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Journal of Advanced Zoology
ISSN: 0253-7214
Volume 44 Issue S-5 Year 2023 Page 1101:1115
_______________________________________________________________________________________________________________________________
- 1101 -
Green Synthesis of Silver Atropa Acuminata Nanoparticles:
Characterization and Anti-Diabetic Potential
Priyanka Thakur1*, Vinay Pandit2
1*Department of Pharmacology, Shiva Institute of Pharmacy, Chandpur, Bilaspur, HP, India- 174004
2Department of Pharmaceutics, Laureate Institute of Pharmacy, Kathog, Kangra, HP, India- 176029
*Corresponding author’s: Priyanka Thakur
Article History
Received: 06 June 2023
Revised: 05 Sept 2023
Accepted: 17 Oct 2023
CC License
CC-BY-NC-SA 4.0
Abstract
Background: Atropa acuminata plant, which is also known as the maitbrand
or Indian belladonna, belongs to the family Solanaceae and is closely related
to the deadly nightshade of Europe and North Africa used in the treatment of
various diseases. Objectives: The main objective is to investigate the
antidiabetic potential of silver nanoparticles of Atropa acuminata. Methods: In
this study green synthesis of silver nanoparticles was carried out. The current
study engross the green synthesis of A. acuminata roots by reducing silver ions,
where ultraviolet (UV) spectral analysis and transmission electron microscopy
confirmed nanoarchitecture. The optimized silver nanoparticles were
characterized for shape, size and morphological features by various techniques
viz., SEM, TEM EDXA and XRD. The optimized formulation was further
subjected for lipid peroxidation assay and in vitro antidiabetic assay in order
to understand the antidiabetic potential of formulated silver nanoparticles.
Results: The Silver nanoparticles of Atropa acuminata roots was successfully
prepared by optimizing different concentration of plant extract at different
temperatures and stirring speed. The Resultant optimized nanoparticles showed
a particle size around 20 nm and -28 mv zeta potential. Further the
characterization of optimized AgNPs was carried out. SEM provides the shape,
size, and morphological features, whereas EDXA confirms the compositions
and distribution of nanoparticles through spectrum and elemental mapping.
XRD diffraction analysis revealed the crystalline structure of nanoparticles.
Further, Nanoparticles showed a maximum scavenging potential of
82.633±0.116 for superoxide anion free radicals at 100 µg/mL concentration.
Among two anti- diabetic assays, the αamylase assay shows a better result of
percent inhibition 63 ± 1.32 at 75 μg/ml concentrations. Lastly, in vitro, drug
release study revealed a 101.50% cumulative release from Ag- NPs formulation
up to 1 hour, which was better than the standard one. Conclusion: This study
explores the novel technology of green synthesis for various biomedical
applications.
Keywords: Ag-NP, Atropa acuminata, Antidiabetic, Biocompatible;
Bioassay
1. Introduction
Atropa acuminata plant, which is also known as the mait-brand or Indian belladonna, belongs to the
family Solanaceae and is closely related to the deadly nightshade of Europe and North Africa used in
the treatment of various diseases [1]. The four species of this plant are A. acuminata Royle, Atropa
belladonna L., Atropa baetica Wilk, and Atropa pallidiflora [2]. Plant species’ dried roots, leaves, and
stems carry numerous phytochemicals like atropine, scopolamine, hyoscyamine, and other tropane
alkaloids [3]. The plant has been medicinally described as an antidote, anodyne, analgesic,
Green Synthesis of Silver Atropa Acuminata Nanoparticles: Characterization and Anti-Diabetic
Potential
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hallucinogenic, Parkinsonism, encephalitis, carcinoma, spastic dysmenorrhoea, mydriatic, narcotic, and
sedative properties, according to literature review
[4] [5] [6]. The field of research known as nanotechnology studies molecular and nanoscale processes.
There are many instances of nanoscale objects in nature, such as DNA, water molecules, viruses, red
blood cells (RBCs), etc [7] [8]. Researchers now have the chance to alter several treatments due to
recent advances in nanotechnology. With the use of this technology, it is now possible to modify the
biological and physicochemical characteristics of nanomaterials to enable more effective medication-
targeted delivery to produce therapeutic effects [9] [10].
As per the literature survey, Singh et al. [11] have described that the A. acuminata plant is used to
manage CNS and memory-related disorders. Majid et al. [12] have found anti-inflammatory potential
due to the active constituents in this plant. Jayakanthi et al. [13] have reported the hepatoprotective role
of the A. acuminata plant in acetaminophen-induced oxidative stress and hepatotoxicity in rat liver.
Rahman et al. [14] analyzed the anti-cancer potential of the plant by inhibiting protein kinase enzymes.
Khan et al. [15] reported that plant tissues are rich in antioxidant molecules. Literature also revealed
that the plant has numerous medicinal and aromatic properties but is declining in wildlife [16]. The
major threat to this species is overexploitation due to sociocultural patterns [17]. I propose that this
plant species should be preserved by seed propagation; otherwise, it will be totally abolished if no action
is taken [18] [19]. Mohammad et al. [20] reported that the propagation of A. acuminata plant should be
optimized through numerous plant growth regulator sources as this plant will be critically endangered
[21]. So, to achieve this rationality, research is focused on highlighting this plant’s worth as it is highly
medicated.
Pharmaceutical and other medical corporations avail themselves of nanotechnology applications
worldwide in numerous biotechnology, bioengineering, and biomechanics disciplines, which swap the
trendy scenario of researchers looking after this field as an alternative drug delivery system [22]. So,
nanotechnology has become one of today’s most exciting research zone. Nanomedicine and
nanodelivery systems are emerging sciences in which Nanoscale materials are used as diagnostic tools
to deliver therapeutic agents to specific targets [23]. Because the quantum energy of these particles can
be easily ascertained due to their smaller size, this field makes it possible to design the biomaterials
with the desired Nanoscale size, shape, and morphology [24].
Nanotechnology is creating nanoparticles of copious metal oxides like gold, silver, zinc, magnesium,
titanium, etc., with sizes ranging from 1 to 100 nm [25]. Among all these oxides, the free radical
scavenging and antioxidant potential of silver oxides grabbed the mind of scientists to think deeply.
Further, the operating procedure of silver nanoparticles inside the body implies releasing silver ions,
generating reactive oxygen species, and penetrating the targeted cell membrane, followed by the
blockage of deoxyribonucleic acid [26]. Furthermore, these particles have outstanding optical properties
due to their small size, large surface area, and distinct colloidal, optical, and electrical properties [27].
In addition, plants have an abundance of phytochemicals that serve as reducing agents in the emergence
of Ag-NP, increasing the probability of synergistic effects within the biological systems [28].
Agricultural nanoparticles are becoming popular because of their outstanding features and practical
versatility. AgNPs are primarily used in antimicrobial and anti-cancer remedies but are also used as a
vaccine adjuvant, an anti-diabetic agent, and a biosensor [29].
In the evident nanotechnology, top-up and bottom-down approaches were used where top-up
approaches included Physical vapor deposition, Chemical vapor deposition techniques, etc., and
bottom-down approaches involved Sol-gel synthesis, Colloidal precipitation techniques, etc. to produce
the nanomaterial [30]. Different physical, chemical, and biological methods (Pulse Laser Ablation, sol-
gel method, microorganism method) were used to synthesize the nanoparticles [31]. Among all these
methodologies, physical methods are expensive, cause radiation exposure, require high energy,
temperature, and pressure, and generate a large amount of waste, whereas, in chemical methodology,
the chemicals used in the procedures are costly and toxic, and making them hazardous to the
environment as well as for human health [32]. However, biological methodology does not involve
hazardous chemicals for reduction and stabilization. Moreover, biological methods are eco-friendly,
biocompatible, synthesized in big batches, economical, safe, and target specific effects due to their
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smaller size and very few side effects [33]. Therefore, this current research preferred the biological
green synthesis method for nanoparticle formulation.
In this current investigation, the research aims to scrutinize this critically endangered plant A. acuminata
roots for their green synthesis of Ag-NP and confirm the formulation by UV spectral analysis and
transmission electron microscopy.
2. Materials And Methods
Chemicals
Fuming nitric acid, methanolic potassium hydroxide, acetone, solution of nitrated residue, 1mM
aqueous solution of silver nitrate, platinum, DMSO, NBT, 5 mM NaOH, Tris-HCl buffer, 78 μM of β-
nicotinamide adenine dinucleotide, 50 M of nitro blue tetrazolium, stock solution, 0.1mM methanolic
solution of superoxide radical, 0.02 M sodium phosphate buffer (pH 6.9), 1U of αamylase, 30-100 g/mL
of ANE, 1% starch solution, dinitrosalicylic acid reagent, , 2.4 U/mL αglucosidase, 2.5 mM pNPG in
the buffer, and 100 mM phosphate buffer (pH 6.8), sodiumcarbonate solution, 4-nitrophenol, phosphate
buffer (pH 8), ethanol, water, PMS-NADH, epigallocatechin gallate, acarbose.
All the chemicals were of analytical grade and procured from Sigma Aldrich. Some ingredients were
purchased from Franco-Indian Pharmaceuticals Pvt. Ltd. The plant material was collected from local
region of Mandi and Kullu district of Himachal Pradesh and authenticated by Dr. Madhava Chetty,
Assistant Professor in the Department of Botany at Sri Venkateswara University in Tirupati, Andhra
Pradesh, India (SVU/SC/76/321/20-21).
Plant roots collection
The Himachal Pradesh districts of Mandi and Kullu are the regions from where the plant material was
gathered. The plant roots were identified and verified by Dr. Madhava Chetty, Assistant Professor in
the Department of Botany at Sri Venkateswara University in Tirupati, Andhra Pradesh.
Preparation ofA. acuminata root extract
After the collection, plant roots were first swept with plain water to get rid of unnecessary waste. After
being shade-dried for up to 2 weeks at room temperature, crush it into a powder using an electric mixer
grinder (Superflame GX 11), and then store it in an airtight container. Crushed powder of plant roots
was strained through sieve number 40. The soxhlet apparatus is suffused with 1000 gm of powdered
medication, and ethanol: water solvent in a 50:50 ratio and subjected to an extraction process (Figure
01). The residual extract solvent is then incubated with continuous stirring, filtered through Whatman
Paper No. 1 and evaporated using the phenomena of decreasing pressure distillation by rota evaporator
(Rota vapour, R-210/215, Buchi, Switzerland). After concentrating the filtrate, the extract was stored
at a low temperature (5˚C) for further experiments.
Figure 01: Soxhlet extract apparatus assembly for extraction of phytochemicals from Plant Root
Extract
Preliminary Screening for Qualitative Phytochemicals of Extract
Atropa acuminata plant root extract lug numerous important phytochemical components that were
qualitatively assessed in accordance with the literature review [35]. Alkaloids, phenolic chemicals,
Green Synthesis of Silver Atropa Acuminata Nanoparticles: Characterization and Anti-Diabetic
Potential
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flavonoids and tannins were thus found by screening evidence, but no inference evidence for glycosides,
terpenoids, proteins, or carbohydrates was found [36] [37]. According to accepted methodologies, the
findings showed that the alkaloids make up the majority of this plant species [38]. For the confirmation
of type of alkaloids Vitali Morin Test was performed. Sample solution was reacted with fuming nitric
acid and then allowed to evaporate until it converted into dried residue. Afterwards, dried residue were
treated with methanolic potassium hydroxide, acetone and solution of nitrated residue which produce
violet colour indicate the presence of tropane alkaloids [39].
Thin Layer Chromatography
Extract sample was applied on TLC plates where different solvents ratio were selected on the basis of
their ascending polarity (Toluene: Ethylacetate= 1:9/ 9:1, Ethylacetate: Butanol: Distilled water= 2:4:4,
Toluene: Ethylacetate: Formic acid= 1:1:1, Ethyl acetate: chloroform:formic acid:water = 7:1.5:1.5:1
etc.) to optimize the solvent system for the elution of phytochemicals effectively on TLC plate. The
yellowish green spots were produced in silica plate which indicates the presence of flavonoids, alkaloids
and phenols in the sample (Figure 02). Chloroform: Methanol: Acetone: 25% Ammonia (75:15:10:1.8
v/v) elutes the phytochemicals most effectively which will be further used for qualitative and
quantitative analysis.
Figure 02: Band Appearance and visualization under UV Chamber in TLC Plate.
Synthesis of Atropa acuminata AgNPs
For the production of AgNPs of root extract, a 1mM aqueous solution of silver nitrate (AgNO3) was
prepared in a 250ml volumetric flask. In a nutshell, according to established procedures, 10 ml of A.
acuminata root extract was dissolved in 90 ml of an aqueous solution containing 1 mM silver nitrate
[40] [41]. The transformation of color from colorless to dark brown demonstrated that AgNO3 had
completely reduced to Ag+ ions. After that, the colloidal mixture was securely packed and kept for later
use. Using UV-Visible spectroscopy (Varian Inc., USA) in range between 300 to 600 nm, the production
of AgNPs of root extract was examined. The solution was diluted 20 times for UV spectroscopic
monitoring. A high peak at 430–450 nm confirms the synthesis of root extract Ag–NPs, which was
confirmed by the use of millipore water as a blank to reset the baseline. All the reactions were repeated
for three times.
Characterizations Surface Plasma Resonance (SPR)
Green synthesized A. acuminata root extract AgNP formulation was inspected for the reduction of silver
ions into nanoparticles using UV-visible spectroscopy Tecan Multimode Microplate Reader
(InfiniteM200). The baseline was calibrated using millipore water, and spectra were monitored between
300 and 700 nm which indicate the bio-reduction of silver ions to produce AgNPs.
Analysis of Particle Size and Zeta Potential
The dilution approach was used to examine the particle size distribution, which is one of the most
important element in determining the quality, efficacy, and safety of a nanoparticle formulation [42].
The mixture was assessed using Malvern Zeta Sizer ZS technology after being diluted up to ten times
with distilled water. In a disposable cuvette, a 1 ml diluted sample is obtained and evaluated between a
250 and 900 angle. Helium was employed as the light source, and Particle diffusion by Brownian motion
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phenomenon was used to determine particle size. Zeta potential is checked using the sample in the
capillary tube [43].
Transmission Electron Microscopy (TEM)
Sample was blended with 10 ml of distilled water after being centrifuged at 20,000 rpm for 30 mins,
and it was subsequently stored at 15-20°C for at least one full day. The entire mixture in suspension is
subsequently kept within the lyophilizer (Jenco CP135-M4-2). By putting a few drops of the processed
sample inside the instrument’s copper grid, the processed sample is combined with double-distilled
water. The process of dry heat sterilization is employed to eliminate any remaining water from the
treated sample for a maximum of two hours at 55 ˚C [44].
XRD Analysis
The attributes of crystalline structure, phase nature, lattice parameters, and crystal size is frequently
accomplished by XRD analytical instrument X’Pert MRD (PAN analytical, Almelo, Netherlands).
When electrons impact nanoparticles, they scatter X-rays with a wavelength of 2 A˚ [45]. A glass slide
was coated with solid AgNPs to serve as the sample container. The XRD machine, which is connected
to an integrated program, houses the holder. Using CuK radiation (=1.54056) in the range of 20° to 80°
at 40keV, the XRD pattern of AgNPs was captured by a Bruker D8 diffractometer. The Powder X
computer program ascertains the lattice parameters.
SEM and EDXA analysis
Even for extremely small particles smaller than 10 nm, scanning electron microscopy is a high
resolution imaging technique. Particle size, shape, and texture are utilized to assess surface fractures,
defects, impurities, and corrosions [46]. Initially, a sample container containing carbon tape and AgNP
was coated with platinum and viewed under S-4200 for 120 secs (Hitachi, Tokyo, Japan) [47].
Afterwards, synthesized AgNPs were examined using a SEM device connected to an EDXA Inc.,
Mahwah, NJ, USA).
Antioxidant Assay of Atropa acuminata Ag-NP Formulation In vitro Free Radical Scavenging
Activity Synthesized Ag-NP in vitro antioxidant activity was determined using standard techniques
which entail the suppression of free radicals. After samples were added up to a system that gives rise to
free radicals, the suppression of free radical activity is perceived by the following formula [48].
The IC50 values calculated represent the sample concentrations required to scavenge 50% of free
radicals.
Superoxide Radical Assay
To evaluate the superoxide anion scavenging activity, a reaction mixture including 0.2 ml of each
AgNP, substance, and standard in DMSO, 0.2 ml of NBT (1 mg ml-1 solution in DMSO), and 1 ml of
alkaline DMSO (5 mM NaOH in 1 ml water) was prepared. In this assay, superoxide radicals emerged
in 3.0 ml of Tris-HCl buffer (16 mM, pH 8.0), which also included 1 mg of test sample (Ag-NP)
dissolved in 50% ethanol, which was added at various concentrations (31.25-1000 μg/ml), 78 μM of
βnicotinamide adenine dinucleotide (reduced form NADH), 50 M of nitro blue tetrazolium (Ransod kit,
Randox Laboratories Ltd, Crumlin, UK) [49]. Numerous concentrations of solutions have been
prepared of concentrations ranging from10-100 μg/ml from stock solution and then were mixed with
0.1mM methanolic solution of superoxide radical. This solution was incubated for 30 min at room
temperature and was recorded at 560 nm.
Antidiabetic potential of A. acuminata AgNPs α-Amylase Inhibition Assay
The α-amylase assay combination includes 0.5 mL of 0.02 M sodium phosphate buffer (pH 6.9), 1U of
α- amylase, and 30-100 g/mL of ANE-AgNPs that had been pre incubated for 250 min at 37 °C. About
20 mins at 37 °C of incubation, 200 L of a 1% starch solution in the previously mentioned buffer was
introduced to the tubes. By administering 0.5 mL of dinitrosalicylic acid reagent and heating in a boiling
Green Synthesis of Silver Atropa Acuminata Nanoparticles: Characterization and Anti-Diabetic
Potential
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water bath for 15 mins, the reaction was ceased. After cooling the tubes, absorbance at 540 nm was
measured [50]. α-
glucosidase enzyme inhibitory activity
The microplate-based approach was slightly modified to assess the α-glucosidase inhibitory activitiesof
the Ag-NP [51]. 75 L of the reaction mixture for the sample group included 15 L of each of thefollowing:
experimental drug, 2.4 U/mL α-glucosidase, 2.5 mM pNPG in the buffer, and 100 mMphosphate buffer
(pH 6.8). The reaction was stopped by adding 75 L of a sodium carbonate solutioncontaining 0.2 mol/L
to each well after the 96-well plates had been incubated at 37°C for 25 min. At405 nm, the absorption
of 4- nitrophenol was observed. The reaction mixture’s composition in thecontrol group was identical
to that in the sample group, with the exception that solvent was employedin place of the test substance.
The Ag-NP sample group’s composition was identical to that of thecontrol blank group’s, with the
exception that α-glucosidase
was employed in place of the buffer.
Using the following formula, the inhibitory activity was determined:
The following formula was used to evaluate the % inhibition of α-glucosidase enzyme activity:
% Inhibition= Abs control- Abs Test/Abs control X 100
Drug Release Studies
In order to establish the effectiveness, therapeutic activity, formulation development and stability, it is
very important to evaluate the in vitro drug release study. Keshary-Chien (K-C) diffusion cell and
dialysis membrane 60 LA390-1MT (Thames chemical, Ludhiana) of molecular weight 13000 Da, pore
size 2.4 nm and surface area 3.14 cm2 was used which is ideal for diffusion and osmosis work. The
temperature of outer jacket of diffusion cell was maintained using water at temperature 37 ± 2˚C
whereas 10 ml of freshly prepared receptor medium was used as phosphate buffer (pH 8) stirred at 100
rpm. 1 gm of A. acuminata plant extract Ag-NP was loaded inside the diffusion cell and during the
whole experiment sink condition, 37± 2 C temperature was maintained. 5ml of sample was withdrawn
at predetermined time intervals. Samples collected in test tubes were then analyzed using UV visible
spectroscopy at 420 nm [52] against the similarly treated blank.
3. Results and Discussion
Analysis of Phytochemicals and Percentage yield
Harvested shade dried powdered material was extracted using ethanol and water as the solvents and
overall percentage yield 19.25% from the 1000 gm weight of powdered material was determined
utilizing extract. Weight of crude extract was considered to be 192.5 gm (Table 01). Preliminary
phytochemical screening test was accompained to reveal the presence of alkaloids, phenolic
compounds, and flavonoids while glycosides, terpenoids, proteins, and carbohydrates were lacking
from the roots extract. Specific confirmatory testing of alkaloids (Tropane alkaloids) was also escorted
by Vitali Morin Test as atropine is the prime phytochemical accountable for antioxidant and antidiabetic
potential According to literature survey [53].
Table 01: Calculation of Percentage Yield of Crude Hydroethanolic Extract of A. acuminata roots
Sr.
No.
Name of Plant
Extract Type
Weight of Powd Plant
Material (g)
Weight
Extract (g)
c
r%age Yie
Extract
1.
Atropa acum
plant roots
i
Hydroalcoholic
1000g
192.5
19.25
A. acuminata root extract Silver Nanoparticles: The Ag-NP from the roots of the Atropa acuminata
plant was initially established by visual inspection of reaction mixture (root extract and AgNO3)
from pale yellow to dark reddish brown color, specifying clearly the reduction of cationic silver to
its metallic counterpart and synthesis of Ag-NPs of different root extract concentrations in an
experimental laboratory (Fig. 02). Reaction mixture of concentration 10 mg/ml has been augmented
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by using two state variable firstly by keeping temperature constant (40 ˚C) and stirring variable,
subsequently stirring constant (500 rpm) and variable temperature (Table 02). Reaction mixture
(10mg/ml) was considered to be as stable colloidal nanoparticles with no lumps or precipitate
formation.
Table 02: Green synthesis of plant extracts Ag-NP of numerous Concentrations and formulation
optimization using two Sate Variables.
Sr. No.
Plant Extract Conc.(mg/ml)
Temperature (0C)
Stirring (rpm)
Speed
1.
2
25
200
2.
4
30
300
3.
6
35
400
4.
8
40
500
5.
10
50
600
6.
12
60
700
7.
14
70
800
8.
16
80
900
9.
18
90
1000
10.
20
100
1100
Surface Plasma Resonance (SPR)
Surface Plasma Resonance (SPR) of green synthesized silver nanoparticles of A. acuminata root extract
was analyzed by employing UV spectroscopy at 420nm. Spectroscopic SPR revealed the arithmetic
sequence meaning at low concentration (Below 6 mg/ml) peaks are less sharp and at very higher
concentration (above 14 mg/ml) peaks were immense (Fig. 03). Spectroscopic plot recommended that
at low concentration of plant root extract peak was obtuse that means concentration is insufficient to
reduce silver ions into Ag-NP while at very higher concentration also become insufficient for synthesis
consequently; an optimum level is needed for its synthesis.
It was noticed from graphical analysis that below the 6 mg/ml concentration the values of λmax were
almost alike that may be due to identical size and shape of nanoparticles in the formulation. All the
samples remained as a quiet stable suspension for up to 1 month with no precipitate or lumps formation.
Fig 03 shows how the blending of optimum concentration of plant root extract and AgNO3 with heating,
stirring and time leads to synthesis of Ag-NP within 2-5 min and it takes maximum up to 25 min for its
complete reduction and synthesis.
Figure 03: UV-visible absorption spectra of Ag-NPs synthesized by different concentrations of A.
acuminata
plant extract Conc. (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20%) against 1mM AgNO3 by Green synthesis
Method.
Particle Size and Zeta Potential Analysis
Green Synthesis of Silver Atropa Acuminata Nanoparticles: Characterization and Anti-Diabetic
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The formulation was processed to determine the particle size and zeta potential using Nano Zeta Sizer
ZS (Malvern,UK) equipment which act in accordance with the laser Doppler electrophoresis and
dynamic light scattering phenomenon. The particle size and zeta potential of optimized formulation (10
mg/ml) was found to be 20 nm and -28 Mv respectively (figure 04). It was observed from report that
the formulation was a stable colloidal suspension with no precipitations.
Figure 04: Particle size, PDI and Zeta Potential distribution Report produced by Nano Zeta Sizer
ZS(Malvern, UK) equipment based on intensity (8 mg/ml, 10 mg/ml, 12 mg/ml, and 14 mg/ml of
different A. acuminata Root Extract Concentration Ag-NPs.
Transmission Electron Microscopy
To ascertain the particle size, shape and morphological attributes of green synthesized Ag-NP of A.
acuminata root extract, UHRTEM instrument was used which analyze spherical, oval shaped
nanoparticles of average size ±20nm (Figure 05). The surface morphological features were recorded at
atomic level by transmitting wave through the set of lattice plane of the crystals in formulation. Bright
and dark stripped images of lattice revealed the fringes of silver metal in nanoparticles (Figure 05).
Figure 05: UHRTEM of Green Synthesized A. acuminata Ag-NPs emerged at optimum concentration
(10 mg/ml)
Dispersive XRD Analysis
XRD analysis of green synthesized Ag-NP of Plant A. acuminata ethanolic root extract revealed the
face centered cubic crystalline arrangement of nanoparticles in the formulation which is reflected by
the peaks in the spectrum with distinct planes (Figure 06, 7a). Size of the nanoparticles are at all times
the determining factor for XRD peak pattern [54]. XRD data displayed in figure 06 9manifest the five
Bragg reflections characteristic 2θpeaksat 32.18°, 38.04°, 46.13°, 54.63°, and 77.08° of Ag-NPs
with respect to the (120),
(206), (115), (207) and (317) planes respectively (Figure 06).
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Figure 06: (a) XRD Pattern of the Green synthesized Ag-NP using A. acuminata Root Ethanolic
extract (b) Crystalline confirmation by Electron Diffraction Pattern.
SEM and EDXA Analysis
Atomic force Microscopic and scanning electron microscopic analysis technique was used to ascertain
the shape and morphological dimensions of silver nanoparticles [55]. Micrograph (Figure 07a) clearly
indicates the approximate 20 nm sized spherical, tubular and some cuboidal shaped Ag-NPs which may
be due to the availability of different reducing agents in extract phytochemicals. Micrograph analysis
in Figure 07b, c and Table 03 revealed 72.43% weight percentage of Ag in formulation.
Figure No. 07: (a) Scanning electron microscopy Micrograph of Green synthesized Ag-NPs of A.
acuminata Ethanolic Root Extract (b) EDXA micrograph Analysis of silver nanoparticles of Atropa
acuminata plant Root Hydroethanolic Extract (c) and Corresponding EDX spectra.
Table 03: Weight and Atomic Percentage of Different Elements in Ag-NP of A. acuminata Ethanolic
Root Extract
Element
Weight %
Atomic %
C, K
3.16
2.53
O , K
27.65
38.9
Na, K
7.6
3
Ca, K
14.7
6.3
Ag
72.43
28.45
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Superoxide radical Antioxidant Assay
The antioxidant potential of Ag-NPs was measured on the basis of ability of Ag ions to quench the
superoxide radical in a reaction mixture of formulation and free radicals. It was observed that
superoxide radicals showed maximum inhibition 82.633±0.116 at 100 µg/mL while minimum
22.124± 0.203 at 10
µg/mL concentration when analyzed at 560 nm (Figure 08 and Table 04). The ability to reduce NBT by
PMS- NADH coupling can determine the superoxide radicals emerged from dissolved oxygen [56].
Figure 08: Antioxidant (Superoxide anion scavenging) activity of A. acuminata Root Extract Ag-NPs
as percentage of superoxide anion radical’s inhibition
Anti-diabetic Potential of AgNPs Nanoparticles Formulation
α-Glucosidase and α-Amylase Enzyme inhibitory activity
The α-glucosidase enzyme was significantly inhibited by the A. acuminata hydroethanolic extract
AgNPs. The varied concentrations of Epigallocatechin gallate, i.e., 0.3, 0.5, and 0.7 µg, shown 51 ±
5.32%, 53 ± 4.43%, and 63 ± 6.76% of α- glucosidase enzyme inhibition, and the IC50 value was
observed 48.4 µg/ml. In contrast, Ag-NPs, at concentrations of 25, 65, and 75 µg/ml, demonstrated 49
± 7.65, 50± 6.54, 59± 7.65% inhibitions respectively at 405 nm (Figure 09, Table 04). While in
αAmylase assay, on alike Acarbose concentrations percent inhibition was 56 ± 4.34, 61 ± 2.34, 68 ±
4.53 µg/ml and IC50 value was 52 .3 µg/ml. And for similar concentrations of Ag-NPs percent
inhibition of α-Amylase was51 ± 3.76, 57 ± 5.43, 63 ± 1.32 µg/ml and IC50 value was 49.3 µg/mlat
540 nm respectively (Figure 09, Table 04).
Table 04:In vitro α-glucosidase and α-amylase enzyme inhibition by comparing %inhibition and I
value of two samples of Standard &A. acuminata Root Hyroethanolic Extract at different concentrat
(μg/ml)
Sample
Concentration
Concentration
IC50/(µg/ml)
Concentration
IC50/(µg/m
(Reference/Test)
(µg/ml)
(µg/ml)
inhibition α-
glycosidase
(µg/ml)
inhibition α-
amylase
α-glycosidase -
Epigallocatechin
gallate
α-amylase
-Acarbose
0.3 µg
51 ± 5.32
48.4
56 ± 4.34
52.3
0.5 µg
53± 4.43
61 ± 2.34
0.7 µg
63± 6.76
68 ± 4.53
Ag-NPs of Plant Root
Hydroethanolic
Extract
25 (µg/ml)
49 ± 7.65
39.3
51 ± 3.76
49.3
65 (µg/ml)
50± 6.54
57 ± 5.43
75 (µg/ml)
59± 7.65
63± 1.32
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Figure 09: The comparison of percent inhibition (μg/ml), and IC50 value shown in the above figure
for both Standard & Ag-NPs.
Drug Release Study
In drug release study, percent cumulative drug release for the both standard (atropine) and test sample
(Ag- NPs) of various concentrations was calculated at different time interval by analyzing the
absorbance under UV visible spectroscopy at 420 nm [57] (Table 05 and Figure 10). It was observed
from the result that with the passage of time in vitro drug release for Reference was 99.60% and for
Ag-NPs was 101.50% respectively (Table 05). All the data is expressed as the mean value plus the
standard deviation minus one. Using SPSS software and the 5% threshold of significance, analysis was
performed using the ANOVA test and the Duncan’s multiple range tests.
Table 05: Percent cumulative release of Reference and Test sample at different Time Intervals
Sr. No.
Time (Min)
PercentCumulativeReference (%)
Release
Percent Cumulative Release for NPs (%)
1.
5
39.80
18.50
2.
10
80.50
41.00
3.
15
97.40
60.60
4.
20
97.80
76.90
5.
25
97.70
90.00
6.
30
98.00
98.10
7.
45
99.60
101.50
Green Synthesis of Silver Atropa Acuminata Nanoparticles: Characterization and Anti-Diabetic
Potential
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Figure 10: Cumulative percent in vitro release of Green Synthesized Silver Nanoparticles of
Standard Atropine and Ag-NPs of Atropa acuminata Hydroethanolic Extract
4. Conclusion
In this research, green synthesis of Silver nanoparticles of A. acuminata hydroethanolic root extract was
carried out to check its antioxidant and anti-diabetic potential. UV spectral analysis, TEM, SEM, EDXA
confirm the spherical, cuboidal shaped 20 nm nanosized particles. XRD revealed the crystalline lattice
of nanoparticles.Synthesis of silver nanoparticles wassupported by the phytochemicals available in
plant extract which work as reducing agent for the reduction of silver ions to nanoparticles. This method
of Nano architecture was very simple, easy, economic, ecofriendly, biocompatible and much superior
to chemical synthesis method. Nanoparticles shows maximum scavenging potential 82.633±0.116 for
superoxide anion free radicals at 100 µg/mL concentration. Among two anti-diabetic assay, α-amylase
assay shows better result of percent inhibition 63 ± 1.32 at 75 μg/ml concentration. Lastly in vitro, drug
release study revealed the 101.50% cumulative release from Ag-NPs formulation up to 1 hour which
was found to be better than the standard one. It indicated that the plant have excellent antioxidant and
anti-diabetic potential. So, there should be more and more germination of this critically endangered
species for medicinal worth prospectives.
Acknowledgement
Authors like to acknowledge IIT, Delhi for TEM Central Instrumental Facility, LPU for Nano Sizer,
SLIET Central University for XRD, SEM, EDXA and Shiva Institute, HPTU for providing facility of
synthesizing formulation and to carry out Drug Release Study.
Funding
This research did not receive any specific grant from funding agencies in the public and commercial
sectors.
Conflict of interest
The authors declare no conflict of interest.
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