1.

ABSTRACT:

Green synthesis in an effective way to synthesize silver

nanoparticles due to its eco-friendly, simple, cost effective and efficient
protocol. The present study included the production of environmentally benign
AgNPs using Viscum orientale leaf extract, in which biomolecules act as a
stabilizing agent. In the present report, bio-reduction of silver nitrate into silver
nanoparticles using the leaf extract of Viscum orientale is explained. In this
report, an aqueous extract of fresh leaves of Viscum orientale was used for the
biosynthesis of silver (Ag) nanoparticles. Different biological methods are
gaining recognition for the production of silver nanoparticles due to their
multiple applications. The use of plants in the green synthesis of nanoparticles
emerges as a cost-effective and eco-friendly approach. Characterization of
nanoparticles was done using two methods, which includes; ultraviolet-visible
spectroscopy (UV-Vis) and size and shape of the silver nanoparticles were
analysed by scanning electron microscope(SEM). UV-Vis spectrum of aqueous
medium containing silver nanoparticles showed absorption peak at around
480nm. Scanning electron microscope micrographs showed the formation of
well separated silver nanoparticles of the size in the range of 119 to 222nm. The
antibacterial activity of AgNPs against generally found bacteria was assessed to
find their potential use in silver-containing anti-bacterial product. The AgNPs
exhibited good anti-bacterial activity against Gram-negative and Gram-positive
bacteria. But it showed higher antibacterial activity Staphylococcus aureus,
Escherichia coli and Bacillus cereus than Salmonella typhi and Bacillus subtilis.
This biosynthesized AgNPs were found to be multifunctional with good
antioxidant activities. Hence, this method can be employed in large-scale
nanoparticles can be synthesized and can be used in many medical and
technological applications.
2. INTRODUCTION

The field of nanotechnology is one of the most active areas of research in

modern material science. Nanoparticles exhibit completely new or improved
properties based on specific characteristics such as size, distribution and
morphology. New applications of nanoparticles and nanomaterials are emerging
rapidly. Nanocrystalline silver particles have found tremendous applications in
the field of high sensitivity biomolecular detection and diagnostics,
antimicrobials and therapeutics, catalysis and micro-electronics. However, there
is still a need for economic, commercially viable as well environmentally clean
route to synthesize silver nanoparticles.[1,2]

A number of approaches are available for the synthesis of silver

nanoparticles for example, reduction in solutions, chemical and photochemical
reactions, thermal decomposition of silver compounds, radiation assisted,
electrochemical, sonochemical, microwave assisted process and recently via
green chemistry route.[4,8,10]

Unfortunately, many of the nanoparticle synthesis or production methods

involve use of hazardous chemicals, low material conversions, high energy
requirements, difficult and wasteful purifications[80]. Biosynthetic methods
employing either biological microorganisms or plant extracts have emerged as a
simple and viable alternative to chemical synthetic procedures and physical
methods[14,15,22]. Most of the methods are still in the developmental stage and
various problems are often experienced with the stability of nanoparticle
preparations, control of crystal growth and aggregation of particles.

Green synthesis provides advancement over chemical and physical

method as it is cost effective, environment friendly, easily scaled up for large-
scale synthesis and further there is no need to use high pressure, energy,
temperature and toxic chemicals[10,31,47]. Using plants for nanoparticle
synthesis can be advantageous over other biological processes because it
eliminates the elaborate process of maintaining cell cultures and can also be
suitably scaled up for large-scale synthesis of nanoparticles under non-aseptic
environment. Silver nanoparticles play a profound role in the field of biology
and medicine due to their attractive physiochemical properties[3,4,5].

Silver products have long been known to have strong inhibitory and
bactericidal effects, as well as a broad spectrum and antimicrobial activities,
which has been used for centuries to prevent and treat various diseases, most
notably infections[6,8,11]. Silver nanoparticles are reported to possess anti-
fungal, anti-inflammatory, anti-viral, anti-angiogenesis and anti-platelet
activity[18,19,20,48,50,51,60].

Several microorganisms, such as bacteria, fungi and yeasts, have come up

as nanofactories, synthesizing metal nanoparticles of Ag and Au[22,33].
However, the use of plants for the fabrication of nanoparticles has drawn
attention, because of its rapid, economical, eco-friendly protocol, and it provides
a single step technique for the biosynthesis process[64]. Biological approaches
using microorganisms and plants or plant extracts for metal nanoparticle
synthesis have been suggested as valuable alternatives to chemical methods[76].
An important branch of biosynthesis of nanoparticles is the application of plant
extract to the biosynthesis reaction.

The present study aims to synthesize silver nanoparticles by a green

biological route, using an extract of Viscum orientale leaves and characterization
of the synthesized silver nanoparticles utilizing UV-Visible spectroscopy.
Besides, their antibacterial activity against different pathogenic microorganisms,
antioxidant activity of AgNPs, Phytochemical screening, protein estimation and
hemagglutination assay are investigated.
Antioxidants:

An antioxidant is a molecule that inhibits the oxidation of other

molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen
from a substance to an oxidizing agent. Oxidation reactions can produce free
radicals[2]. In turn, these radicals can start chain reactions. When the chain
reaction occurs in a cell, it can cause damage or death to radical intermediates
and inhibit other oxidation reactions[7]. They do this by being oxidized
themselves, so antioxidants are often reducing agents such as thiols, ascorbic
acid, or polyphenols. Antioxidants have an extra electron, so they can donate
their extra electron to a free radical. Antioxidants donate an electron to a free
radical, by neutralizing them[38,39,40,41,42].

Antibacterial activity

An antimicrobial (antibacterial) molecule kills or inhibits the growth of

microbes such as bacteria, fungi, protozoans or viruses[20,43,49]. Antimicrobial
drugs either kill microbes or prevent the growth of microbes.

Antibiotics are most important weapons in fighting microbial infections

benefitting the human health. But the emergency of antibiotics resistant
microbial species limited the use of antibiotics[56,58,61]. The secondary
metabolites of higher plants may give a new source of antimicrobial agents with
possibly novel mechanism of action.
Target Microbial Species for antibacterial activity:

Escherichia coli:

E.coli is a Gram-negative, rod shaped bacterium that is commonly found

in the lower intestine of warm blooded organisms (endotherms). Most E.coli
strains are harmless, but some serotypes can cause serious food poisoning in
humans, and are occasionally responsible for product recalls due to food
contamination. The harmless strains are part of the normal flora of the gut, and
can benefit their hosts by producing vitamins K2 and by preventing the
establishment of pathogenic bacteria within the intestine.

Bacillus subtilis:

Bacillus subtilis is one of the oldest named bacteria. It is non-pathogenic,

Gram-positive, aerobic, sporulating bacteria commonly found in soil. A member
of the genus Bacillus, B.subtilis is rod shaped and has the ability to form a
tough, protective endospore, allowing the organism to tolerate extreme
environment conditions. It is also been called hay bacillus or Grass bacillus. It
may contaminate food but rarely causes food poisoning. B.subtilis produces the
proteolytic enzyme subtilisin. B.subtilis spore can survive the extreme heat
during cooking. B.subtilis is responsible for causing ropiness-asticky, stringy
consistency caused by bacterial production of long chain polysaccharides in
spoiled bread dough.

Staphylococcus aureus:

Staphylococcus aureus is a facultative anaerobic, Gram-positive coccus

and is the most common cause of staph infections. It is frequently part of the
skin flora found in the nose and on skin. The carotenoid pigment
staphyloxanthin is responsible for S. aureus characteristic golden colour, which
may be seen in colonies of the organism. This pigment acts as a virulence factor
with an antioxidant action that helps the microbe evade death by reactive oxygen
species used by the host immune system. S. aureus can cause a range of illnesses
from minor skin infections, such as pimples, impetigo, boils (furuncles),
carbuncles, scalded skin syndrome.
Bacillus cereus:

Bacillus cereus is a Gram-positive, rod shaped, aerobic, facultative

anaerobic, motile, beta haemolytic bacterium commonly found in soil and food.
Some strains are harmful to humans and cause foodborne illness, while other
strains can be beneficial as probiotics for animals. It is the cause of “fried rice
syndrome”, as the bacteria are classically contracted from fried rice dishes that
have been sitting at room temperature for hours. B. cereus bacteria are
facultative anaerobes, and other members of the genus Bacillus, can produce
protective like endospores. Its virulence factors include cereolysin and
phospholipase C.

Salmonella typhi:

Salmonella is a genus of rod-shaped (bacillus) gram-negative bacteria of

the family Enterobacteriaceae. The two species of Salmonella are Salmonella
enterica and Salmonella bongori. Salmonella enterica is the type species and is
further divided into six subspecies that include over 2500 serotypes.
 Salmonella species are non-spore forming, predominantly motile
enterobacteria with cell diameter between about 0.7 and 1.5μm, lengths from 2
to 5μm, and peritrichous flagella(all around the cell body). They are
chemotrophs, obtaining their energy from oxidation and reduction reactions
using organic sources. They are also facultative anaerobes, capable of generating
ATP with oxygen when it is available, or when oxygen is not available, using
other electron acceptors or fermentation (anaerobically). S.enterica subspecies
are found worldwide in all warm-blooded animals and in the environment.
S.bongori is restricted to cold-blooded animals, particularly reptiles.

Salmonella species are intracellular pathogens. Certain serotypes cause

illness. Nontyphoidal serotypes can be transferred from animal to humans and
from human to human. They usually invade only the gastrointestinal tract and
cause Salmonella food poisoning. Typhoid fever occurs when Salmonella
invades bloodstream- the typhoidal form, or in addition spreads throughout the
body, invades organs, and secretes endotoxins.

Selected medicinal plant for the green synthesis of silver nanoparticles:

Viscum orientale.

Viscum is a genus of about 70-100 species of mistletoes, native to

temperate and tropical regions of Europe, Africa, Asia and Australia.
Traditionally, the genus has been placed in its own family Viscaceae, but recent
genetic research by the Angiosperm Phylogeny Group shows this family to be
correctly placed within a larger circumscription of the sandalwood family,
Santalaceae.

They are woody, obligate hemiparasitic shrubs with branches 15-80cm

long. Their hosts are woody shrubs and trees. The foliage is dichotomously or
verticillately branching, with opposite pairs or whorls of green leaves which
perform some photosynthesis but with the plant drawing its mineral and water
needs from the host tree. Different species of viscum tend to use different host
species.

The flowers are inconspicuous, greenish-yellow, 1-3 millimetres

diameter. The fruit is a berry, white, yellow, orange, or red when mature,
containing one or more seeds embedded in very sticky juice, the seeds are
dispersed when birds eat the fruit, and remove the sticky seeds from the bill by
wiping them on tree branches where they can germinate.

Scientific classification:

Kingdom : Plantae

Division : Angiosperms

Class : Eudicots

Sub-class : Core eudicots

Order : Santalales

Family : Santalaceae

Genus : Viscum

Species : Orientale
Common name: Blunt leaved mistletoe, orientale mistletoe.

Botanical features:

A small semi parasitic features bushy shrub of 30-50cm. Stem is

cylindrical but branchlets are four angled, jointed and green. Flowers are
unisexual, small, inconspicuous, sessile and often forms a crowded cluster at the
node. Floral lobes are four and are deciduous. Stamens are adnate to the floral
lobes. Fruits are a globose green berry

Habit: Parasite

Native: India, Sri Lanka, China, Australia

Common comments: A common parasite on Pongamia trees.

The leaf extract of Viscum orientale is well known for its different types of
pharmacological properties such as haemostatic, antihelminthic, anti-parasitic,
anti-dysentric, antipyretic and anticancerous. It has long been used to treat pain,
neuropharmacological disorders and various forms of tumor. The methanol
extract of the V.orientale leaves are used as anti-nociceptive, CNS depressant.
Other species of Viscum such as Viscum album is reputed for its nervous system
relaxing effect and believed to protect from high blood pressure and stroke. A
secondary use of V.album has been reported for fevers, measles and whooping
cough. V.album combined with Cardamine pratensis also has a reputation for
nervous afflictions. Viscum Oriental is reported for cytotoxic and Antibacterial
activity. It is reported to contain reducing sugar, Alkaloid ,Tannins and
flavonoids. Viscum album has been reported for Anti-tumor activity and
stimulation of the immune function against several forms of cancer. A wide
range of biological activities have been reported including antiviral,
Antihypertensive, cell proliferation inhibition activities, immunemodulating, and
inflammation modifying effects from other species. Medicinal preparations
containing various species of Viscum are also listed in official pharmacopeias
and used in homeopathic, phytotherapeutic , or anthroposophical remedy.
3.REVIEW OF LITERATURE

The green environment friendly processes in chemistry and chemical

technologies are becoming increasingly popular and are much needed as a result
of worldwide problems associated with environment concerns. Silver is the one
of the most commercialised nanomaterial with five hundred tons of silver
nanoparticles production per year and is estimated to increase on next few years.

The green synthesis of nanoparticles and application are gaining intense

importance in biomedicine,the smaller size of nanoparticles (1-100nm), high
surface area and reactivity provide them the ability for therapeutic purpose in
different dosage forms and dosing forms and dosing routes[20,48,51,56,60 and
66]. Nanoparticles could be derived from various sources of gas,liquid or solid
phases they can be synthesized using different synthesis methods like
physical,chemical,and biological synthesis.

Recent years researches are interested on developing efficient method for

the large scale synthesis of nanoparticles (NPs). Nanomedicine is a rapidly
developing and promising field that makes best use of inert metals like silver,
gold and platinum to synthesize metallic nanoparticles with high therapeutic
potential for various biomedical applications[5,14,50]. Among all metal
nanoparticles, silver nanoparticles (AgNPs) have much attention due to the
surface plasmon resonance (SPR) (strong absorbtion in the visible region),which
can be easily observed in UV-visible spectrometer. Silver with its potent
antimicrobial activity has been used in the synthesis of silver nanoparticles
which finds extensive use in the preparation of food processing, topical
ointments and medical implants.[52]

The synthesis of silver nanoparticles has been carried out by various

methods such those based on reduction in solution, chemical and photochemical
reactions.[4,12]
 Green synthesis of silver nanoparticles using various medicine plants
including, Saraca indica, Pedalium murex, Nelumbo nucifera, Cydonia oblong,
Nyctanthes arbor-tristis, Azadirachta indica, Boerhaavia diffusa has been
reported.[8,21,23,26]

Such green synthesized silver nanoparticles from Helicteres isora, Ruta

graveolens, Aristolochia indica L. and antibacterial activities. With these
evidences, This study was designed to synthesize AgNPs using aqueous Viscum
orientale leaf extract and assess their antioxidant and antibacterial activity.

Considering the vast potentiality of plants as sources this work aims to

apply a biological green technique for the synthesis of silver nanoparticles as an
alternative to conventional methods. In this regard, fruit extract of Tabebuia
rosea, popularly known as ‘Trumpet tree’ a species of family Bignoniaceae was
used for bioconversion of silver ions to nanoparticles. It used to decoration, and
its leaves cure the breast cancer, and its roots used to treat tonsil inflammation,
fever, abdominal pain, diarrhea and various other disorder.[43,81]

The green chemistry method is based on the mechanism of plant assisted due
to the presence of phytochemicals. The main phytochemicals involved are
carbohydrates, proteins and amino acids.

The effect of concentration of metal ions and concentrations of concentrations of

fruit extract quantity were also evaluated to optimize route to synthesis of silver
nanoparticles. The method applied here is simple, cost effective, easy to perform
and sustainable.[16]

The present study aims to synthesize silver nanoparticles by a green

biological route, using an extract of Viscum orientale and characterization of the
synthesized silver nanoparticles utilizing UV-visible spectroscopy. Besides,
their antimicrobial activity against pathogenic microorganism and antioxidant
activity of AgNPs solution was investigated. In the presence work, a leaf extract
of Viscum orientale used as reductant and stabilizer for silver nanoparticles.
4. OBJECTIVES:

1. Phytochemical screening of Viscum orientale leaf extract

2. Green synthesis and characterization of silver nanoparticles(AgNPs)
3. Protein estimation of synthesized AgNPs solution by Lowry’s method
4. Antibacterial activity of synthesized AgNPs of leaf of Viscum
orientale
5. In vitro antioxidant activity of synthesized AgNPs solution:
a) DPPH radical scavenging assay
b) Hydroxyl radical scavenging assay
c) Reducing power capacity
d) Nitric oxide radical scavenging assay
6. Hemagglutination assay
7. Antihelminthic assay
5. MATERIALS AND METHODS
5.1 Chemicals:

Silver nitrate, Ethyl acetate, Chloroform, Petroleum ether, NaOH, Sodium

carbonate, FC reagent, Glacial acetic acid, Sulfanilamide, Potassium iodide,
Ammonium sulphate, Methanol, are obtained from Finar chemical limited,
Ahmadabad. EDTA, NaCl, Ascorbic acid, TCA, BHT, Iodine, FeCl3, CuSO4,
are from SRL. Sodium nitroprusside, Nitric acid, Potassium ferricyanide, are
from Rankem limited, Haryana. BSA, Acetone, Napthylene diamine
dihydrochloride, DNPH, Nutrient broth, are from High media laboratories
private limited, Mumbai. DMSO, H3PO4, Sodium potassium tartarate, are from
Merck specialities private limited, Mumbai.

5.2 Plant collection and Authentication:

The leaves of Viscum orientale was collected from University of Mysuru,

Manasagangothri campus.it was primarily authenticated by
5.3 Preparation of aqueous and solvent extract[62]:

20g of leaves sample for different extract was weighed and leaves are
broke into small pieces. The leaves were added to a beaker containing 100ml of
double distilled water and boiled for 20-30 mins. Then the boiled sample was
filtered using Whatman filter paper to obtain aqueous extract.

The leaves are shade dried and powdered for the preparation of
phytochemical screening sample. 10g of the powdered sample was weighed and
added to 50ml of Ethanol to get ethanol extract, 50ml of Hexane to get Hexane
extract, 50ml of Chloroform to get Chloroform extract and 50ml of Ethyl acetate
to obtain Ethyl acetate extract was added, mixed and covered with aluminium
foil. Then keep it in a stirrer for 4 to 6 hours at room temperature. Then filter by
using Whatman filter paper to get clear solution. The filtrate is used for analysis.

5.4 Phytochemical screening of extracts[5,7,19]:

Qualitative tests:

The aqueous extract, Ethanol extract, Ethyl acetate, Hexane extract,

Chloroform extract were subjected to the following qualitative tests for the
identification of its constituents.

a. Test for alkaloids: In this test, 1ml of leaf extract was shaken with 5ml of
27%HCL and heated gently in a steam bath for a minute. Then 0.5ml of
Wagner’s reagent was added and observation was made for a brick red color.

b. Test for saponins (form test): This was carried out according to the method
of Horbone.1ml of leaf extract were taken in separate test tubes and shaken
vigorously. It was then allowed to stand for about a minute and observation was
made for the formation of stable forms.
c. Test for flavonoids(alkaline reagent test): The extract was treated with few
drops of NaOH solution. The observation was made for the formation of any
precipitate.

d. Test for phenols and tannins(ferric chloride test): To 1ml of the leaf
extract, 3 drops of 10% Ferric chloride was added and observation was made for
the appearance of blackish-green or blackish-blue colouration.

e. Test for carbohydrates (Benedict’s test): The extracts were treated with
Benedict’s reagent and heated gently. The observation was made for the
formation of orange red precipitate.

f. Test for glycosides (legal’s test): The extracts were treated with sodium
nitroprusside in pyridine and sodium hydroxide. Observation was made for the
formation of pink to blood red colour.

g. Test for phytosterols (Salkowski’s test): The extracts were treated with
chloroform and filtered. The filtrates were treated with the few drops of
concentrated sulphuric acid. The observation was made for the appearance of
golden yellow colour.

h. Test for Vitamin-C (DNPH test): The test solutions were treated with
Dinitrophenyl hydrazine dissolved in concentrated sulphuric acid. The
observation was made for the formation of yellow precipitate.

i. Test for Proteins and amino acids (Xanthoproteic test): The extracts were
treated with few drops of concentrated Nitric acid. The observation was made
for the formation of yellow colour.

j. Test for Terpenoids: The crude extracts were dissolved in 2ml of chloroform
and evaporated to dryness. To this, add 2ml of concentrated sulphuric acid was
added and heated for about 2 minutes. The observation was made for the
formation of grayish colour.
5.5 Synthesis of silver nanoparticles (AgNPs)[11,12,16]:

Preparation of Silver nitrate solution:

0.01697g of Silver nitrate was dissolved in 100ml of triple distilled

water to get 1mM AgNO3.

Preparation of leaf extract for Silver nanoparticles synthesis:

20g of fresh leaves were added in 100ml of double distilled water and
boiled for about 20 minutes and filtered by using Whatman No. 1 filter paper.

Principle:

The preparation of silver nanoparticles by using plant extract is a

bioreduction process; the silver ions on bioreduction to form silver metal in
nanometer range forming dark brown colour is measured at 350-700 nm.

Procedure:

1mM of 100ml Silver nitrate solution was prepared in a 250ml beaker

covered with aluminium foil, and kept in a magnetic stirrer. With vigorous
stirring, 10-15ml of leaf extract was added dropwise to the silver nitrate
solution. This process was continued until the colour of the solution changes
from pale green to dark brown for about 6 hours. After the brown colour
formation it is confirm that by characterization using UV-Vis
spectrophotometer. This solution was then centrifuged at 3000rpm for 20
minutes and filtered to get AgNPs and the supernatant was collected and stored
for further studies. The AgNPs are washed 3 times with methanol, vaccum dried
and stored in an eppendorf tube for further characterization and biological
studies.
Synthesis of silver nanoparticles at varying stirring time intervals[46]:

1mM of 100ml Silver nitrate solution was prepared in a 250ml beaker

covered with aluminium foil, and kept in a magnetic stirrer. With vigorous
stirring, 10-15ml of leaf extract was added dropwise to the silver nitrate
solution. To study the stirring time effect, on silver nanoparticles synthesis, the
bio-reduced silver nanoparticles solution was run on a UV-Visible
spectrophotometer at regular time intervals (0, 1, 2, 3, 4 hours) and ʎmax was
noted. Then the graph is plotted by ʎmax on x-axis and absorbance on y-axis.

5.6 Protein estimation by Lowry’s method[57]:

The protein was estimated by using Bovine Serum Albumin (BSA) as

working standard (75μg/ml). The working solution from 0-1ml was pipette out
into 6 different test tubes and the volume was made upto 1ml by adding distilled
water. 0.5ml and 1ml of undiluted synthesized AgNPs solution was taken in
separate test tubes, incubate for 5 minutes at room temperature. Add 0.5ml of
FC reagent to all the test tubes and the obtained solution was read at 570nm.

5.7 ANTIBACTERIAL ACTIVITY[69]:

Preparation of the bacterial sub-culture:

The different bacterial culture used for present investigation are

Escherichia coli, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and
Salmonella typhi were obtained from P.G. Department of Microbiology,
Manasagangothri Campus, University of Mysuru. 100μl of the bacterial culture
was added to 5ml of sterile nutrient broth taken in 5 different sterile test tube
and incubated at 370c for 16 hours to obtain a bacterial subculture.

Preparation of Nutrient Agar medium:

2.8g of nutrient agar in 100ml of distilled water, boil until the air solution
forms.
Procedure:

Antibacterial activity of the synthesized silver nanoparticles was

determined using agar disc diffusion assay method. This was confirmed by the
inhibitory effect on bacterial growth as reflected by the inhibition zone
compared to known antibiotics. Nutrient agar medium was prepared and
sterilized by autoclave. Then sterilized medium was poured over on sterile petri
plates and it was allowed to solidification for 30 minutes. That test bacterial
cultures of 100μl were then swabbed on the surface of the media using sterile L-
shape glass rod. Disc diffusion assay was carried out using standard protocol.
An antibiotic, Gentamicin is taken as positive control. 10μl of Gentamicin, 10μl,
15μl and 20μl of the synthesized silver nanoparticles was applied on separate
sterile discs of diameter 6mm (whatman filter paper discs) and allowed to dry
before being placed on the agar medium. The plates were incubated at 370 c for
24 hours and the resulting zone of inhibition was measured.

5.8 ANTIOXIDANT ASSAYS

5.8.1 DPPH radical scavenging assay[37]:

DPPH radical scavenging assay by the method of Blois (1958)

Principle:

DPPH radicals are scavenging by antioxidant through the donation of a

protein forms the reduced DPPH. The colour change from purple to yellow after
reduction can be quantified by its decrease in absorbance at wavelength 517nm.

Reagent:

1. 0.1mM DPPH (2,2 diphenyl-1-picryl-hydrazyl)

2. 90% Methanol
3. 0.05mM BHT
Procedure:

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging potential

of the AgNPs was determined using the method by Blois (1958). Various
concentrations (20, 40, 60, 80 and 100 μl) of AgNPs and standard butylated
hydroxytoluene (BHT) were taken in different test tubes. In the above samples,
1ml of freshly prepared DPPH (0.1mM) dissolved in methanol was added and
vortexed thoroughly. Finally, the solution was incubated in dark place for 30
min. The absorbance of stable DPPH was recorded at 517nm. The DPPH
(containing no sample) was used as a control prepared using the same
procedure. The free radical scavenging activity was expressed as the inhibition
percentage. The inhibition percentage was calculated using the following
formula.

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷−𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷
Percentage of radical scavenging activity = × 100
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷

BHT was taken as reference standard. The percentage inhibition vs

concentration was plotted and the concentration required for 50% inhibition of
radicals was expressed as IC50 value.

5.8.2 Hydroxyl radical scavenging assay[38]:

Hydroxyl scavenging radical assay was determined by the method of Klein

et al. (1991).

Principle:

Hydroxyl radicals were generated from ferrous ammonium sulphate and

EDTA. This was detected by their ability to react with ascorbic acid to produce
yellow colour complex which was measured at 412nm.
Reagents:

1. 0.13% Ferrous ammonium sulphate

2. 0.26% and 0.018% EDTA
3. 0.85% Dimethyl sulfoxide (DMSO)
4. 0.1 M Phosphate buffer pH 7.4
5. 0.22% Ascorbic acid
6. 17.5% TCA
7. Nash reagent: 75g of ammonium sulphate, 3ml of glacial acetic acid
and 2ml of acetyl acetone were mixed and make up to 1 L with
distilled water.

Procedure:

Various concentration (20, 40, 60, 80 and 100μl) of extract were added
with 1ml of Iron ethylene diamine tetra acetic acid (EDTA) solution (0.13%
ferrous ammonium sulphate and 0.26% EDTA), 0.5ml of EDTA solution
(0.018%), and 1ml of DMSO (0.85% v/v in 0.1 M phosphate buffer, pH 7.4).
The reaction was initiated by adding 0.5ml of ascorbic acid (0.22%) and
incubated at 80-900c for 15 mins in water bath. After incubation the reaction was
terminated by the addition of 1ml of ice cold TCA (17.5% w/v). 3ml of Nash
reagent was added and left at room temperature for 15 min. The reaction mixture
without sample was kept as control. The identity of the colour formed was
measured spectrometrically at 412 nm against reagent blank. The percentage of
hydroxyl radical scavenging activity is calculated by the following formula

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷−𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷
Percentage of radical scavenging activity = × 100
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷
5.8.3 Reducing power capacity[39]:

Reducing power was estimated by the method of Oyaizu (1986)

Principle:

Reducing power was measured by direct electron donation in the

reduction of Fe3+ (CN- )6 to Fe2+ (CN- )6. The product was visualized by the
addition of free Fe3+ ions after the reduction reaction by forming the intense
Prussian blue colour complex, Fe43+[Fe2+(CN-)6]3 with trichloro acetic acid
(TCA) and quantified by absorbance at 700 nm. The absorbance of the reaction
mixture indicates the reducing power.

Reagents:

1. 0.2 M Phosphate buffer

2. 1% Potassium ferricyanide
3. 10% TCA
4. 0.1% Ferric chloride

Procedure:

Various concentrations of the extracts (20, 40, 60, 80 and 100μl) were
prepared. To all the extracts in test tubes, 2.5ml of sodium phosphate buffer was
added followed by 2.5ml of 1% Potassium ferricyanide solution. The content
were vortexed well and the incubated at 50 0c for 20 min. After incubation ,
2.5ml of 10% TCA was added to all the tubes and centrifuged at 3000 g for 10
min. To 5ml of the supernatant, 5ml of deionized water was added. To this 1ml
of 1% Ferric chloride was added to each test tube and incubated at 35 0c for 10
min. the reducing power of the extract was linearly proportional to the
concentration of the sample. Increasing absorbance of the reaction mixture
indicated reducing power. Butylated hydroxyl toluene (BHT) was taken as
reference standard. The absorbance of these solutions was measured at 700nm.
Percentage inhibition was calculated as follows.
 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷−𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷
Percentage of radical scavenging activity = × 100
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷

5.8.4 Nitric oxide radical scavenging assay[40]:

Nitric oxide radical scavenging assay was determined by the method of

Garrat (1964).

Principle:

Sodium nitroprusside in aqueous solution at physiological pH

spontaneously generate nitric oxide, which interact with oxygen to produce
nitrite ions, which can be estimated by the use of Griess Illosvoy reaction.
Scavengers of nitric oxide compete with oxygen leading to decreased production
of nitric oxide.

Reagents:

1. Phosphate buffered saline, pH 7.4

2. 100mM Sodium notroprusside
3. Griess reagent: (1% Sulphanilamide, 2% H3PO4 and 0.1%
naphthylethylene diamine dihydrochloride)

Procedure:

Various concentrations of the extracts (20, 40, 60, 80 and 100μl) were
prepared. Then the volume is made up to 1ml using distilled water. To all the
extracts in test tubes, 1ml of Sodium nitroprusside was added followed by 1ml
of Griess reagent. The content were vortexed well and then incubated at room
temperature for 1 hour. A pink coloured chromophore is formed in diffused
light. The absorbance of these solutions was measured at 540 nm against the
corresponding blank solutions. Percentage inhibition was calculated as follows.

𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷−𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷
Percentage of radical scavenging activity = × 100
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷
6.Hemagglutination assay[73,79]:

Principle:

The interaction(immune reaction) between antibody and a particulate

antigen resulting a visible clumping called agglutination. Antibodies that
produce such reactions are called agglutinins. Agglutination reactions are similar
in principle to precipitation reactions. Method is simplest method. They depend
on the cross linking of polyvalent antigens. Specimen’s were detected by testing
for hemagglutination activity against human red blood cells.

Reagents:

1. 8% Sodium citrate
2. Blood samples from healthy individual
3. 0.01M PBS (pH 7.2)
4. U-shaped micro-titer plates

Procedure:

A serial dilution of the silver nanoparticles solution in U-shaped micro-

titer plate (100μl) was mixed with 100μl of 2% suspension of human erythrocyte
in phosphate buffer, pH 7.2 at room temperature. The plates were left
undisturbed for 1 hour for agglutination to take place. The assay was carried in a
96 well round bottom micro-titer plates. The first well of each column was
served as negative control to which 100μl of blood and PBS was added. For the
second well of the row, 100μl of the nanoparticles solution sample was poured
and it was serially diluted till 7th row using 100μl PBS. Finally 100μl of
processed blood sample was placed in a plane surface without disturbing it.
After 1-2 hours hemagglutination assay results were observed visually.
7.Antihelminthic assay[25,26]:

Collection of earthworms:

The earthworms are collected from Gayathripuram, Mysuru

Procedure:

Adult motility in-vitro assay was conducted on mature live earthworms.

The assay was performed on adult earthworms. Due to it’s anatomical and
physiological resemblance with the intestinal round worm parasite of human
beings. Because of easy availability, earthworms have been used extensively for
the preliminary in-vitro evaluation of antihelminthic compounds in-vitro. The
earthworms of 6-9cm in length were used in the experimental protocol. The
earthworms ranged in live wet mass between 820-1500g. the earthworms were
washed with distilled water to remove the fecal matter.

Assay:

The study was conducted by exposing the worms to different

concentrations of synthesized silver nanoparticles solution. In-vitro studies were
performed according to the standard method. 15ml formulations containing 3
different concentrations, each of aqueous extract, Silver nitrate solution and
Silver nanoparticles solution of it’s various fractions (5, 10, 15ml) were
prepared and worms were placed in it. The control was prepared with saline and
water. The standard was prepared using Silver nitrate solution. Each saline,
water, Silver nitrate solution and Silver nanoparticles solution was taken in
different concentrations (5, 10 and 15ml). Time of paralysis was noted when no
movement of any sort could be observed except when the worms were shaken
vigorously. Time for death of worms was recorded after ascertaining that the
worms neither moved when shaken vigorously nor when dipped in warm water
and thus worm lost their motility followed with fading away of their body
colour. The mean paralysis time and mean lethal time of each concentration was
recorded.
8.RESULTS AND DISCUSSIONS

8.1 Phytochemical screening of Viscum orientale:

The phytochemical analysis obtained qualitatively by all the extracts of

Viscum orientale is present in the following table 1.

Table-1. Phytochemical screening of Viscum orientale.

Constituents Hexane Chloroform Ethyl Ethanol Water

acetate
Alkaloids + + + + +
Saponins + - - + +
Flavonoids + + + - -
Phenols and - + + + +
tannins
Carbohydrates + + - - +
Glycosides + - + - -
Phytosterols + - + + +
Vitamin-C - - + - -
Protein and - + - - +
amino acid
Terpenoids + - - + +

‘+’ indicates presence and ‘-’ indicates absence

The phytochemical screening of Viscum orientale was performed as per

procedure[5,7,19]. The leaf extract of Viscum orientale revealed the presence of
the major chemical constituents of the extract were flavonoids, alkaloids,
tannins, Phytosterols, Saponins, glycosides, carbohydrates, amino acids and
proteins.
8.2 Synthesis of silver nanoparticles (AgNPs)

Fig 1-Visual observation

AgNO3

A B
Fig.1 Visual observation of the solution before bio-reduction (A) and after bio-reduction (B)

In accordance with the literature studies, AgNPs solution has

dark brown colour. The colour of the Viscum orientale was pale green (fig.1)
before its treatment with silver nitrate solution, but after the reaction it turned to
dark brown(fig.1), indicating the formation of AgNPs due to reduction of silver
ions by active molecules present in the extract. This colour is attributed to
surface plasmon resonance, which is a size-dependent property of
NPs[11,12,16].
Figure-2: UV-Visible spectra analysis

UV-Visible spectroscopy was employed to understand the biosynthesis

of silver nanoparticles by Viscum orientale (fig.2). It is generally recognized that
UV-Visible spectroscopy could be used to examine size and shape of controlled
NPs in aqueous suspensions. This analysis showed the sharp absorbance at
around 480nm (fig.2). Which was specific for AgNPs. The UV-Visible
absorption band in the current visible light region (420-500nm ) is an evidence
of the presence of surface Plasmon resonance (SPR) of AgNPs[22,47,53].

Fig.1 indicates the formation of silver nanoparticles which turn pale green
solution to reddish brown colour. Fig.2 indicates the silver nanoparticles formed
after stirring of 4 hours in the Viscum orientale leaf extract solution exhibited
an absorption spectrum ranging from 350–700nm with a peak at 480nm and
giving the yield of 30mg/15ml of extract.

8.3 Synthesis of silver nanoparticles at varying stirring times:

Figure.8.3.1: 0 hour

Time(0 hour) Time(1 hour)

1.8

1.6

1.4

1.2
Absorbance

0.8

0.6

0.4

0.2

0
360380400420440460480500520540560580600620640660680700
Wavelength(nm)

Figure.8.3.2: 1 hour

Time(1 hour) Time(1 hour)

1.8
1.6
1.4
1.2
Absorbance

1
0.8
0.6
0.4
0.2
0
360380400420440460480500520540560580600620640660680700
Wavelength(nm)
Figure.8.3.3: 2 hours

Time(2 hours) Time(2 hour)

2
1.8
1.6
1.4
Absorbance

1.2
1
0.8
0.6
0.4
0.2
0
360380400420440460480500520540560580600620640660680700
Wavelength(nm)

Figure.8.3.4: 3 hours

Time(3 hours)
Time(3 hours)
2.5

2
Absorbance

1.5

0.5

0
360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
Wavelength(nm)
Figure.8.3.5: 4 hours

2.5
Time(4 hours) Time(4 hours)

2
Absorbance

1.5

0.5

0
360380400420440460480500520540560580600620640660680700
Wavelength(nm)

The stirring time effect on the synthesis of silver nanoparticles is shown

in the above five graphs. The colour intensity and monodispersity increased
peak (SPR) at a wavelength range of 400-500nm corresponds to AgNPs which
absorb radiations intensely at a wavelength of 480nm due to the transition of
electrons[46].
 Figure.8.4: SEM images of silver nanoparticles synthesized from
Viscum orientale leaf extracts at various magnifications.

SEM characterizations of the synthesized silver nanoparticles are shown

in the figure ( 8.4 ), it shows relatively spherical-shaped nanoparticles formed
with diameters that range from 119-222nm[66,78].

8.5 Protein estimation of AgNPs solution by Lowry’s reagents[57]:

The protein estimation of synthesized silver nanoparticles solution by

Lowry’s reagents contains 7.5 mg/dL.
8.6 Antibacterial activity[69]:

Figure.8.6.1 Antibacterial activity study of AgNPs using different

bacteria
Table.8.6.1 Effect of synthesized AgNPs on different species of
bacteria:

Inhibition zone(mm)
Sl. Name of the
Gentamicin AgNO3 AgNPs AgNPs AgNPs
no organism
(10µg/ml) (10µg/ml) (10µg/ml) (15µg/ml) (20µg/ml)

1 E.coli 14±1 3.1±0.28 5.5±0.5 6.3±0.28 8.1±0.28

2 Staph.aureus 15.5±0.5 3.1±0.28 6.1±0.28 7.2±0.25 10.3±0.3

3 B.subtilis 10.8±0.76 5.5±0.5 5.5±0.5 6.26±0.25 7.3±0.26
4 B.cereus 13.1±0.28 4.3±0.32 6.3±0.28 7.3±0.26 8.2±0.25
5 S.typhi 14.3±0.86 5.5±0.5 6.1±0.28 7.4±0.05 7.1±0.15

Figure.8.6.2: Antibacterial activity:

18
Gentamicin(10μg/ml)
16
AgNPs(10μg/ml)
14
AgNPs(15μg/ml)
Inhibition zone in mm

12 AgNPs(20μg/ml)

10 AgNO3(10µg/ml)

0
E.coli Staph.aureus B.subtilis B.cereus Salmonella
typhi

Graphical representation of zone of inhibition for bacterial species

against synthesized AgNPs from Viscum orientale.
 The AgNPs exhibited good antibacterial activity against both gram
negative and gram positive bacteria. But it showed higher antibacterial activity
against Staphylococcus aureus (gram positive), Escherichia coli (gram negative)
and Bacillus cereus (gram positive) than Bacillus subtilis (gram positive) and
Salmonella typhi (gram positive), these results indicate that AgNPs show very
less antibacterial activity against these microorganisms. Peptidoglycan is
composed of a thick layer of bacterial cell wall, consisting of linear
polysaccharide chains cross linked by short peptides thus forming more rigid
structure leading to difficult penetration of the AgNPs. This high bactericidal
activity is certainly due to the silver cations released from AgNPs that act as
reservoirs for the Ag bactericidal agent. Therefore, AgNPs were widely used in
antibacterial coatings in processing of medical instruments and food industries
for packing. The biologically synthesized AgNPs using different plant extracts
also showed a similar potent bactericidal activity [24,43,47,78].

Silver nanoparticles and silver nitrate inhibited the growth of all

gram-positive and gram-negative bacteria nevertheless; Silver nanoparticles had
superior antibacterial activity than silver nitrate.
8.7 Antioxidant assays
Figure.8.7.1: DPPH radical scavenging assay [37]
14
BHT
12
AgNPs
10
% of scavenging

0
20 40 60 80 100
Concentration of AgNPs solution in μg

The value of the 50% inhibition concentration (IC50)

BHT: 600μg/ml
Viscum orientale : 747.38μg/ml
Figure.8.7.2: Hydroxyl radical scavenging assay [38]
70
BHT
60
AgNPs
50
% of scavenging

40

30

20

10

0
20 40 60 80 100
Concentration of AgNPs solution in μg

The value of the 50% inhibition concentration (IC50) of

BHT: 98.03μg/ml

Viscum orientale: 86.95μg/ml

Figure.8.7.3: Reducing power capacity [39]
120
BHT
100 AgNPs
% of scavenging

80

60

40

20

0
20 40 60 80 100
Concentration of AgNPs solution in μg

The value of the 50% inhibition concentration (IC50) of

BHT: 47.16μg/ml

Viscum orientale: 67.66μg/ml

Figure.8.7.4: Nitric oxide radical scavenging assay [40]

90
80 BHT
70 AgNPs
60
% scavenging

50
40
30
20
10
0
20 40 60 80 100
Concentration og AgNPs solution in μg
The value of the 50% inhibition concentration (IC50) of
BHT: 63.29μg/ml

Viscum orientale: 65.53μg/ml

8.8 Antihelminthic assay[25,26]:

Figure.8.8.1 Antihelminthic study of AgNPs using earthworms
Figure.8.8.2 Antihelminthic assay

80
AgNO3
70 Aqueous extract

60 AgNPs
Death time in mins

50

40

30

20

10

0
5ml 10ml 15ml

Table.8.8.2:Time of Death:
5ml 10ml 15ml
AgNO3 22.8 Mins 18.3 Mins 15 Mins
Aqueous extract 68 Mins 60 Mins 48 Mins
AgNPs 11 Mins 8.2 Mins 6.5 Mins

No paralysis and death occurs in the plates containing worms

with water and saline. The worms placed in 5ml, 10ml, and 15ml
standard AgNO3 solution were paralyzed and died within 20-30
minutes. The worms placed in 5ml, 10ml, and 15ml aqueous extract
take more than 1 hour for paralysis and death. The worms placed in
5ml, 10ml and 15ml synthesized AgNPs solution were paralyzed and
died within 10-15 minutes[25,26].
8.9 Hemagglutination assay[73,79]:

Figure.8.9.1: Hemagglutination study of AgNPs using Human blood

9.Conclusion:
Green synthesis is an effective way to synthesize silver nanoparticles due
to its eco-friendly, simple, cost-effective and efficient protocol. The present
study included the production of environmental benign AgNPs using Viscum
orientale leaf extract, in which biomolecules act as stabilizing agent. AgNPs
have emerged typical antibacterial nanomaterial applied in industry, daily life,
and medicine. Due to the strong activity of AgNPs and release of Ag +, the
biological properties and safety there of have attracted tremendous attentions
from scientists in recent era. A sample, stable and eco-friendly method of
biosynthesizing AgNPs was successfully developed using Viscum orientale leaf
extract. Viscum orientale leaf contain more phenols, alkaloids that play major
roles as reducing agents for using in synthesis of AgNPs. The extract acts as
both reducing and stabilizing agent which was confirmed by UV-Visible
spectrophotometer and SEM techniques reports revealed that synthesized
AgNPs were crystalline in nature with an average particle size 119 to 222nm.
This biosynthesized AgNPs were found to be multifunctional with good
antioxidant activities. This biosynthesized method facilitates best alternative for
both chemical and other physical methods. Hence, this method can be employed
in large-scale nanoparticles can be synthesized and can be used in many medical
and technological applications.
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