with an earlier report on
the leaves extract. the H2O2 scavenging effect of leaf extract of Abutilon indicum [46].
Accumulation of uninhibited H2O2 leads to development of Nitric oxide (NO) is an important bioregulatory molecule in the
oxygen free radicals (Peroxide and hydroxyl) which causes heavy nervous, immune and cardiovascular systems [47]. At higher con-
damage to cell membranes in living systems. Using spectrophoto- centration 100 ll/ml the biosynthesized CtAgNPs showed concen-
metrically, quantified the hydrogen peroxide scavenging activity of trated dependent NO scavenging activity of 78.46% (Fig. 4C) and it
CtAgNPs (Fig. 4B). The concentrations at 100 lg/ml inhibition were was less than standard BHT (79.11%). The interaction between
found to be 83.94 and 79.68% for the AgNPs and ascorbic acid, AgNPs and NO under anhydrous, anaerobic conditions at room
 M. Govindappa et al. / Results in Physics 9 (2018) 400–408 405

Table 2
Antibacterial activity of CtAgNPs in zone of inhibition (mm).

S. No. Bacterial strains (1) Control (2) Standard (3) AgNPs Plant extract
1 Pseudomonas aeruginosa – 8 8 –
2 Escherichia coli – 7 7 –
3 Staphylococcus aureus – 13 16 –
4 Klebsiella aerogenes – 13 16 –

Control-double distilled water; AgNPs-Silver Nanoparticles, Standard-Taxim; Plant Extract- Calophyllum tomentosum AgNPs.

Fig. 3. Antibacterial activity of AgNPs synthesized by C. tomentosum using AgNO3 Control, B. Standard, C. AgNPs, D. plant extract.

temperature (26 ± 2 °C) favours to easily accept electron from sil- The CtAgNPs have ability to inhibit the membrane stabilization
ver nanoparticles [48]. The biosynthesized AgNPs of leaves extract (84.18 ± 1.4) and it is near to standard aspirin (85.89 ± 1.4)
of C. tomentosum was dose dependent in reducing powers. (Table 3). Denaturation of proteins leads to inflammation. The
Increased concentration of AgNPs consistently increased the reduc- CtAgNPs strongly inhibited the denaturation of protein in
ing power activity. The CtAgNPs have shown almost equal reducing membrane stabilization test. The CtAgNPs had inhibited the
power activity (74%) of standard BHT (83%) (Fig. 4D). The reducing release of RBC lysosomal to various levels and effectively induced
power activity was due to presence of phytochemicals in the the hemolysis of anti-inflammatory activity effect. The CtAgNPs
extract [49]. The obtained result was correlated with the results effectively inhibited the induced albumin denaturation was
of Dipankar and Murugan [50] (2012) and Bhakya et al. [51]. observed. The CtAgNPs strongly inhibited the heat induced hemol-
The CsAgNPs has potentially inhibited the activity of ysis in RBCs membrane stabilization. The CtAgNPs inhibited the
a-amylase, a- glucosidase and dipeptidyl peptidase IV in vitro release of neutrophils lysosomal content at inflammation area
assay (Fig. 5). The b-glucosidase and DPPIV are greatly inhibited (Table 3). The CtAgNPs was showed 84.64 ± 1.4 in albumin
by CtAgNPs compared to a-amylase. The CsAgNPs decreased the denaturation was compared with aspirin 86.52 ± 1.4. The CtAgNPs
levels of enzymes, which are responsible for catalyzing the hydrol- also showed protein inhibitory activity of 89.17 ± 1.4 was
ysis of complex carbohydrates and increased the consumption rate compared with standard aspirin 92.90 ± 1.4 (Table 3). Our results
of glucose reported by Rajaram et al. [52], Abideen and Sankar are confirmatory with the reports of Pretsch et al. [55] and
[53], Sengottainyan et al. [54]. Naz et al. [56].
406 M. Govindappa et al. / Results in Physics 9 (2018) 400–408

Fig. 4. Antioxidant activity of biosynthesized AgNPs, A. DPPH method, BH2O2 assay, C. NO scavenging assay, D. Reducing power.

Table 4
Xanthine oxidase and acetyl cholinesterase inhibition by CtAgNPs.

Test sample Xanthine oxidase (%) Acetyl cholinesterase (%)

CtAgNPs 93.87 ± 1.4 28.41 ± 1.4
Allopurinol (lg/ml) 98.48 ± 1.4 50.00 ± 1.36

Data represented the arithmetic mean and standard error of three determinants.
According to Duncan’s multiple range test (DMRT) the values provided with dif-
ferent superscripts remains significant at P  0.05.

Table 5
Fig. 5. Anti-diabetic activity of Calophyllum tomentosum mediated silver
Antilipoxygenase and tyrosinase inhibitory activity by CtAgNPs.
nanoparticles.
Test extract Tyrosinase inhibition (%) Lipoxygenase inhibition (%)
CtAgNPs 98.81 ± 1.4 71.52 ± 1.4
The CtAgNPs had inhibited the xanthine oxidase at maximum Ascorbic acid 99.99 ± 1.4 –
level (93.87 ± 1.4) was compared with standard aspirin (98.48 ± Arbutin – 52.68 ± 1.4
1.4) (Table 4) [56]. The CtAgNPs significantly inhibited the acetyl
Data represented the arithmetic mean and standard error of three determinants.
cholinesterase activity by 28.41 ± 1.4 (Table 3) [56]. The maximum According to Duncan’s multiple range test (DMRT) the values provided with dif-
antilipoxygenase activity was observed with CtAgNPs (71%) ferent superscripts remains significant at P  0.05.
(Table 4). Reports are available on plants extracts on inhibition of
lipoxygenase [57]. The potent inhibition of tyrosinase enzyme

was observed with CtAgNPs (98.81 ± 1.4) was compared with arbu-
Table 3 tin (52.68 ± 1.4) (Table 5). Tyrosinase enzyme is involved in mela-
Effect of C. tomentosum mediated silver nanoparticles on membrane stabilization, nin formation and catalyzes the tyrosine oxidation process to
albumin denaturation and protein inhibitory activity.
dihydroxy-phenylalanine (DOPA) and from DOPA to DOPA qui-
Test sample MS (%) AD (%) PI (%) none. Tyrosinase is a metalloenzyme containing copper at an active
CtAgNPs 84.18 ± 1.4 84.64 ± 1.4 89.17 ± 1.4 site and it catalyzed to change the oxidative site of copper atoms
Aspirin (lg/ml) 85.89 ± 1.4 86.52 ± 1.4 92.90 ± 1.4 [58]. The AgNPs synthesized from C. tomentosum leaf extract had
shown potent antibacterial, antioxidant, anti-diabetic, anti-
Data represented the arithmetic mean and standard error of three determinants.
According to Duncan’s multiple range test (DMRT) the values provided with dif- inflammatory and anti-tyrosinase activity and it may be due to
ferent superscripts remains significant at P  0.05. capping of functional groups on AgNPs.
 M. Govindappa et al. / Results in Physics 9 (2018) 400–408 407

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