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Green Synthesis and Characterization of silver nanoparticles using Kinnow

mandarian peels extract and its application in Shampoo Formulation

Article  in  Research Journal of Pharmacy and Technology · August 2017

DOI: 10.5958/0974-360X.2017.00435.8

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 Research J. Pharm. and Tech. 10(8): August 2017

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE
Green Synthesis and Characterization of silver nanoparticles using Kinnow
mandarian peels extract and its application in Shampoo Formulation
Rajni Bala*, Reecha Madaan, Vibhu, Sandeep Arora
Chitkara College of Pharmacy, Chitkara University, Rajpura, Punjab
*Corresponding Author E-mail: rajni.bala@chitkara.edu.in

ABSTRACT:
Nanoparticles are being viewed as fundamental building blocks of nanotechnology. The most important and
distinct property of nanoparticles is that they exhibit larger surface area to volume ratio. The most effectively
studied nanoparticles today are those made from noble metals, in particular Ag, Pt, Au, and Pd. Silver
nanoparticles have various applications such as biomedical devices, biosensors, catalysis, electronics and
pharmaceuticals. In this study rapid, simple and eco-friendly approach was applied for synthesis of silver
nanoparticles (AgNPs) using Kinnow mandarian aqueous peel extract as the reducing agent as well as capping
agent. The reaction process is simple for the formation of stable silver nanoparticles at room temperature by
using the bio waste of the fruit peel. The synthesis of nanoparticles has been monitored by UV–Visible
spectroscopy (UV–Vis). Synthesized nanoparticles are characterized by Fourier transform infrared spectroscopy
(FTIR), Scanning electron microscopy (SEM). In addition the synthesized nanoparticles were incorporated in the
shampoo formulation. The shampoo formulations containing synthesised silver nanoparticles were evaluated for
physical appearance, solid content, pH, foamability, surface tension and dirt dispersion test. Anti microbial
activity was tested against pathogenic microbes Escherichia coli. and Candida albicanas. It was concluded that
shampoo formulation containing silver nano particles showed enhanced antimicrobial activity as compared to
shampoo formulation not containing silver nano particle and standard antimicrobial agents.

KEYWORDS: biogenic nanoparticles, antimicrobial activity, shampoo, Citrus reticulata, kinnow mandarian
peel.

INTRODUCTION: An eco friendly plant mediated synthesis of in organic

Nanoparticles are fundamental building block of
nanotechnology. The most important and distinct
property of nanoparticles is their exhibit large surface
area to volume ratio. The metallic nanoparticles are
useful as they have showed good antimicrobial efficacy
against bacteria, viruses, and other eukaryotic
microorganisms. A number of approaches are available
for the synthesis of silver nanoparticles for example
facile method, thermal decomposition of silver
compounds, electrochemical, sono chemical, and
microwave assisted process and recently via green
chemistry path[1].
Received on 14.04.2017 Modified on 09.05.2017
Accepted on 28.06.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(8): 2461-2466.
DOI: 10.5958/0974-360X.2017.00435.8
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nanoparticles is a fast growing research in the field of
nanotechnology. Biological organisms like bacteria,
fungi and actinomycetes were also utilized for the
synthesis of AgNPs. Biosynthetic methods employing
plant extracts have drawn attention as a simple and
viable alternative approach to chemical and physical
method. Silver nanoparticles (AgNPs) can be
synthesized from various parts of the herbal plants like
bark of Cinnamomum zeylanicum, Azadirachta indica
(neem) leaves, Citrus limon, tannic acid, Parthenium
hysterophorus leaf, Murraya koenigii (curry leaf),
Lantana camara leaf and various plant leaves[2,3].
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, aqueous extract
of Kinnow mandarian (Citrus reticulate) peels family
 Research J. Pharm. and Tech. 10(8): August 2017

Rutaceae was used for bioconversion of silver ions to Formulation of antidandruff shampoo:
nanoparticles. The mechanism behind the synthesis of Herbal anti-dandruff shampoo was formulated by adding
silver nanoparticles was not known exactly but it is the weighed amounts of herbal ingredients as per the
hypothesized that the enzymes NADH-dependent nitrate composition given in the [Table 1]. All ingredients were
reductase were involved in the reduction of silver ions [4]. taken in motar and pestle mixed to form a homogenous
blend. Methi seeds extract was used to prevent hair fall,
MATERIAL AND METHODS: amla, shikakai and reetha extract was added as cleanser,
All chemicals used in the experimentation were of lemon oil to give aroma, gaur gum was added to
highest purity grade and were obtained from sigma, maintain consistency and Ag nanoparticles were added
Bangalore, India and Merck, Mumbai, India. Kinnow to impart antidandruff property to the shampoo
fruits for the preparation of silver nanoparticles were formulation [6].
purchased from the local market of Rajpura, Patiala,
India and were authenticated from Punjabi university Table 1: Composition of silver nanoparticles loaded antidandruff
shampoo.
Patiala. Ingredients F1 F2 F3 F4
Methi seed extract 2g 2g 2g 2g
Green synthesis of silver nanoparticles: Amla extract 1g 1.5g 2 2.5g
The fresh peels of kinnow fruits were taken and Shikakai extract 1g 1.5g 2 2.5g
Reetha extract 1g 1.5g 2 2.5g
thoroughly washed with distilled water and cut into Lemon oil 1ml 1ml 1ml 1ml
small pieces. 4gm of these peels were weighed and taken Gaur gum 1% 1% 1% 1%
in 40 ml of distilled water and boiled for 2minutes. The Methyl paraben 1ml 1ml 1ml 1ml
extract so obtained was filtered using Whatman filter AgNP 100 uL 80 uL 40 uL
-
paper. 1ml of the prepared extract was added to 5ml of
1Mm solution of silver nitrate and kept in microwave
oven for 1minute at room temperature for the required Evaluation of Shampoo containing silver
colour change. The sample was subjected to several nanoparticles:
short burst of microwave irradiation at frequency of 2.45 Physical appearance:
GHz, at power output of about 100 W [5]. The formulated shampoo formulations were evaluated
for the colour, transparency, odor and foam producing
Characterization of silver nanoparticles: ability.
UV Spectral analysis:
The reduction of pure Ag+ ions was monitored by Determination of pH:
measuring the UV Visible spectra of the resulting The higher pH level may damage the hairs and make
reaction mixture at different interval of time within the them rough, the ideal pH level for shampoos is between
wave length range between 300nm- 600nm using 5-7. The pH of the shampoo formulations was measured
Systronic UV double beam spectrophotometer- 2202. by preparing 10% v/v shampoo solution in distilled
water pH was determined using deluxe pH meter-101 at
Fourier Transform Infrared Spectroscopy: room temperature.
Infrared spectroscopy gives information on the
Determination of percentage of solid contents:
vibrational and rotational modes of motion of a molecule
and hence an important technique for identification andFor the estimation of percentage solid content in a
previously clean, dry and weighed evaporating dish, 4
characterization of a substance. The silver nanoparticles
were analyzed under FTIR for the size conformation. Allgrams of formulated shampoo were taken. It was then
samples of the synthesized nanoparticles were scanned weighed again to confirm the exact weight of the
in the range of 3500– 500 nm by using Bruker Alpha-T shampoo. The liquid portion of the shampoo was
instrument. evaporated by placing the dish on hot plate. The weight
and percentage of the solid contents of shampoo left
Scanning Electron Microscopy (SEM): after complete drying was taken to estimate percentage
[7]
This technique was employed to visualize the size and of solid content .
shape of silver nanoparticles synthesized using kinnow
fruit peel extract. The colloidal dispersion of silver Foaming ability:
nanoparticles was mounted on a copper coated grid, Cylinder shake method was used to test for the foaming
extra solution was removed using filter paper and then ability. 50 ml of the 1% formulated shampoo solution
allowed to dry prior measurement. Finally images of was placed into a 250 ml graduated cylinder, covered
prepared silver nanoparticles were taken using Joel, with one hand and shaken for 10 times. After 1 min of
Japan, Model- LV 6501 at excitation voltage of 2 KV, shaking, the total volume of the foam content was
magnification at 5000X.
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 Research J. Pharm. and Tech. 10(8): August 2017

recorded. Foam stability was valued by recording the

foam volume after 1 min and 4 min of shake test.

Surface tension measurement:

Surface active agents are added into shampoo
formulations to lower down the surface tension and to
adsorb oil and dirt from the formulation so as to increase
the efficiency of formulation. The surface tension of
10% w/v solution of shampoo in distilled water was
measured using stalagmometer at room temperature
using drop count method.
Figure 1: Silver nano particles synthesized using fresh peels of
Dirt dispersion test: kinnow fruit
To measure the dirt dispersion ability of shampoo two
drops of formulated shampoo were added to 10 ml of The bioreduction of Ag+ ion in aqueous solution was
distilled water taken in a test tube. To this solution, one monitored with the help of UV– visible spectroscopic
drop of India ink was added and the test tube was analysis represented in [Figure 2]. The UV-visible
stoppered and shaken ten times. The amount of ink in the spectrum of silver nanoparticles synthesized with the
foam was indicated as None, Light, Moderate or Heavy help of kinnow fruit peel extracts as a reducing agent
[8]
. gave characteristics surface Plasmon absorption band at
412-449 nm.
Wetting time test:
FTIR analysis was carried to check the possibility of any
A canvas paper was cut into 1-inch diameter discs
interaction between the bio molecules and silver ions
having an average weight of 0.44 g. The smooth surface
during the formation of silver nanoparticles by bio
of disc was placed on the surface of 1% v/v shampoo
reduction reactions. The FTIR images of kinnow
solution and the stopwatch was started. The time
mandarian peel extract and silver nanoparticles
required for the disc to sink into shampoo solution was
containing kinnow peel extract are shown in the [Figure
noted down as the wetting time. [8, 9]
3and 4] respectively. The FTIR of silver nanoparticles
showed intense peaks at 3245.38, 2937.77, 1637.13,
Antimicrobial assay of silver nanoparticles loaded
1301.12, 1082.27 and 1019.98 cm-1 the FTIR of kinnow
shampoo:
peel extract showed intense peaks at 3243.28, 2933.70,
The antimicrobial activity of the prepared silver
2895.89, 1635.65 and 1456.63 cm-1 the shift in the
nanoparticles and shampoo loaded with silver
positions of bands clearly indicates that flavonoids,
nanoparticles was studied against various pathogenic
proteins and sugar present in the extract play important
microorganisms such as E. coli and C. albicans using
role in the bioreduction of silver ions and have strong
well diffusion method [10]. Penicillin and amphotericine
ability to bind with silver nanoparticles
was used as the positive control for the study. Sterile
nutrient agar plates were prepared using 24 hr. grown SEM images of green synthesized silver nanoparticles
cultures of test micro organisms. The wells of 10mm are shown in [Figure 5] which indicated the formation of
diameter were cut into plates using sterile cork borer. polydispersed silver nanoparticles and confirmed the
The prepared silver nanoparticles, plain shampoo and formation of silver nanostructure.
shampoo formulation loaded with silver nanoparticles
were loaded into the wells and incubated at 37ºC for 24
hrs; the antimicrobial activity was estimated by
measuring the diameter of zone of inhibition produced
by the penicillin G pellet used as control, shampoo
formulation and shampoo loaded with silver
nanoparticles[11].

RESULTS AND DISCUSSIONS:

The formation of brownish colored solution [Figure 1]
indicated the formation of silver nanoparticles.

Figure 2: UV visible spectra of green synthesized silver

nanoparticles

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 Research J. Pharm. and Tech. 10(8): August 2017

Characterisation of shampoo containing silver

Figure 3: FTIR spectrum of green synthesized silver nanoparticles
nanoparticles:
The results of physical appearance of all indicated that
all formulation have good appearance with uniform
dispersion. All formulations had the good foam forming
ability. The pH of shampoos has been shown to be
important for improving the qualities of hair and to
minimizing irritation to the eyes and scalp. As seen from
[Table 2], all the shampoos were acid balanced with pH
ranged 5.0-5.5, which is near to the skin pH. If the
shampoo has too many solids it will be hard to work into
the hair or too hard to wash out. The result of percent of
solids contents was found between 19-24% which means
good consistency and spreadibility which is easy to wash
out [12]. It has been mentioned that a proper shampoo
should be able to decrease the surface tension of pure
water. The reduction in surface tension of water from
72.8 dynes/cm to 32.4 dynes/ cm by the herbal shampoos
is an indication of their good detergent action. Dirt
dispersion test was carried out to evaluate the cleansing
action of the shampoo formulations; all shampoos ensure
satisfactory cleansing action. Results of evaluation of
silver nanoparticles loaded shampoo are given in [Table
2].

Table 2. Characterization of silver nanoparticles loaded shampoo

formulation.
Parameter F1 F2 F3 F4
s
pH 5.0 5.1 5.5 5.2
Foam 158 165 160 148
ability(ml)
Solid 19 24 20 22
content%
Surface 33.3 32.4 32.2 34.1
tension(dyn
es/cm)
Dirt satisfactory satisfactory satisfactory satisfactory
dispersion
Figure 4: FTIR spectrum of kinnow mandarins peel extract Wetting 142 180 172 151
time

Results of antibacterial and antifungal activity measured

in term of zone of inhibition measured[Figure 7] at
different interval of time indicated that shampoo
formulations containing different concentrations of silver
nanoparticles exhibit enhanced antimicrobial activity as
compared with plain shampoo formulation and control
penicillin G pellet. The enhanced anti microbial activity
may be due to the release of silver cations from silver
nanoparticles which act as reservoir for bactericidal
agent [13]. Results of zone of inhibition are shown in the
[Figure 5 and 6] which indicated that formulation F2
showed maximum antibacterial and antifungal activity
against the studied pathogens.

Figure 5: SEM image of silver nanoparticles

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 Research J. Pharm. and Tech. 10(8): August 2017

After 24 hrs After 24 hrs

After 48 hrs After 48 hrs

After 120 hrs After 120 hrs

After 10 days After 10 days

Figure 5: antimicrobial activity of silver nanoparticles loaded Figure 6: antifungal activity of silver nanoparticles loaded
shampoo against Escherichia coli shampoo against Candida. albicans

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 Research J. Pharm. and Tech. 10(8): August 2017

Figure 7: zone of inhibition of different shampoos formulations and control

CONCLUSION: 10. T Tuutijarvi, J Lu, M Sillanpaa. As (v) adsorption on maghemite

In conclusion it was found that biogenic silver nano nanoparticles. J Hazard Mater. 2009, 166: 1415-1420.
particles using peel extract of Citrus reticulate showed 11. DR Bhumkar, HM Joshi, M Sastry. Chitosan reduced gold
potential antimicrobial activity against bacterial and nanoparticles as novel carriers for transmucosal delivery of
insulin. Pharmaceutical Research. 2009, 24: 1415-1426.
fungal pathogens when formulated as shampoo. The 12. M J Fridhouse, P Lalitha. Biogenic silver nanoparticles-
formulation F2 containing 100µl of silver nanoparticles synthesis, characterisation and its potential against cancer
showed maximum zone of inhibition against bacterial inducing bacteria. Journal of molecular Liquids.
and fungal pathogens. Further the proposed approach 13. A Ahmad, P Mukherjee , S Senapati , D Mandal , MS Khan , R
Kumar, M Sastry. Extracellular biosynthesis of silver
was found to be cost effective, eco friendly simple and is nanoparticles using the fungus Fusarium oxysporum. Colloids
devoid of possibility of development of anti biotic Surf B. 2003; 28:313–318.
resistance. Finally antimicrobial and antifungal activity
of silver nano particle containing shampoo formulation
opens the door for new range of pharmaceutical
products.

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