Instrumentation 3. Literature Review 4. Materials and Methods 5. Result and Discussion 6. Conclussion 7. Reference

Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant and anti Oxidant
activity of synthesized Nikel Nano particles

- MEDIDI RANI PRIYA (2005020)
CONTENTS
1. INTRODUCTION
2. INSTRUMENTATION
3. LITERATURE REVIEW
4. MATERIALS AND METHODS
5. RESULT AND DISCUSSION
6. CONCLUSSION
7. REFERENCE
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CHAPTER-1
INTRODUCTION
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1. INTRODUCTION
Nanotechnology is one of the most significant and fast growing area in the field of science and
engineering, involving the combination of knowledge from chemistry, biology, materials science
and other related branches. The term “nano” is derived from a Greek language meaning dwarf or
extremely small, ranging in dimension from 1−100 nm. Nanoparticles (NPs) have a very high
surface-to-volume ratio because of their very small size. NPs are also called Nano-crystals.
Based on their shape, nanoparticles are known as nanocube, nanoflower, nanotube and nanowire
etc. [1] While based on their structure, nanoparticles are called as cluster, core shell and bimetallic
etc. [2, 3]
NPs have a wide range of applications in different commercial areas such as food,
cosmetics, agriculture, drug delivery, cancer detection/diagnosis, cancer therapy and many more.
[4]
The extent of NPs applications is due to unique and fascinating properties (magnetic, optical,
chemical, electronic, mechanical, sensing properties). Metallic nanoparticles (MNPs)
significantly vary in many properties from that of their bulk materials such as size, shape, surface
effect, electrical and magnetic properties.
Although, numerous metals nanoparticles and metal oxide nanoparticles exist in nature such as
silver, platinum, gold, copper, magnesium, cobalt, cesium oxide and zinc oxide etc. [4, 7]
Among
4|Page
the different MNPs, NiONPs have attracted the attention of scientific community due to
multifunctional and tunable nature. NiONPs are one of the most engineered NPs with a wide
band gap of (3.7-40 eV) and an intrinsic p-type semiconductor with an average size of 34 nm.
NiONPs possess a wide range of applications in lithium ion batteries, electrochromic test
devices, super-capacitors, smart windows, water remediation through photocatalysis,
electrochemical sensing and catalysis of chemical processes. [8, 9,6]

NiONPs also have
applications in the adsorption of toxic environmental pollutants and dyes. [10]

Because of their
anti-inflammatory property, NiONPs can also be used in biomedicines. [11]

The previous research
work of NiONPs have revealed cytotoxic effects by releasing ROS and Ni++ to oxidative
damage. [12]
NiONPs are prepared by various physical and chemical routes including sol-gel, galvanostatic
anodization, electro-deposition, hydrothermal, solvothermal and co-precipitation. [13, 14, 15, 16]
However, these physicochemical approaches have numerous harmful side effects which limit
their wide range biological applications. [17]

Chemical synthesis most frequently generate toxic
chemical waste leading to environmental toxicity and non-biodegradable products while that of
physical methods needs massive energy input. [18, 19]
In contrast, biological method to synthesize MNPs including NiONPs have been emerged as cost
effective option in the field of “green chemistry”. [6, 20, 21, 22]
Biofabrication of MNPs is
considered to be simple, eco-friendly, economically feasible, easily scaled up and accomplished
at room temperature and pressure in an aqueous environment. [21, 22] Biogenic synthesis of MNPs
5|Page
can be achieved using plants, bacteria, fungi and algae. [23, 24]
Among these, plant-extract
mediated synthesis of nanoparticles have significantly gained the attention due to its simplicity.
[25, 26]
The plant extract can act as a strong reducing, stabilizing and capping agent and has drawn
the attention of scientific community due to its simple, fast, cost effective and eco-friendly
nature. [25, 21]

The phytochemicals available in the plant extracts function as a strong reducing
agent and result in the development of capped NPs. Thus, plant extract function as both natural
reducing and capping agent, eliminate multiple steps and reduces costs and chemicals utilization.
[24, 25, 26]
Furthermore, the phytocompounds in aqueous environment replace chemical reagents
and organic/inorganic solvents. [20]

Currently, plant extracts of various parts (such as flower,
fruit, seed powder, leaf, peel, bark) have been used for the formation of NPs. [27, 28, 26]
Plant
extracts have medicinal values due to presence of different phytocompounds such as alkaloids,
flavonoids, terpenoids, polyphenols, amino acids and vitamins. [24, 25, 26]
The purpose of this study was to synthesize NiONPs using leaves extract of Rhamnus virgata.
The Rhamnus virgata is a rich source of alkaloids, flavonoids and anthraquinones compounds
and can be used as a natural source in the formation of biogenic nanoparticles. The plant is
comprised of (physcion, emodin, kaempferol, rhamnocitrin, quercetin, herbacetin, maesopsin)
which may act as a strong reducing, stabilizing and capping agents. [29, 30]
The ethnobotanical
survey and literature review has reported the medicinal values of Rhamnus virgata and have
shown strong therapeutic potentials such as antioxidant, antimicrobial, laxative, emetic and
6|Page
Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant and
anti Oxidant activity of synthesized Nikel Nano particles
purgative mostly used in the treatment of parasitic infections, spleen affection and in leg
swelling.
Green synthesis of NiONPs has been achieved in current years and there is a growing interest in
the synthesis of NiONPs for different biological applications from different other medicinal
plants. Plant-mediated synthesis of NiONPs have already been reported for Moringa oleifera
(Lam.), [34] Agathosma betulina (Berg.), Callistemon viminalis (Sims.), [8] Tamarix serotine
(Bunge. ex Boiss.) [35] and Nephelium lappaceum (L.). [36] In this study, we reported the synthesis
of NiONPs using aqueous leaves extracts of Rhamnus virgata without using surfactant or
organic/inorganic solvents. Furthermore, green NiONPs were characterized using UV, XRD,
SEM, TEM, EDS, DLS, Raman spectroscopy and FTIR. In addition, diverse in vitro biological
activities such as antibacterial, antifungal, biocompatibility assays against RBCs and
macrophages, cytotoxicity assays against brine shrimps, leishmanial parasites and HepG2 cell
line are also investigated.
UV Visible spectrophotometer:
Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis)

refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral
region. This means it uses light in the visible and adjacent (near-UV and near-infrared [NIR])
ranges. The absorption or reflectance in the visible range directly affects the perceived color of
the chemicals involved. In this region of the electromagnetic spectrum, molecules
undergoelectronic transitions. This technique is complementary to fluorescence spectroscopy, in
that fluorescence deals with transitions from the excited state to the ground state, while
absorption measures transitions from the ground state to the excited state.
Principle of ultraviolet-visible absorption:
7|Page
Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb the energy
in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding
molecular orbitals. The more easily excited the electrons (i.e. lower energy gap between the
HOMO and the LUMO), the longer the wavelength of light it can absorb.
Beer–Lambert law:
The method is most often used in a quantitative way to determine concentrations of an absorbing
species in solution, using the Beer-Lambert law:
where A is the measured absorbance, in Absorbance Units (AU), is the intensity of the incident
light at a given wavelength, is the transmitted intensity, L the pathlength through the sample,
and c the concentration of the absorbing species. For each species and wavelength, ε is a constant
known as the molar absorptivityor extinction coefficient. This constant is a fundamental
molecular property in a given solvent, at a particular temperature and pressure, and has units of
or often.
The absorbance and extinction ε are sometimes defined in terms of the natural logarithm instead
of the base-10 logarithm.
The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a
universal relationship for the concentration and absorption of all substances. A 2nd order
polynomial relationship between absorption and concentration is sometimes encountered for very
large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red, for example).
8|Page
Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant
and anti Oxidant activity of synthesized Nikel Nano particles
CHAPTER-2
INSTRUMENTATION
9|Page
2.Instrumentation:
The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction
grating in a monochromator or a prism to separate the different wavelengths of light, and a
detector. The radiation source is often a Tungsten filament (300-2500 nm), a deuterium arc lamp,
which is continuous over the ultraviolet region (190-400 nm), Xenon arc lamp, which is
continuous from 160-2,000 nm; or more recently, light emitting diodes (LED)[8] for the visible
wavelengths. The detector is typically a photomultiplier tube, a photodiode, a photodiode array
or a charge-coupled device (CCD). Single photodiode detectors and photomultiplier tubes are
used with scanning monochromators, which filter the light so that only light of a single
wavelength reaches the detector at one time. The scanning monochromator moves the diffraction
grating to "step-through" each wavelength so that its intensity may be measured as a function of
wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of these
devices consist of many detectors grouped into one or two dimensional arrays, they are able to
collect light of different wavelengths on different pixels or groups of pixels simultaneously.
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SPECTROPHOTOMETER
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Schematic of UV- visible

spectrophotometer
A spectrophotometer can be either single beam or double beam. In a single beam instrument
(such as the Spectronic 20), all of the light passes through the sample cell. Must be measured by
removing the sample. This was the earliest design and is still in common use in both teaching
and industrial labs.
In a double-beam instrument, the light is split into two beams before it reaches the sample. One
beam is used as the reference; the other beam passes through the sample. The reference beam
intensity is taken as 100% Transmission (or 0 Absorbance), and the measurement displayed is
the ratio of the two beam intensities. Some double-beam instruments have two detectors
(photodiodes), and the sample and reference beam are measured at the same time. In other
instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The
detector alternates between measuring the sample beam and the reference beam in synchronism
with the chopper. There may also be one or more dark intervals in the chopper cycle. In this case,
the measured beam intensities may be corrected by subtracting the intensity measured in the dark
interval before the ratio is taken.
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Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of gases
and even of solids can also be measured. Samples are typically placed in a transparent cell,
known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width
of 1 cm. (This width becomes the path length, , in the Beer-Lambert law.) Test tubes can also be
used as cuvettes in some instruments. The type of sample container used must allow radiation to
pass over the spectral region of interest. The most widely applicable cuvettes are made of high
quality fused silica or quartz glass because these are transparent throughout the UV, visible and
near infrared regions. Glass and plastic cuvettes are also common, although glass and most
plastics absorb in the UV, which limits their usefulness to visible wavelengths.[9]
Specialized instruments have also been made. These include attaching spectrophotometers to
telescopes to measure the spectra of astronomical features. UV-visible microspectrophotometers
consist of a UV-visible microscope integrated with a UV-visible spectrophotometer.
A complete spectrum of the absorption at all wavelengths of interest can often be produced
directly by a more sophisticated spectrophotometer. In simpler instruments the absorption is
determined one wavelength at a time and then compiled into a spectrum by the operator. By
removing the concentration dependence, the extinction coefficient (ε) can be determined as a
function of wavelength.
Applications of UV-Visible Spectrophotometry:
1. Chemical Applications of UV-visible spectrophotometry:

a. Quantitative Analysis (Identification of the concentration of the unknown
substance):
UV-visible spectrophotometry is used to measure the concentration of an unknown sample. For

this, first, the choice of the absorption band is to be made where the absorbance measurements
will be taken (meaning in what range of wavelength will the sample absorb). The sample of
interest’s absorption spectrum may be available in the literature if it has already been researched.
If not, then double-beam spectrophotometry has to be performed to know where its absorption
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band will lie.
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A suitable absorption band is now selected. Generally all the organic compounds will absorb in
the UV-visible range of the spectrum and so a number of biological compounds may be
measured using UV-visible spectrophotometer. Unknown concentration of nucleic acid and
proteins are a good example. Nucleic acids absorb at 254nm (or 260nm) and proteins at 280nm.
Nucleic acids absorption depends on the aromatic rings of purines and pyrimidines while that of
proteins at 280nm depends on the number of amino acids - tyrosine and tryptophan content and a
little due to phenylalanine content.
b. Qualitative Analysis (Identification of an unknown substance):
UV-visible spectrophotometry may be used to identify various classes of compounds in both

pure state and as well as in biological preparations. This is done by plotting the absorption
spectrum curves. These curves represent specific class of compounds and a knowledge of these
curves will help in identification of any substance. For example, the substances which do not
absorb in 220-280nm range are usually aliphatic or alicyclic hydrocarbons or their derivatives.
Also, the complex systems will give rise to absorption curves with several maxima and each of
them will have a characteristic shape and range indicating the presence of a particular functional
group.
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The graph on the right side shows the absorption spectrum of several plant pigments. As we can
see each of the pigment has its own peculiar absorption spectrum which will help it identify in a
mixture of compounds.
c. Enzyme Assay:
The enzyme activity can be easily, quickly and conveniently be calculated when the substrate or
the product is colored or absorbs light in the UV range. In these cases. The rate of appearance or
disappearance of light absorbing product or substrate can be measured with the help of
spectrophotometer (which can also give the continuous record of the progress of reaction). We
will take an example of the enzyme lactate dehydrogenase to understand how the enzyme assay
is carried out or how enzyme activity is measured. Lactate dehydrogenase is an enzyme involved
in the transfer of electrons from lactate to NAD+. The reaction is shown as follows:
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So, as we can see here, the products are pyruvate, NADH and a proton. Here, one of the
products, NADH absorbs radiation in the ultraviolet range at 340nm and its counterpart NAD+
does not. Neither any of the other substrate nor the product absorbs at 340nm. Thus, the progress
of the reaction in the forward direction can be followed by measuring the increase in absorption
at 340nm in spectrophotometer. Here, comes the role of optical assays which prompts their use
in following the time course of an enzymatic reaction in which neither the substrate nor the
product have a characteristic absorption spectrum.
Such reactions are then coupled to another enzymatic reaction (hence also called Coupled Assay)
which can be measured easily optically. Example of such reaction is that of phosphoenopyruvate
and ADP reacting to yield pyruvate and ATP catalyzed by pyruvate kinase.
Here, as neither any of the substrates nor the products absorb radiation, hence, this reaction can
be coupled to the above mentioned first reaction. Here, if lactate dehydrogenase and NADH are
added in excess, the system will be a little manipulated and we will get the coupled reaction as
follows:
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As we have added excess of NADH to the reaction, the system will now absorb at 340 nm. Thus,
for each molecule of pyruvate formed in the first reaction, a molecule of NADH is oxidised to
NAD+ in the second reaction where pyruvate in converted to lactate. As mentioned earlier, NAD
does not absorb at 340nm, the absorbance goes on decreasing as pyruvate gets converted to
lactate.
Structural Applications of UV-Visible Spectrophotometry:
a. Control of Purification:
This is one of the most important application of UV-visible spectrophotometry. Impurities can be
detected very easily by testing if the compound is not showing its characteristic absorption
spectrum. Example: Benzene impurity in absolute alcohol can be detected by this method. This
can be detected by measuring the absorbance at 280nm. As at 280nm, benzene will absorb,
whereas alcohol (210nm) will not absorb.
b. Study of Cis-Trans Isomerism:
The trans-isomer is more elongated as compared to its counterpart cis-isomer. Hence, this
structural difference will be reflected in absorbance spectrum. The trans-isomer will have a
higher wavelength of maximum absorption.
The graph at the adjacent shows the absorption spectrum of the azobenzene dye, 4-n-butyl-4'-
methoxyazobenzene (BMAB) where both cis-BMAB and trans-BMAB have different absorption
spectrum.
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c. Molecular Weight Determination:

Suppose a compound forms a derivative with a reagent which has a characteristic absorption
band. Suppose a compound forms a derivative with a reagent; now it will give the absorption
band of a high intensity at a wavelength where the compound does not absorb, then the
extinction coefficient of the derivative is usually the same as that of the reagent. Although the
extinction coefficient will remain same for any of the derivatives formed, the optical density is
different for the compounds of different molecular weight. The molecular weight of the
compound can then be calculated readily on the basis of absorption data.
M = awb/OD
Where a – absorption coefficient
w – Weight of the compound in g/l
b - path-length.
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d. Turbidimetry:
Any particulate matter (or even bacteria) makes the solution look turbid. This is due to
Tyndall effect which is because of the light scattering by the colloidal particles.
The particles in this solution will absorb at a particular wavelength and these particles will
scatter the incident light. If this happens, then the radiation of a wavelength which is
not absorbed by the solution is made to pass through the suspension and the apparent
absorption will be solely because of the scattering by the particles. So, the transmitted light
will have lower intensity as compared to that of the incident light. As a result, if the intensity
of the transmitted light is measured, it will give an idea of the number of particles in the
suspension. This technique is turbidimetry. By using this technique, we can find out an
approximate number of particles in a given suspension.
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CHAPTER-3
LITERATURE
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Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant and anti
Oxidant activity of synthesized Nikel Nano particles
Abstract
In the present report, Nickel oxide nanoparticles (NiONPs) were synthesized using Rhamnus
virgata (Roxb.) (Family: Rhamnaceae) as a potential stabilizing, reducing and chelating
agent. The formation, morphology, structure and other physicochemical properties of
resulting NiONPs were characterized by Ultra violet spectroscopy, X-ray diffraction (XRD),
Fourier Transform Infrared analysis (FTIR), Scanning electron microscopy (SEM), Energy-
dispersive-spectroscopy (EDS), Transmission electron microscopy (TEM), Raman
spectroscopy and dynamic light scattering (DLS). Detailed in vitro biological activities
revealed significant therapeutic potential for NiONPs. The antimicrobial efficacy of biogenic
NiONPs was demonstrated against five different gram positive and gram negative bacterial
strains.
Klebsiella pneumoniae and Pseudomonas aeruginosa (MIC: 125 µg/mL) were found to be
the least susceptible and Bacillus subtilis (MIC: 31.25 µg/mL) was found to be the most
susceptible strain to NiONPs. Biogenic NiONPs were reported to be highly potent against
HepG2 cells (IC50: 29.68 µg/mL). Moderate antileishmanial activity against Leishmania
tropica (KMH23) promastigotes (IC50: 10.62 µg/mL) and amastigotes (IC50: 27.58 µg/mL)
cultures are reported. The cytotoxic activity was studied using brine shrimps and their IC50
value was recorded as 43.73 µg/mL. For toxicological assessment, NiONPs were found
compatible towards human RBCs (IC50: > 200 μg/mL) and macrophages (IC50: > 200 μg/mL),
deeming particles safe for various applications in nanomedicines.
Moderate antioxidant activities: total antioxidant capacity (TAC) (51.43 %), 2,2- diphenyl-1-
picrylhydrazyl (DPPH) activity (70.36 %) and total reducing power (TRP) (45 %) are
reported for NiONPs.
LITERATURE
Plant sampling and identification
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Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant and anti
Oxidant activity of synthesized Nikel Nano particles
Causonis trifolia commonly known as bush Grape, fox-grape, three-leaved wild vine or three leaf
cayratia is a species of liana plant native to Australia and Asia. It has black-colored berries, and its
leaves contain several flavonoids, such as cyanidin and delphinidin. Hydrocyanic acid is present in the
stem, leaves and roots.
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Preparation of plant extract
10-15 g of cleaned Causonis trifolia (dried leaves powder) was added in 300 mL of deionized
water under magnetic stirrer and was placed on hot plate at 80°C for 2 hr. The plant extract was
cooled and was than three times filtered using Whatman filter paper to achieve pure aqueous
extract. The pH of aqueous extract was recorded 7.3 at room temperature. The plant extract was
kept in refrigerator at 4°C for future studies.
Synthesis of nickel oxide nanoparticles
Previously described green synthesis protocol with slight changes was used for the formation of
NiONPs. [9]
The plant extract was shifted to a reaction flask and 6.0 g of precursor salt NiNO 3
(purchased from Sigma Aldrich) was added to synthesize NiONPs. The resulting solution (pH.
6.89) was followed by heating at 60°C for 2 hr. The collected precipitate was allowed to cool,
followed by three time washing at 3,000 rpm for 25 min. After washing, the sample was dried at
100°C using incubator (K & K Scientific and Medical equipments, Korea) for 2 hr followed by
calcination at 500°C for 3 hr in open air furnace (KSL-1100X, MTI Corporation, China). The
particles formed were considered as NiONPs and were characterized using diverse
characterization techniques. A general study layout is shown in (Figure 1)
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Characterizations techniques
Diverse characterization techniques were applied to study the physical, chemical, optical,
thermal and electrochemical properties of NiONPs. The synthesis of NiONPs was confirmed
using a UV-Vis spectrophotometer (UV-4000 UV-Vis spectrophotometer Germany) in the range
of 200−400 nm wavelength. The composition and the functional groups which are involved in
capping and efficient stabilization of the NiONPs were identified by Fourier transformed infrared
spectroscopy (FT-IR). The spectral range used for scanning sample was 400–4000 cm -1. The FT-
IR spectra were observed using FT-IR spectrometer (Alpha, Bruker, Germany). To further
evaluate nanoparticles, Raman spectrum was recorded for NiONPs calcined at 500°C. Moreover,
size and shape were studied using TEM. Particle size distribution and morphological features of
nickel oxide nanoparticles were determined using scanning electron microscopy (SEM (NOVA
FEISEM-450 equipped with EDX detector). XRD analysis was carried out for the thermally
annealed sample on X-ray diffractometer (PANalytical, Netherland) equipped with a Cu
radiation source at 45 kV and 40 mA of current voltage and their corresponding size was
calculated using Scherrer equation. The hydrodynamic size distribution, ζ-potential and
polydispersity index (PDI) were evaluated by dynamic light scattering (DLS) system (Malvern
Zetasizer Nano). In addition, the elemental composition was confirmed by EDS analysis.
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Cytotoxic assays:
Brine shrimp cytotoxicity Eassay
To confirm the cytotoxic effect of green NiONPs, brine shrimps cytotoxicity assay was
performed using Artemia salina larvae (Ocean Star, USA). Eggs of Artemia salina were
incubated for 48 hr under light at 30°C in sea water (3.8 g/L). After 24 hr incubation 20 mature
nauplii were harvested through micropipette and were shifted into glass vials containing sea
water and NiONPs. Different doses of NiONPs were used. The vials with mature nauplii,
vincristine sulphate and sea water was considered as positive control while the vials containing
nauplii, DMSO and sea water was used as negative control. After 24 hr incubation dead shrimps
were counted and % inhibition was calculated. Median lethality dose (LD 50) was recorded using
GraphPad software.
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Antileishmanial assay
The Leishmania tropica “KWH23 strain” (both promastigotes and amastigotes cultures) were
used to study the cytotoxic activity of NiONPs via MTT cytotoxic assays. [37]
The Leishmanial
parasites were culture on MI99 media added with 10% fetal bovine serum (FBS). The
Leishmania tropica KWH23 parasites were cultured to a density of 1 × 106 cells/mL.
Amphotericin B was used as a positive control and DMSO as negative control. NiONPs in the
range of 1−200 μg/mL was studied for their cytotoxic potential. The 96 well plate with tested
NPs was placed in 5% CO2 incubator for 72 hr at 24°C. Reading were recorded at 540 nm using
spectrophotometer. All the survived promastigote and amastigotes were counted under an
inverted microscope and IC50 value was calculated using GraphPad software. The percentage
inhibition was measured as:
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1 − (absorbance of sample)
% age inhibition = [ ]×100
(absorbance of control)
Anticancer activity
Previously optimized protocol [38] was used to study the in vitro cytotoxic potential of bioinspired
NiONPs against HepG2 cancer cell line. Cells were grown on DMEM media added with 10 %
FBS, 1% penicillin and 1% streptomycin. In addition, cells were grown in humified 5 % CO 2
incubator at 37°C. The MTT assay was demonstrated in 96-well microplate by incubating at
different test concentration of the NiONPs (500−3.9 µg/mL) for 48 hr. 20 µL of MTT solution
was loaded in each well and was placed in incubator for 3 hr. The culture media was replaced
with 100 µL of DMSO followed by further incubation for 20 min. The quantity of formazan
formed by survived cells was monitored by means of plate reader at wavelength of 570 nm with
a reference wavelength of 630 nm.
Antimicrobial activities:
Antibacterial activity
Antimicrobial activity of Rhamnus virgata synthesized NiONPs was evaluated via disc diffusion
method against gram-positive bacterial strains including Bacillus subtilis (ATCC 6633) and
Staphylococcus aureus (ATCC 25923) and gram negative strains Escherichia coli (ATCC 15224),
Pseudomonas aeruginosa (ATCC 9721) and Klebsiella pneumoniae (ATCC 4617). For this
purpose, 200 µl of bacterial cultures were utilized to get uniform bacterial lawns. Filter discs (6
mm) loaded with 10 µl of test samples were carefully placed on uniform lawns while 10µg
28 | P a g e
oxytetracycline disc was used as a positive control. After 24 hr incubation at 37°C, zones of
inhibition were measured and their respective MIC values were calculated in the concentrations
range of (700−21.875 µg/mL). Furthermore, NiONPs were exposed for about 20 min to UV light
illumination (germicidal 6 Watt UV rod 6GT5) to determine their antibacterial potential.
Antifungal assay
The antifungal activity of NiONPs was also determined using different fungal strains such as
Candida albicans (FCBP 478), Mucor racemosus (FCBP 0300), Aspergillus niger (FCBP 0918),
Fusarium solani (FCBP 0291) and Aspergillus flavus (FCBP 0064). Sabouraud dextrose agar
media (Oxoid CMO147) was prepared and autoclaved for the growth of fungal strains and was
allowed to solidify. Filter discs were loaded with 10 µL of sample solution and were placed on
media plates. 10 µg of amphotericin B was used as a positive control while DMSO as a negative
control. Furthermore, these plates were placed in incubator overnight at 28°C for 48 hr. The
zones of inhibition were measured via Vernier caliper. Different doses of NiONPs were used in
range of 700−10.9375 µg/mL. MICs values were calculated at lowest concentration of NiONPs.
Enzyme inhibition potentials:
Protein kinase (PK) inhibition
Protein kinase inhibition assay for green NiONPs was performed as discussed earlier. [9]
The
whole experiment was performed under sterile conditions. Streptomyces 85E strain was culture
on ISP4 minimal media to achieve uniform lawns. After that 10 µL of NiONPs was added on 6
mm autoclaved filter disc and was placed on microbial lawns. Surfactin was used as positive
control and DMSO was used as negative control. The incubation period was executed for 24−36
hr at 30°C to target the growth of strain. The appearance of clear and bald zones around discs
indicates the inhibition of spore and mycelia formation.
Alpha amylase (AA) inhibition
Alpha amylase inhibitory potential of green NiONPs was performed using previously optimized
protocol. [9]
The reaction mixture was comprised of 25 μL of AA enzyme, NiONPs (10 μL),
starch solution (40 μL) and PBS (15 μL). Reaction mixture with all components was placed in
incubator for 30 min at 50°C followed by adding 20 µl of 1 M HCL and 90 µl of iodine solution.
Acarbose was used as positive control while deionized water was used as negative control.
Following formula was used to calculate percentage inhibition
% inhibition = [(ODS – ODN) ÷ (ODB – ODN)} × 100
Biocompatibility assays:
Biocompatibility against human RBCs
Haemolytic assay was performed to determine the biocompatible nature of NiONPs with freshly
extracted human RBCs. For this purpose, 1 mL fresh human RBCs was isolated and centrifuged
at 12,000 rpm for 12 minutes. The erythrocyte suspension was prepared in a test tube by mixing
200 µL of erythrocytes with 9.8 mL of phosphate buffer saline (pH 7.2). Erythrocytes suspension
of 100 µL was added with different doses of NiONPs and were incubated at 35°C for 50 min.
Moreover, sample was centrifuged at 12,000 rpm for 20 min. The supernatant was collected and
shifted in a 96-well plate and hemoglobin release was studied at 540 nm. DMSO and Triton X-
100 were used as negative and positive control respectively. The results were recorded as %
hemolysis caused by NiONPs dilution which can be calculated using the formula below:
[Absorbance of sample – Absorbance of negative control

% hemolysis ]×100
Absorbance of positive control − Absorbance of negative control
Biocompatibility against macrophage
The biocompatible nature of NiONPs was studied against freshly isolated macrophages. For this
purpose, Ficoll−Gastrografin based method was applied to isolate macrophages. The freshly
isolated blood cells were diluted with Hank’s buffer salt solution, layered on
Ficoll−Gastrografin, centrifuged at 12,000 rpm for 25 min and was purified using percoll
gradient adjusted with autoclave distilled water. The cells isolated were grown on RPMI media
with addition of 10% FBS, 0.1 mg/mL streptomycin, 25 mM HEPES and 100 U/mL Penicillin.
Th macrophages were cultured and incubated with 5% CO 2 to a density of 1 × 105 cells/well. The
below formula was utilized to calculate % inhibition of macrophages.
1 – Absorbance of sample
% inhibition = ×100
Absorbance of control
Antioxidant assays:
DPPH assay
DPPH free radical-scavenging potential was performed using different doses of NiONPs (1−200
µg/mL) through microplate reader to determine the antioxidant potential of NiONPs. [39] For this
purpose, reagent solution was set by adding 2.4 mg of DPPH to 25 mL of methanol. Ascorbic
acid was used as positive and DMSO as negative control. The procedure involves the addition of
20 µL of test sample into 180 µL of reagent solution to prepare 200 µL of reaction mixture. After
incubation for 1 hr, readings were taken at 517 nm using microplate reader to determine the %
scavanging of DPPH applying the formula below:
Absorbance of sample
% scavanging = 1-[ ]×100
Absorbance of control
Total reducing Power (TRP)
Previously described method was used to determine the TRP of green NiONPs [9]
The 40 µL test
sample was added into 50 µL of PBS followed by incubation for 20 min at 50°C. 50 µL of
tricholro-acetic acid (10%) was mixed with mixture and was centrifuged at 3000 rpm for 1 min.
33.3 µL of 0.1% FeCl3 was added to 166.6 µL of the collected supernatant in a 96 well-plate.
Ascorbic acid was used as a positive control while DMSO as a negative control. Absorbance was
recorded at 630 nm using microplate reader and the results are shown as ascorbic acid equivalent
per/mg of the test sample.
l antioxidant capacity (TAC)
TAC test was performed using phosphomollybdenum method. [9]

The reaction mixture was made
by mixing 0.6 M H2SO4, 28 mM of NaH2PO4 and 4 mM (NH4) 6 Mo7O24 4H2O and was
incubated at 95°C for 90 min. Readings were taken at 695 nm. Results are shown as microgram
equivalent of ascorbic acid per/mg of test sample.
Physical characterizations of the NiONPs
Different physical characterization techniques were used to confirm the synthesis of NiONPs.
The progress of NiONPs formation was monitored by a color change after salt (Nickel nitrate)
addition at 60°C. The change in solution (black color) demonstrates the presence of NiONPs. To
determine the stability of NiONPs, 1 mg/mL solution of NiONPs was made and sonicated for 25
min. The turbid colloidal suspension was allowed to stand for 48 hr and surface plasmon
resonance was checked from time to time using spectrophotometers. Absorption at 332 nm
shows that colloidal suspension remained stable for 24 hr. The results are given in Figure 2a, b.
Figure 2c represents the FTIR spectra of NiONPs synthesized at 60°C. The presence of various
chemical bondings in NiO were determined by FTIR analysis. The FTIR spectrum of NiO shows
vibration at ~1030 cm−1, ~ 1380 cm−1, ~1630 cm−1 indicating the presence of –OH groups and
vibration at ~3425 cm−1 is for H2O which play important role in both stabilization and reduction
of NiONPs. The bands in the region 800–470 cm−1 give information about NiO. For further
confirmation of NiONPs, Raman spectrum was observed in the range of 200 cm −1 to 2000 cm−1
as shown in Figure 2d. The distinguished the major modes of Raman spectra were centered at
~561 cm−1 (1 P), ~1047 cm−1 (2 P) and ~1616 cm−1 (2 M). From the high intensity sharp peak at
~561 cm−1, it can be concluded that NiONPs is defect rich. The presence less intense yet broader
peak at 1616 cm-1 corresponds to decrease in antiferromagnetic spin correlation among
individual Ni++ and is additional evidence for the nanosized nature of NiONPs (crystal size ~ 24
nm).
The morphology of NiONPs was further observed by scanning electron microscopy (SEM) and
transmission electron microscopy (TEM). Figure 3 a,b,c,d indicates typical SEM and TEM
images of NiONPs incubated at 100°C and annealed at 500°C. In SEM images shape of the
nanoparticles appeared to be agglomerated. TEM images revealed the spherical morphology of
nanoparticles with an average size of ~24 nm. The crystalline nature of the sample was study by
XRD analysis calcined at 500°C. The XRD spectrum has confirmed the successful formation of
NiONPs. XRD spectrum further revealed the pure crystalline nature of NiONPs along with 2
theta (2Ø) values of 27.33, 37.14, 43.22, 62.81, 76.95 and 79.27 which are indexed to (110),
(111), (200), (220), (311) and (222) Bragg’s reflection of the face centered cubic (fcc) crystalline
phase of NiONPs. The average size was ~24 nm using Debye Scherer’s equation (D= k λ/ β½
cosθ) and is in agreement with TEM results. No Bragg peaks for other related compounds were
found confirming the pure crystalline nature of biogenic NiONPs. Results of the XRD analysis
are given in Figure 4a, b.
The hydrodynamic size distribution, PDI and ζ- potential of NiONPs calcined at 500°C was
measured by DLS. The data have shown larger particle aggregates of 173.7 nm. The zeta
potential and PDI of NiONPs was -13 mV and 1.000 respectively (Figure 5a, b. DLS is mainly
used to confirm nanoparticles size in colloidal suspensions in range of nano and submicron. The
average hydrodynamic particle diameter in water shows particle aggregation. Our
characterizations results are consistent with Sudhasree et al. [11]

for aggregation of NPs in
aqueous medium. Furthermore, elemental composition of NiONPs was studied using EDS,
which shows the presence of both Ni and O in the sample. The EDS spectrum is shown in Figure
5c.
Cytotoxic assays
Brine shrimp lethality assay (BSLA)
The cytotoxicity potential of green NiONPs was studied using brine shrimps as a model
organism. Figure 6a shows the % mortalities at different doses (200 to 1 μg/mL) of
biosynthesized NiONPs. The cytotoxicity of NiONPs was confirmed through dose dependent
manner and was increasing as the concentration of NPs was increasing while their median
lethality dose (IC50) was calculated as 43.73 µg/ml.
Antileishmanial activity (ALA)
The ALA of biosynthesized NiONPs was determined against Leishmania tropica (KMH23) as
indicated in Figure 6b. Leishmanial parasites were exposed to NiONPs at different
concentrations (1 to 200 μg/mL) for 72 hr and has shown dose dependent inhibition. The
antileishmanial potential was increasing as concentration of the NiONPs was increasing. The
NiONPs have shown strong antileishmanial potential against L. tropica promastigotes (IC50:
10.62 µg/mL). Similarly, strong ALA against L. tropica amastigotes was shown by NiONPs
(IC50: 27.58 µg/mL). The dose dependent and lower IC50 values of the NiONPs showed that
these materials can be used for strong antileishmanial drug delivery in the future medicines.
Anticancer activity
The anticancer potential of NiONPs was also performed against HepG2 cell line. Cancer cells
exposed to different concentrations of NiONPs (500 to 3.9 µg/mL) for 24 hr resulted in a dose
dependent inhibition of cancer cells. Our results have shown significant anticancer potential for
NiONPs. However, ~84% mortality of cancer cells was observed at 500 µg/mL and the
anticancer potential was decreasing as the concentration was decreasing. The IC50 value
calculated was 29.68 µg/mL. The data is given in (Figure 6c). The plausible schematic
representation of cytotoxicity is shown in (Figure 7).
Antimicrobial assay
Antibacterial activity
Newly synthesized green NiONPs were used against five different bacterial strains across six
different concentrations (1000−31.25 µg/mL) with and without UV illumination. The Gram
positive bacteria strains that were used are S. aureus and B. subtilis while gram negative strains
are P. aeruginosa, K. pneumonia and E. coli. Our study reported that the antibacterial potential
was increasing with increase in NPs concentrations. Many strains were found susceptible to these
NPs. K. pneumonia and P. aeruginosa were found least susceptible bacterial strains with MIC
values of 125 µg/ml. MIC values are given in table 1. The bacterial strain that was most
susceptible to biogenic NiONPs was B. subtilis with MIC values of 31.25 µg/mL. 10 µg
oxytetracycline drug was used as a positive control. No single concentration was found effective
than oxytetracycline drug. The antibacterial activity against different test concentration are
shown in (Figure 8a). Figure 8b shows the antibacterial potential of the NiONPs after UV
exposure. Increase was observed in the antibacterial potential after being exposed to the UV
illumination.
Antifungal activity
Antifungal activities of biogenic NiONPs were performed against different fungal strains in the
concentration range of (1000−15.625 µg/mL). The Amphotericin B was used as a positive
control. The different fungal strains used in the study include M. racemosus, A. flavus, A. Niger,
C. albican, and F. solanai (Figure 8c). Our results concluded direct relation between
concentration and test sample of NiONPs. M. racemosus (MIC: 31.25 µg/mL) was the most
susceptible strain and that of A. flavus (MIC: 125 µg/mL) was found to be least susceptible
strain. MIC values for different fungal strains are given in table 2.
Enzyme inhibition potential
Figure 9a depicts protein kinase enzyme inhibition potential of NiONPs. Protein kinase
inhibitors play significant role with respect to cancer therapeutics. [40]

These enzymes are playing
crucial role in phosphorylation of important amino acids and regulate vital biological pathways:
cell division, differentiation and apoptosis etc. Deregulated phosphorylation by PK enzymes at

serine-threonine and tyrosine residues can lead to tumor development. Hence, chemicals
substances with ability to inhibit PK enzyme is highly important. In the Streptomyces-85E strain,
PK play significant role in the development of hyphae. The same strain was used to determine
the PK inhibitory potential of biogenic NiONPs. Following disc diffusion method, maximum
zone of inhibition (17 mm) was observed at highest concentration (1000 μg/mL), which
indicated strong PK inhibition potential of biogenic NiONPs.
Figure 9b shows the inhibition potential of alpha amylase enzyme (AA) by biogenic NiONPs.
Furthermore, the biogenic NiONPs has shown insignificant amylase inhibition (36.1 %). The
alpha amylase is responsible to convert carbohydrates into glucose therefore associated with
postprandial blood glucose excursion in a person suffering from diabetes. Hence, block starch
conversion to glucose can be blocked by inhibiting alpha amylase enzyme and therefore,
represent main area of diabetic research. Moderate inhibition is reported to be 36.1 % at a highest
concentration of 1000 μg/mL. However, no inhibition was seen up to concentration of (125
μg/mL).
Biocompatibility tests
The biocompatible nature of bioinspired NiONPs and their toxicological properties were studied
against freshly isolated human RBCs and macrophages. Results were examined in the range of
200−l μg/mL. According to American Society, biological substances showing % hemolysis of >
5% is known as hemolytic, between 2−5% are slightly hemolytic, while < 2% is non-hemolytic.
[27]
In this regard, NiONPs are non-hemolytic at lower concentrations (< 5 μg/mL), while highest
hemolysis (28.23 %) was reported at the concentration of 200 μg/mL. The LD 50 value for
NiONPs against RBCs was calculated to be > 200 μg/mL.
For further investigation of the biocompatible nature of NiONPs, MTT cytotoxicity assay was
performed against human macrophages. The results have shown that these particles are non-toxic
towards human macrophages. In this regard, NiONPs are non-toxic at lower doses of 2 μg/mL,
slightly toxic at 5–100 μg/mL, while macrophages were found more effected with % mortality of
40.2 % at 200 μg/mL. The IC 50 value of NiONPs was recoded to be > 200 μg/mL. Generally, the
test samples can be recognized as safe at low level. The biocompatibility results for NiONPs are
summarized in Figure 9c. The IC50 values for different assays are given in table 3.
3.6 Antioxidant activities
Biogenic NiONPs were found to have strong antioxidant potentials. Antioxidant potentials of
biogenic NiONPs were studied via DPPH activity, total reducing power (TRP) and total
antioxidant capacity (TAC). The results revealed highest scavanging capacity of 70.36 % at the
concentration of 200 µg/mL, while the scavanging potential was decreasing as the concentration
was decreasing. The highest TAC (51.43 %) and reducing powers (45 %) were observed on
highest concentration (200 µg/mL). In short, good radical scavanging potential, moderate TAC
and TRP potential have been shown by NiONPs (Figure 9d).

4. Materaials and Methods
Among the different medicinal plants that can be utilized in the biofabrication of
nanoparticles, Rhamnus virgata was selected due to high content of bioactive compounds
(physcion, emodin, kaempferol, rhamnocitrin, quercetin, herbacetin and maesopsin) which can
function as strong reducing, stabilizing and capping agents in the biosynthesis of nanoparticles.
[29, 30]
These phytocompounds are rich source of electrons and are responsible for reduction and
stabilization of NiONPs. [21, 25]

A schematic representation of the biosynthesis of NiONPs is
given in Figure (10). Mostly phenolic compounds are responsible for the biogenic synthesis of
NPs, however the clear mechanism by which NPs get synthesized is an open area of research for
the scientific community. Successful plant mediated synthesis of NiONPs using plant extracts
have been reported in the literature. [8, 34, 35]

Previously NiONPs have been synthesized but their
extensive biological potentials like antibacterial, antifungal, antioxidant, anticancer, enzyme
inhibition, antileishmanial and biocompatibility assays are rarely reported.
Biogenic synthesized NiONPs were characterized by different techniques. Bragg peaks for these
NPs are of pure NiONPs in XRD pattern as no other peaks were found for other compounds. The
TEM results are consistent with XRD data. Our results are in accordance to the XRD pattern of
NiONPs using Moringa oleifera (Lam.). [34]

EDS analysis has further explored the presence of
Ni-O in the sample. The absorption bands in FTIR were found having stretching vibrations of
Ni_O in NiONPs and are in agreement with previous results [41, 9, 6]

The FT-IR spectrum was
consistent with the results obtained by XRD data as no other impurity phase was observed.
Considering the difference in Raman shifts (that could be because of stress/strain effect, size,
intensity and relative positions) of 5 peaks, spectra are in agreement with reported values of
photon modes. Overall, Raman scattering support the purity of NiONPs and our results are
consistent with the earlier reports. [17, 8]

DLS is primarily used to confirm NPs size in the
colloidal suspensions in the range of nano and submicron. The measurement of zeta potential is
based on the movement of particles under electric field. Moreover, surface charge and local
environment of the particles also play significant role in the measurement of zeta potential. [42]
Our hydrodynamic size distribution and zeta potential results are in correspondence with the
earlier studies performed on NiONPs. [43, 44]
Bacterial infection is a public health problem in both developed and developing countries in
terms of mortality. Traditional medicines (antibiotics) are often used for bacterial infections but
certain problems are associated with them such as antibiotic resistance. Scientific community is
engaged to develop new approaches to resolve the issue of antibiotic resistance and minimize the
risk of its development. [45, 46]

In current study, NiONPs has shown strong antibacterial and
antifungal potentials. The antimicrobial activities were found to be dose-dependent. The
antibacterial and antifungal potential was increasing as the concentration of NiONPs was
increasing.
Generally, there was also increase in the antibacterial potential of NiONPs after UV exposure.
For example, the MIC values calculated for P. aeruginosa was found to be 125 µg/mL while
with UV exposure, the MIC was reduced to 31.5 µg/mL. Previously, Khalil et al. [9]
has also
reported that UV illumination of Sageretia thea (Osbeck) mediated NiONPs has significantly
increased antibacterial potential where P. aeruginosa was found to have MIC value of 250
µg/mL while with UV exposure, the MIC was reduced to 15.6 µg/mL. The strong antibacterial
potential of NiONPs due to UV exposure might be due to high production of ROS. This high
production of negative ROS due to UV exposure is already reported by scientists. [47]

The ROS
such as, H2O2 intrude inside bacterial cells and initiate genotoxic effect. Further, research studies
have reported the formation of novel complexes due to UV irradiation that enhances antibacterial
potential against different pathogenic bacterial strains. [47]

The oxytetracycline and amphotericin
B were used as positive control and have shown greater zone of inhibition compared with
different doses of nanoparticles. Recent research study has also investigated that reference drugs
were more effective than NiONPs. [9] The new advancement in nanotechnology will provide new
opening for novel tools and will develop new substances with strong antimicrobial properties. [48]
Additionally, NiONPs have shown strong antitumor potential against HepG2 cell line by
inhibiting cancer cell progression. Our results are in correspondence with the previous studies
using Hep-2, MCF-7 and HT-29 cell lines, where NiONPs have shown strong cytotoxicity using
MTT measurement assays. [49,50]

NPs take advantage of the unique tumor microenvironment
(angiogenesis, permeable vasculature, poor lymphatic system etc) which are not associated with
normal cells/tissues thus act as a potential candidate for targeted drug delivery in tumor cells.
NPs in combination with other anticancer drugs have shown synergistic effect by targeting
cancer lesion without damaging normal cells. [51]

Furthermore, NPs used in smart imaging has
significantly decreased the death rate of cancer. [52]

BSLA and ALA assays were performed to investigate the cytotoxic effect of NiONPs. BSLA is
the most appropriate test to investigate the biological effect of any naturally occurring
compound. [53]
The present study concluded dose-dependent response of NiONPs. Our test
sample was found to be in category of general cytotoxicity with IC 50 value of 43.73 µg/mL and
are consistent with the previous results of Ates et al. [44]

. Leishmaniasis is a neglected tropical
disease caused by Leishmania tropica (KMH23). The antileishmanial drugs conventionally used
are often toxic, expensive and less effective against leishmanial parasites. NPs have shown
potential antileishmaniasis activity. [54,

. In current study, NiONPs have shown strong
55]
antileishmanial effect against amastigotes compared to promastigote and have shown dose-
dependent inhibition of leishmanial parasites. Antileishmaniasis results revealed that amastigotes
are more vulnerable compared to promastigotes and is in accordance with the previous report. [9]
The applications of different nano-formulations have also confirmed potential results in the
treatment of leishmaniasis. [56]

Biocompatibility assays revealed that NiONPs are biocompatible
and can be used in different therapeutic treatments. Similar results have been reported for
NiONPs in the earlier studies using Sageretia thea. [9]

Current research findings are premature
and need further research with normal human cells. Our results have also revealed strong
antioxidant potentials which increases with increase in NiONPs concentration and are consistent
with previous studies.

CHAPTER-7
REFERENCE
References:
[1] M. Khatami, R.S. Varma, N. Zafarnia, H. Yaghoobi, M. Sarani, V.G.., Kumar, 2018e. Sustain. Chem. Pharm,
2018e, 10, 9.
[2] K. Karthik, S. Dhanuskodi, C. Gobinath, S. Prabukumar, S. Sivaramakrishnan, J. Mater. Sci. Mater. Electron,
2018b, 7, 5459.
[3] M. Khatami, H. Alijani, M. Nejad, R. Varma, Appl. Sci. 2018a, 8, 411.
[4] J. Iqbal, B.A. Abbasi, R. Ahmad, T. Mahmood, B. Ali, A.T. Khalil, A. Munir, Appl. Microbiol. Biotechnol, 2018
(b), 102, 9449.
[5] Y. Han, S. Zhang, N. Shen, D. Li, X. Li, Mater. Lett. 2017, 188, 1.
[6] N. Mayedwa, N. Mongwaketsi, S. Khamlich, K. Kaviyarasu, N. Matinise, M. Maaza, Appl. Surf. Sci, 2018,
446, 266.
[7] Y. Yang, W. Zhang, F. Yang, B. Zhou, D. Zeng, N. Zhang, G. Zhao, S. Hao, X. Zhang, Nanoscale, 2018,
[8] B.T. Sone, X.G. Fuku, M. Maaza, Int J Electrochem Sci. 2016, 11, 8204.
[9] A.T. Khalil, M. Ovais, I. Ullah, M. Ali, Z.K. Shinwari, D. Hassan, M. Maaza, Artif. Cells. Nanomed.
Biotechnol, 2018, 46, 838.
[10] Pandian, C. J., Palanivel, R., S. Dhananasekaran, Chin. J. Chem. Eng, 2015, 23, 1307.
[11] S. Sudhasree, A. Shakila Banu, P. Brindha, G.A. Kurian, Toxicol Environ Chem. 2014, 96, 743.
[12] N. Gong, K. Shao, W. Feng, Z. Lin, C. Liang, Y. Sun, Chemosphere. 2011, 83, 510.
[13] Y. Zhang, Appl Surf Sci, 2015, 344, 33.
[14] M. Kundu, L. Liu, Mater. Lett, 2015, 144, 114.
[15] R.A. Soomro, Z.H. Ibupoto, M.I. Abro, M. Willander, Sens. Actuator B-Chem, 2015, 209, 966.
[17] F.T. Thema, E. Manikandan, A. Gurib-Fakim, M. Maaza, J Alloys Compd, 2016, 657, 655.
[18] N. Thovhogi, A. Diallo, A. Gurib-Fakim, M. Maaza, J Alloys Comp, 2015, 647, 392.
Without UV illumination With UV illumination Positive control
Bacterial strain MIC (µg/mL) Bacterial strain MIC (µg/mL)
TABLE 1 MIC calculations of different bacterial strains.
Gram positive Gram positive
Bacillus subtilis 15.6 Bacillus subtilis 7.8
Staphylococcus aureus 62.5 Staphylococcus aureus 15.6
Gram negative Gram negative Oxytetracycline
Pseudomonas aeruginosa 125 Pseudomonas aeruginosa 31.5
Escherichia coli 62.5 Escherichia coli 15.6
Klebsiella pneumonia 125 Klebsiella pneumonia 62.5

TABLE 2 MIC values of different fungal strains
Fungal strain MIC (µg/mL) Positive control

Aspergillus niger 31.25
Aspergillus flavus 125
Candida albicans 62.5
Amphotericin B
Candida glabrata 87.5
Fusarium solani 31.25
Mucor racemosus 31.25
TABLE 3 IC values calculation for different assays using Graph Pad software.
S. No Assay name IC50 value

1 Brine shrimp cytotoxicity 43.73
2 Anticancer activity 29.68
3 Antileishmanial (promastigotes) 10.62
4 Antileishmanial (amastigotes) 27.58
5 Alpha amylase > 200
6 Human RBCs > 200
7 Human macrophages > 200
AOC_4950_F1.tif
AOC_4950_F4.tif
AOC_4950_F5.tif

Instrumentation 3. Literature Review 4. Materials and Methods 5. Result and Discussion 6. Conclussion 7. Reference

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Instrumentation 3. Literature Review 4. Materials and Methods 5. Result and Discussion 6. Conclussion 7. Reference

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Green synthesis of nickel Nano Particles using aqueous extract of CAUSONIS TRIFOIIA Plant and anti Oxidant

activity of synthesized Nikel Nano particles

4. MATERIALS AND METHODS

5. RESULT AND DISCUSSION

chemical, electronic, mechanical, sensing properties). Metallic nanoparticles (MNPs)

effect, electrical and magnetic properties.

devices, super-capacitors, smart windows, water remediation through photocatalysis,

electrochemical sensing and catalysis of chemical processes. [8, 9,6]

applications in the adsorption of toxic environmental pollutants and dyes. [10]

anti-inflammatory property, NiONPs can also be used in biomedicines. [11]

their wide range biological applications. [17]

physical methods needs massive energy input. [18, 19]

considered to be simple, eco-friendly, economically feasible, easily scaled up and accomplished

nature. [25, 21]

and organic/inorganic solvents. [20]

comprised of (physcion, emodin, kaempferol, rhamnocitrin, quercetin, herbacetin, maesopsin)

activities such as antibacterial, antifungal, biocompatibility assays against RBCs and

line are also investigated.

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis)

Principle of ultraviolet-visible absorption:

Schematic of UV- visible

Applications of UV-Visible Spectrophotometry:

1. Chemical Applications of UV-visible spectrophotometry:

UV-visible spectrophotometry is used to measure the concentration of an unknown sample. For

b. Qualitative Analysis (Identification of an unknown substance):

UV-visible spectrophotometry may be used to identify various classes of compounds in both

Structural Applications of UV-Visible Spectrophotometry:

b. Study of Cis-Trans Isomerism:

c. Molecular Weight Determination:

virgata (Roxb.) (Family: Rhamnaceae) as a potential stabilizing, reducing and chelating

agent. The formation, morphology, structure and other physicochemical properties of

dispersive-spectroscopy (EDS), Transmission electron microscopy (TEM), Raman

deeming particles safe for various applications in nanomedicines.

reported for NiONPs.

Preparation of plant extract

kept in refrigerator at 4°C for future studies.

Synthesis of nickel oxide nanoparticles

characterization techniques. A general study layout is shown in (Figure 1)

using a UV-Vis spectrophotometer (UV-4000 UV-Vis spectrophotometer Germany) in the range

annealed sample on X-ray diffractometer (PANalytical, Netherland) equipped with a Cu

Brine shrimp cytotoxicity Eassay

Leishmania tropica KWH23 parasites were cultured to a density of 1 × 106 cells/mL.

inhibition was measured as:

FBS, 1% penicillin and 1% streptomycin. In addition, cells were grown in humified 5 % CO 2

a reference wavelength of 630 nm.

illumination (germicidal 6 Watt UV rod 6GT5) to determine their antibacterial potential.

Enzyme inhibition potentials:

Protein kinase (PK) inhibition

indicates the inhibition of spore and mycelia formation.

Alpha amylase (AA) inhibition

Following formula was used to calculate percentage inhibition

% inhibition = [(ODS – ODN) ÷ (ODB – ODN)} × 100

Biocompatibility against human RBCs

[Absorbance of sample – Absorbance of negative control

Biocompatibility against macrophage

below formula was utilized to calculate % inhibition of macrophages.

scavanging of DPPH applying the formula below:

Total reducing Power (TRP)

per/mg of the test sample.

l antioxidant capacity (TAC)

TAC test was performed using phosphomollybdenum method. [9]

equivalent of ascorbic acid per/mg of test sample.